Using Chimeric Viruses to study Kaposi’s sarcoma-

associated herpesvirus pathogenesis in mice

André Filipe Coimbra e Seixas

Thesis to obtain the Master of Science Degree in

Microbiology

Supervisors:

Professor Doutor João Pedro Monteiro e Louro Machado de Simas

Professor Doutor Jorge Humberto Gomes Leitão

Examination Committee

Chairperson:

Professora Doutora Isabel Maria de Sá Correia Leite de Almeida

Supervisor:

Professor Doutor João Pedro Monteiro e Louro Machado de Simas

Members of the committee:

Doutora Sofia Pinto Guia Marques

November 2016

i

Acknowledgements

I would like to thank Dr. Pedro Simas for accepting me in his lab, for believing in me and for all the

good advices that he gave me during the development of my work. Thank you also to Dra. Isabel Sá-

Correia for letting me choose this master thesis theme. A special thanks to Dra. Marta Pires de Miranda

for the tremendous help that she provided me and for all the patience to teach me and to advise me.

Thank you to all the former and present members of the Herpesvirus Pathogenesis lab, especially Ana

Quendera, Francesca Martin, Sofia Cerqueira and Diana Fontinha, for the friendship and help provided.

Thank you in general to the Instituto de Medicina Molecular for providing a great environment for me to

develop my work and to Instituto Superior Técnico for accepting me in the Microbiology master.

Queria agradecer aos meus colegas de mestrado por toda a amizade e ajuda que me proporcionaram

ao longo de todo o mestrado, em especial à Inês Leonardo por ser uma amiga sempre presente.

Obrigado também a todos os meus amigos, que são a minha 2ª família com a qual eu posso sempre

contar.

Obrigado a toda a minha família por estar sempre presente e por me apoiar sempre em qualquer

momento, sem hesitação. Não poderia deixar de fazer um agradecimento especial ao meu avô, que

sem ele nada disto seria possível.

ii

Abstract

Herpesvirus are one of the most ubiquitous group of viruses in the world. Kaposi’s sarcoma-

associated herpesvirus (KSHV) is one of the eight herpesvirus that infects humans, and it is the

etiological agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric

Castleman’s disease in our species. Without a good model to study KSHV, the solution resides on a

mouse equivalent virus, murine herpesvirus-68 (MHV-68). Previous work done in our lab aimed to

generate chimeric recombinant viruses, where an essential protein in KSHV latency, latency-associated

nuclear antigen (kLANA) was cloned in a MHV-68 background, substituting the mouse herpesvirus

original LANA (mLANA). It was possible to have a chimeric virus where the influence of kLANA in vivo

was addressed, despite establishing latency in the spleen with lower levels when compared to the wild

type virus. kLANA protein sequence contains an internal acidic repeat region that is poorly

characterized. Only in vitro studies were performed so far regarding this part of the protein. The objective

of this work was to test the importance of these internal regions in vivo, by generating two chimeric

recombinant viruses’: vkLANAΔ465-929 and vkLANAΔ332-929, lacking part or the entirety of the repeat

region, respectively. In vivo it is clear that vkLANAΔ465-929 has the same latency levels as the virus

expressing full length vkLANA, whilst vkLANAΔ332-929 completely failed to established latency in the

spleen. Thus, data indicates that the aspartate and glutamate (DE) (a.a. 330-464) is crucial for the

infection and for the correct latency in the spleen.

Keywords: kLANA; KSHV; internal repeat region; MHV-68; chimeric viruses; in vivo.

iii

Resumo

Herpesvírus são dos vírus mais ubíquos do mundo. O herpesvírus associado ao sarcoma de

Kaposi é um dos oito herpesvírus que afetam humanos, e é o agente etiológico de doenças como o

sarcoma de Kaposi, linfoma primário das cavidades e a doença de Castleman multicêntrica. Sem um

modelo animal para estudar o KSHV, a solução reside no uso de um herpesvírus de ratinho, o

herpesvírus murídeo tipo 68. Previamente no nosso laboratório criou-se um vírus quimérico, no qual

uma proteína essencial na latência de KSHV, o antigénio nuclear associado à latência, foi clonada no

genoma de MHV-68, substituindo a LANA original do herpesvírus de ratinho. Obtiveram-se assim vírus

quiméricos onde a influência de kLANA pode ser analisada in vivo apesar de estabelecer latência com

níveis mais baixos que o vírus MHV-68. kLANA é crucial para o estabelecimento de latência do vírus.

Uma das partes menos bem caracterizadas de kLANA é a sua região interna repetitiva. Apenas estudos

in vitro foram realizados em relação a esta parte da proteína, sendo que o objetivo deste trabalho era

testar a importância desta região interna de kLANA in vivo. Dois vírus quiméricos foram criados,

vkLANAΔ465-929 e vkLANAΔ332-929 onde parte, ou a totalidade da região interna repetitiva for

removida, respetivamente. In vivo é possível observar que vkLANAΔ332-929 tem mais impacto no

estabelecimento de latência no baço, enquanto vkLANAΔ465-929 tem menos impacto, indicando que

a região interna de aspartato e glutamato (DE) é essencial para a infeção e para o estabelecimento de

latência no baço

Palavras-chave: kLANA; KSHV; região interna repetitiva; MHV-68; vírus quiméricos; in vivo.

iv

Abbreviations

%GC – Percentage of guanine and cytosine

AIDS – Acquired Immune Deficiency Syndrome

BAC – Bacterial artificial chromosome

BHK – Baby hamster kidney

cDNA – Complementary DNA

CPE – Cytopathic effect

Ct – Threshold cycle

CWS – Cell working stock

D.p.i. – Days post-infection

DBD – DNA-binding domain

DMEM – Dulbecco's Modified Eagle Medium

DNA – Deoxyribonucleic acid

EBNA-1 – Epstein-Barr nuclear antigen-1

EBV – Epstein-Barr virus

EDTA - Ethylenediamine tetraacetic acid

FACS – Fluorescence-activated cell sorting

GC – Germinal center

GFP – Green fluorescent protein

GMEM – Glasgow Minimum Essential Medium

HCMV – Human cytomegalovirus

HHV-6 – Human herpesvirus-6

HHV-7 – Human herpesvirus-7

HHV-8 – Human herpesvirus-8

HIV – Human Immunodeficiency Virus

HSV-1 – Herpes simplex virus-1

HSV-2 – Herpes simplex virus-2

IE – Immediate early

kLANA – KSHV latency-associated nuclear

antigen

KS – Kaposi’s sarcoma

KSHV – Kaposi’s sarcoma-associated

herpesvirus

LANA – Latency-associated nuclear antigen

LB – Luria-Bertani

LUR – Long unique coding region

LZ – Leucine-zipper

MHV-68 – Murine herpesvirus 68

Min – Minutes

mLANA – MHV-68 latency-associated nuclear

antigen

MOI – Multiplicity of infection

mRNA – Messenger ribonucleic acid

MuHV-4 – Murine herpesvirus-4

MZ – Marginal zone

ORF – Open reading frame

PBS – Phosphate buffered saline

PBS-T – PBS-Tween 20

PCR – Polymerase chain reaction

PEL – Primary effusion lymphoma

pfu – Plaque forming unit

qPCR – Quantitative PCR

RNA – Ribonucleic acid

RT – Room temperature

v

RTA – Replication and transcription activator

SDS-PAGE - Sodium dodecyl sulphate

polyacrylamide gel electrophoresis

SE – Standard error

SHPM – Single-hit Poisson model

SOB – Super optimal broth

SOC – SOB plus glucose

TAE – Tris-acetate-EDTA

TEMED – Tetramethylethylenediamine

TGS – Tris-glycine-SDS

TR – Terminal repeat

vBcl-2 – Viral B-cell lymphoma 2

v-FLIP – Viral Fas-associated death domain-

like interleukin-1β-converting enzyme-inhibitory

protein

v-GPCR – Viral G protein-coupled receptor

VZV – Varicella-zoster virus

WSM – Working stock media

WT – Wild type

1

Table of contents

Acknowledgements ...................................................................................................................................i

Abstract..................................................................................................................................................... ii

Resumo ................................................................................................................................................... iii

Abbreviations ........................................................................................................................................... iv

Table of contents ..................................................................................................................................... 1

1. Introduction ...................................................................................................................................... 3

1.1 Herpesvirus ................................................................................................................................... 3

1.1.1 Gammaherpesvirus ................................................................................................................ 4

1.2 Kaposi’s sarcoma-associated herpesvirus (KSHV) ....................................................................... 4

1.2.1 KSHV latency-associated nuclear antigen (kLANA) .............................................................. 7

1.2.2 The kLANA internal repeat acidic region ................................................................................ 8

1.3 Murine herpesvirus 68 (MHV-68) .................................................................................................. 9

1.3.1 MHV-68 latency-associated nuclear antigen (mLANA) .......................................................... 9

1.4 MHV-68 as a model to study Kaposi’s sarcoma-associated herpesvirus pathogenesis in mice ........ 10

2. Aim of the project ........................................................................................................................... 12

3. Materials and Methods .................................................................................................................. 13

3.1 Plasmids and cloning................................................................................................................... 13

3.2 Ethics statement .......................................................................................................................... 13

3.3 Mice ............................................................................................................................................. 13

3.4 Bacteria ....................................................................................................................................... 14

3.5 Cell lines ...................................................................................................................................... 14

3.6 Transformation of competent bacteria ......................................................................................... 14

3.7 Control Viruses ............................................................................................................................ 14

3.8 Generation and characterization of recombinant viruses ............................................................ 14

3.9 BAC DNA Preps .......................................................................................................................... 15

3.10 Reconstitution of MHV-68 virus on BHK-21 cells ...................................................................... 16

3.11 Production of viral stocks .......................................................................................................... 16

3.12 Plaque assay (virus titration using suspension assay) .............................................................. 16

3.13 Multi-step growth curves............................................................................................................ 17

3.14 Immunoblotting .......................................................................................................................... 17

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3.15 Transcription analysis of kLANA mutants.................................................................................. 18

3.16 In vivo Assays ............................................................................................................................ 19

3.16.1 Infection of mice ................................................................................................................. 19

3.16.2 Single cell suspensions. ..................................................................................................... 19

3.16.3 Infectious Center Assay (ex vivo explant co-culture assay) ............................................... 20

3.16.4 Sorting of GC B cells .......................................................................................................... 20

3.16.5 Limiting dilution assay and Real-time PCR of viral DNA positive cells .............................. 20

3.17 Statistical analysis ..................................................................................................................... 21

4. Results ........................................................................................................................................... 22

4.1 Generation and characterization of MHV-68 recombinant viruses ............................................. 22

4.2 The chimeric recombinant viruses display normal in vitro growth ............................................... 23

4.3 Expression of kLANA mutant proteins ......................................................................................... 24

4.4 Transcription analysis of kLANA mutants .................................................................................... 25

4.5 The internal acidic repeat region has impact in the establishment of latency in the spleen ....... 26

4.6 Quantification of the frequency of viral DNA-positive total splenocytes and GC B-cells ............ 27

5. Discussion ..................................................................................................................................... 29

6. Conclusion ..................................................................................................................................... 31

7. References .................................................................................................................................... 32

3

1. Introduction

1.1 Herpesvirus

Herpesvirus are amongst the most ubiquitous group of viruses in the world. They are widely spread

across the spectrum of vertebrate species and at least in one invertebrate species. These viruses have

co-evolved with their hosts over long periods of time, being extremely well adapted to each one of them

(Davison et al., 2002). Herpesvirus family can be distinguished by having a large double-stranded DNA

genome (130-250 kb) inside a highly ordered icosahedral-shape nucleocapsid (125-130nm in diameter),

surrounded by a semi-ordered tegument that, in turn, is enclosed within a lipid bilayer envelope with

different viral glycoproteins that are responsible for viral attachment and entry to host cells (Figure 1).

Figure 1: Schematic diagram illustrating the multilayer organization of human herpesvirus. Adapted from Human

Herpesviruses: Biology, Therapy, and Immunoprophylaxis; Chapter 3, Comparative virion structures of human

herpesviruses.

Herpesvirus are also characterized by having two different life cycles: a lytic phase and a latent

phase. In the lytic phase more than 80 genes are expressed in a specific order which leads to the

production of infectious viral particles (virions) that infect and lyse other cells (Wu et al., 2010). The

latent phase is characterized by maintenance of the viral genome in the nucleus as a multicopy, non-

integrated circular form designated episome. In latency, there are no infectious virions produced and

viral gene expression is strongly reduced, allowing the virus to escape unnoticed from the host immune

system (Wu et al., 2010).

As mentioned above, herpesvirus are very well adapted to their hosts, with a fine-tuned

equilibrium between the host’s immune system and the viral spread that, in the vast majority of the

cases, results in the absence of pathogenicity. Only when this equilibrium is disturbed, that is, the

immune system is affected by other diseases, stress or changes in life-style, pathogenicity is higher

(Davison et al., 2002).

4

According to the International Committee on Taxonomy of Viruses, the order Herpesvirales

currently has three families: Alloherpesviridae, Malacoherpesviridae and Herpesviridae, the latter being

divided in three subfamilies: Alphaherpesvirinae, Betaherpesvirinae and Gammaherpevirinae.

Alphaherpesvirinae can infect a wide number of mammal species whereas Betaherpesvirinae and

Gammaherpevirinae are more restricted in their host range. Herpesvirus establish life-long latent

infections. Alphaherpesvirinae remain latent in neurons, Betaherpesvirinae remain latent in the

monocyte cell lineage and Gammaherpevirinae remain latent mainly in lymphocytes (Davison et al.,

2009).

Humans are one of the mammal species that can be infected by Herpesvirus. There are eight

known viruses that can infect humans: Herpes simplex virus type-1 (HSV-1), Herpes simplex virus type-

2 (HSV-2) and Varicella-zoster virus (VZV) from the Alphaherpesvirinae; Human cytomegalovirus

(HCMV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7) from the Betaherpesvirinae;

Epstein-Barr virus (EBV) and Human herpesvirus 8/Kaposi’s sarcoma-associated herpesvirus (HHV-

8/KSHV) from the Gammaherpevirinae (Wu et al., 2010).

1.1.1 Gammaherpesvirus

Gammaherpesvirus can be found widely in the natural environment, infecting several mammal

species. The Epstein-Barr virus (EBV) and the Kaposi’s sarcoma-associated herpesvirus (KSHV) are

representative viruses of the gamma-1-herpesvirus and gamma-2-herpesvirus subfamily division,

respectively (Simas & Efstathiou, 1998 and Chang et al., 1994).

These viruses are distinguished among other herpesviruses for their association with human

cancers (Wen & Damania, 2009). They are able to establish latency in lymphocytes, where the viral

episome is maintained in the nucleus and replicated in step with cellular mitosis. They induce

lymphoproliferative diseases and lymphomas in hosts with a compromised immune system (Nash et al.,

2001).

1.2 Kaposi’s sarcoma-associated herpesvirus (KSHV)

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a blood-borne gammaherpesvirus first

discovered in 1994 by Yuan Chang and colleagues. It is the etiological agent of Kaposi´s sarcoma (KS)

a tumour of endothelial-cell origin mostly found in immunosuppressed HIV infected patients (Coscoy,

2007). Besides KS this virus is also an etiological agent in primary effusion lymphoma (PEL), a B cell

lymphoma and multicentric Castleman’s disease, a B cell lymphoproliferative disorder (Moore & Chang,

2003).

Before the acquired immunodeficiency syndrome (AIDS) epidemics, KS was considered

relatively rare, only affecting elderly man in Mediterranean and Eastern Europe regions. AIDS epidemic

transformed KS in the most severe AIDS-associated cancer (Cai et al., 2010). KSHV infected individuals

5

are found around the world. The seroprevalence of KSHV is below 10% in Northern Europe, Asia and

North America, but in the sub-Saharan Africa the values exceed 50% (Figure 2) (Uppal et al., 2014 and

Mesri et al., 2010).

Figure 2: Geographical seroprevalence of KSHV. Adapted from Mesri et al., 2010.

The genome of KSHV consists in a long unique coding region (LUR) of ≈ 140kb with 53,5% GC

content flanked by approximately 40 non-coding terminal repeats (TR) with 84,5% of GC content (Russo

et al., 1996; Lagunoff and Ganem, 1997).

Like other gammaherpesvirus, the KSHV life cycle is divided in two different phases of infection,

the transient lytic infection and the persistent latent infection phases (Figure 3), with different patterns

of viral gene expression in each phase (Ye et al., 2011).

6

Figure 3: Schematic representation of KSHV life cycle in infected cell with the different set of genes expressed in

each phase. Adapted from Purushothaman et al., 2016.

Maintaining a perfect balance between the two life cycle phases is crucial for the long-term

persistence of the virus in the host (Purushothaman et al., 2016). The lytic phase begins with the

expression of an immediate early (IE) gene, ORF50 or replication and transcription activator (RTA), that

leads to the expression of other early genes and the consequential triggering of virus production. In the

latent phase, KSHV expresses a small number of genes located in the major latency locus: ORF73 or

latency-associated nuclear antigen (LANA), ORF72 or v-Cyclin, ORF71 or v-FLIP (Fas-associated death

domain-like interleukin-1β-converting enzyme-inhibitory protein), ORFK12 (Kaposins A, B and C) and

12 microRNAs (Figure 4) (Dittmer et al., 1998 and Uppal et al., 2014). Together these genes help the

establishment of life-long latency and maintenance of viral episomes in host cells.

KSHV relies on some mechanisms to evade both adaptive and innate immune surveillance.

During latency, most viral protein synthesis is silenced to reduce the chance of being recognized by the

immune system. During lytic replication some viral proteins are expressed to block innate and adaptive

immune system like vIL-6, vIRF1 and ORF45 (Kwun et al., 2007).

7

Figure 4: The KSHV episome. Adapted from Mesri et al., 2010.

1.2.1 KSHV latency-associated nuclear antigen (kLANA)

KSHV LANA is a dimer protein encoded by the open reading frame 73 (ORF73). It is a 1162

aminoacid protein constituted by several parts, namely a proline-rich N-terminal domain, central acidic

and glutamine-rich repeat elements, a predicted leucine zipper region and a C-terminal DNA binding

domain (DBD) (Komatsu et al., 2004) (Figure 5).

Figure 5: Schematic representation of complete kLANA protein. The different parts of the protein are indicated,

namely the proline-rich (P), the aspartate and glutamate (DE), the glutamine (Q), the leucine zipper (LZ), the

glutamate and glutamine (EQE) and the DNA binding domain (DBD) regions.

Among KSHV strains, the size of the internal repeat region may change, resulting in kLANA

molecules with different sizes (Gao et al., 1999). This protein is also consistently expressed at high

levels in tumours cells (Komatsu et al., 2001).

8

During the latent phase of the viral cycle, LANA is responsible for maintaining the KSHV genome

as a multicopy persistent non-integrated circular episome in the host cell nucleus (Decker et al., 1996 &

Ballestas et al., 1999). It achieves that by binding directly to the terminal repeats (TR) of KSHV through

its C-terminal DBD domain and by attaching to the cell chromosomes via histones H2A and H2B through

its N-terminal domain (Ballestas & Kaye, 2001 & Barbera et al., 2006). This allows passive segregation

of viral DNA to progeny nuclei during mitosis and maintaining a stable copy number in latently infected

cells (Ballestas & Kaye, 2001 & Uppal et al., 2014). LANA is thus essential for latency. In addition to its

role in episome segregation, LANA dimers assemble into higher order oligomers and are also required

for replication of the episome by recruiting host cell DNA replication machinery (Sun et al., 2014).

Besides its role in the replication and maintenance of the viral episomes, LANA has other

functions such as modulation of host cell gene expression by interacting with several transcription

factors (Uppal et al., 2014). It regulates cell survival and growth by interacting with cellular proteins

(Verma et al., 2007). It contributes also for the maintenance of viral latency by repressing the

transcriptional activity of ORF50/RTA (Lan et al., 2004).

LANA is an incredible multifunctional protein that mediates tumorigenesis by regulating host and

virus transcription, episomal maintenance, viral genome replication, lytic reactivation and ultimately

KSHV latency (Verma et al., 2007).

1.2.2 The kLANA internal repeat acidic region

The internal acidic repeat region of kLANA is the most uncharacterized part of kLANA. It

includes the aspartate and glutamate (DE) regions from aminoacid residue 340 to 431, the glutamine

(Q) region from aminoacid residue 440 to 756 and from aminoacid 765 to 931, leucine (L), valine (V),

glutamate and glutamine (EQE) spaced in a leucine zipper-like pattern (LZ) (Figure 5) (Alkharsah and

Schulz, 2011). Only in vitro studies have been made in order to unveil the functions of this internal repeat

region. In 2003 Viejo-Borbolla and colleagues showed that part of the internal region of kLANA

contributes to the activation of heterologous promoters through heterochromatin modifications (Viejo-

Borbolla et al., 2003). In 2006 Zaldumbide and colleagues reported that the internal repeat region is a

good candidate for inhibition of the presentation of antigen peptides and antigen processing in cis, which

could constitute a good strategy to escape immune surveillance (Zaldumbide et al., 2006). Kwun et al

in 2007 suggested that LANA’s central aminoacid region (QED region) has a role similar to that of

Epstein-Barr nuclear antigen (EBNA-1) glycine-alanine repeats, by inhibiting proteosomal degradation

and retarding protein synthesis (Kwun et al., 2007). Alkharsah and Schulz in 2011 showed that in the

context of the entire viral genome, deleting aminoacid residues 329 to 931 led to a loss of persistence

of latent viral DNA in vitro, although the ability to replicate the viral DNA is maintained (Alkharsah and

Schulz, 2011). Also in 2011 De León Vasquez and Kaye presented a similar result where in short and

long-term episome maintenance assays, LANA mutants lacking the entire internal sequence were highly

deficient in episome persistence (DNA segregation and DNA replication) (De León Vasquez and Kaye,

2011). These authors also presented data suggesting that different cellular proteins with a role in KSHV

9

episome persistence might interact with LANA’s internal region. The same authors, in 2013, generated

a panel of kLANA mutants and showed, in vitro, that a mutant kLANA protein lacking aminoacid residues

465 to 929 had 56,7% of the WT kLANA DNA replication capacity and another mutant lacking aminoacid

residues 332 to 929 had only 36,7% of the WT kLANA DNA replication capacity, suggesting that

aminoacid residues 331 to 464 have a contribution in episome replication in addition to the already

identified important episome replication region (aminoacid residues 262-320) (De León Vasquez and

Kaye, 2013). Overall, a defect in episome maintenance was observed in this two mutants showing that

internal aminoacid residues contribute to maintaining the episomes in in vitro assays.

1.3 Murine herpesvirus 68 (MHV-68)

MHV-68 is an isolate of murine herpesvirus 4 (MuHV-4) and is also a gamma-2-herpesvirus. It

was first isolated from free-living rodents like bank voles (Myodes glareolus) and yellow-necked field

mice (Apodermus flavicollis) in Slovakia (Blaskovic et al., 1980). However, the wood mice (Apodermus

sylvaticus) is the natural reservoir of this virus (Blasdell et al., 2003). The MHV-68 genome consists in

118kb of a unique sequence of DNA flanked by multiple copies of 1,2kb terminal repeats and has a GC

content of approximately 45% (Efstathiou et al., 1990). The virus encodes at least 80 gene products, 63

of them collinear with herpesvirus saimiri (HVS) and human herpesvirus 8/Kaposi’s sarcoma-associated

herpesvirus (HHV-8/KSHV) (Simas and Efstathiou, 1998) and some of them homologous to cellular

genes (Nash et al., 2001). MHV-68 is able to establish a life-long latent infection (Sunil-Chandra et al.,

1992a). Mice that are intranasally inoculated develop an acute lytic infection in the lungs, affecting

alveolar epithelial cells (Sunil-Chandra et al., 1992a). This lytic infection is resolved by the host’s immune

system 9 to 12 days post-infection (Sunil-Chandra et al., 1992b). The virus then spreads to lymph nodes

via dendritic cells and reaches the spleen by infecting marginal zone (MZ) macrophages first, and then

MZ B-cells (Frederico et al., 2014), followed by a marked expansion of latently infected B-cells in

lymphoid germinal centers (Simas et al., 1999), ultimately gaining access to the long-lived memory B

cell pool, the principal long-term reservoir of the virus (Flãno et al., 2002). The peak of latency

(approximately 14 days post-infection) is coupled with splenomegaly and followed by a decrease in the

number of infected cells that ultimately reach a steady-state level (Simas and Efstathiou, 1998). MHV-

68 latent infection is established predominantly in B-cells (Marques et al., 2003), but macrophages,

dendritic and epithelial cells can also be latently infected (Stewart et al., 1998; Weck et al., 1999; Flãno

et al., 2000).

1.3.1 MHV-68 latency-associated nuclear antigen (mLANA)

MHV-68 latency-associated nuclear antigen (mLANA) is a 314 aminoacid protein encoded by

MHV-68 ORF73 that is homologous in sequence and in function to KSHV LANA (Grundhoff and Ganem,

2003). Despite these similarities, mLANA is significantly shorter than kLANA. mLANA is similar to kLANA

in the C-terminal domain (viral DNA binding domain) and in the N-terminal proline rich region, but it lacks

10

the long internal repeat region and the amino-terminal domains present in kLANA (Figure 6) (Barton et

al., 2011 and Habinson et al., 2012).

Figure 6: Schematic representation of the comparison between KSHV latency-associated nuclear antigen (LANA)

and MHV-68 latency-associated nuclear antigen (mLANA). Homologous regions are shown with similar shading.

Unshaded regions lack homology. Aminoacid residues are indicated with numbers. P, proline-rich region; LZ,

predicted leucine zipper. Adapted from Habinson et al., 2012.

Previous studies have demonstrated that mLANA is essential for the establishment and

maintenance of latency in the spleen (Fowler et al., 2003 and Moorman et al., 2003). This protein is

selectively expressed in GC B-cells (Marques et al., 2003). mLANA binds terminal TR elements of the

viral genome to mediate episome persistence by tethering them to the mitotic host chromosomes and

allowing the correct segregation to the daughter cells, analogously to the effect of kLANA on KSHV TR’s

(Habinson et al., 2012). Another common feature between mLANA and kLANA is the regulation of viral

lytic gene expression that is likely to facilitate the establishment of latency (Barton et al., 2011). mLANA,

despite lacking the central repeat region, also confers limited presentation of linked epitopes, reduced

protein synthesis and protein degradation, thus having a role in immune evasion and surveillance

(Bennet et al., 2005).

Given all the common characteristics of mLANA and kLANA, it becomes pertinent to substitute

mLANA for kLANA in the MHV-68 genome in order to study KSHV related properties in a viable in vivo

mouse model.

1.4 MHV-68 as a model to study Kaposi’s sarcoma-associated herpesvirus

pathogenesis in mice

Herpesvirus are thought to have coevolved with their hosts during speciation, with each virus

having a very narrow host range. This aspect is essential when it comes to human gammaherpesvirus,

because no amenable laboratory model exists to study them. An adequate small animal model to study

basic immunological and virological aspects of both phases of infection, as well as evaluate

chemotherapeutic and vaccination strategies, was needed (Barton et al., 2011 and Efstathiou et al.,

1990). MHV-68 represents a good model system to use. It is relatively simple to introduce mutations in

the genome (Simas and Efstathiou, 1998), it encodes some genes involved in latency and reactivation

11

that are conserved between some rodent and primate herpesvirus (v- cyclin, vBcl-2, vGPCR, LANA and

RTA) and despite some differences in the viral pathogenesis between distant related

gammaherpesvirus, MHV-68 has the core pathogenic strategies conserved. Furthermore, the highly

conserved innate and adaptive immune systems between mice and humans helps to reinforce the power

of this murine virus as a good model (Barton et al. 2011).

12

2. Aim of the project

KSHV is an oncogenic gammaherpesvirus that affects humans and for which there is no model

of infection. MHV-68 is a gammaherpesvirus related with KSHV that can function as a mice virus

equivalent model. Previous work done in the lab generated chimeric viruses, that is, MHV-68

recombinant viruses expressing KSHV LANA (kLANA) instead of its original LANA (mLANA). It was

demonstrated that kLANA is able to support MHV-68 latency, despite being with lower levels (Pires de

Miranda and Simas, personal communication). This proved that the chimera model works.

The aim of this project was to access the impact of kLANA internal repeat deletions in vivo.

Collaborators of our laboratory have already expressed kLANA proteins with internal repeat deletions in

vitro, namely kLANAΔ465-929 and kLANAΔ332-929 and assessed their ability to maintain an artificial

episome. Both these mutant proteins presented deficiencies in episome maintenance in vitro. By

introducing these two mutant proteins in a MHV-68 background, we created chimeric mutant viruses

where is possible to understand the importance of kLANA internal repeat regions in vivo.

Ultimately, infection of mice with MHV-68/KSHV chimera will provide a mouse model to

investigate an important part of an essential KSHV latency protein. These studies may constitute the

basis for in vivo testing drug inhibitors to block KSHV latency or tumorigenesis.

13

3. Materials and Methods

3.1 Plasmids and cloning

pT7 kLANAΔ465-929 and pT7 kLANAΔ332-929 containing DNA encoding kLANA with

aminoacid residues 465-929 and 332-929 deleted respectively, were kindly provided by Professor

Kenneth M. Kaye. pSP72 PCR 1_5 was constructed by Marta Miranda. It contains DNA encoding the

full length kLANA ORF and its 5’ untranslated region (KSHV genome coordinates 123,808-127,886,

U75698), flanked by MHV-68 sequences. The BamHI-G shuttle plasmid was constructed by Sofia

Marques. This plasmid contains the MHV-68 genomic BamHI-G fragment (genome coordinates 101,654

to 106,903, U97553) cloned in the pST76K-SR (shuttle) plasmid. The BamHI-G fragment contains

mLANA coding sequence (coordinates 103,927-104,868). DNA fragments encompassing internal

kLANA deletions were excised from pT7 kLANAΔ465-929 and pT7 kLANAΔ332-929 using BamHI and

StuI and subcloned into BamHI/StuI sites of pSP72 PCR 1_5, thus substituting full length kLANA by the

deleted kLANA sequences. The resulting plasmids were termed pSP72Δ465-929 and pSP72Δ332-929,

respectively. (pSP72Δ465-929 and pSP72Δ332-929 were constructed by Ana Quendera). BglII

fragments encompassing Δ465-929 or Δ332-929 kLANA sequences and 5´-UTR, flanked by MHV-68

genomic sequences, were excised from pSP72Δ465-929 and pSP72Δ332-929, respectively, and

subcloned into the BglII sites of BamHI-G shuttle replacing mLANA coding sequence by the deleted

kLANA constructs. The resulting recombinant plasmids were termed Shuttle BamHI-G Δ465-929 and

Shuttle BamHI-G Δ332-929. (Shuttle BamHI-G Δ465-929 was constructed by Ana Quendera).

3.2 Ethics statement

The study was in accordance with the Federation of European Laboratory Animal Science

Associations guidelines on laboratory animal welfare.

3.3 Mice

Female C57BL/6 were purchased from Charles River Institute in France. Animals were housed

and subjected to experimental procedures in specific pathogen-free conditions, at the Instituto de

Medicina Molecular animal facility, Lisbon, Portugal.

14

3.4 Bacteria

To produce the recombinant viruses competent E. coli DH10B with a MHV-68 bacterial artificial

chromosome were used. Standard Competent E. coli DH5α were also used. For transformation of the

ligation reactions XL10-Gold® ultracompetent cells (Agilent Technologies) were used.

3.5 Cell lines

NIH-3T3 (mouse embryonic fibroblasts) stably expressing Cre recombinase were maintained in

DMEM (Dulbecco’s Modified Eagle Medium) containing 10% of heat inactivated fetal bovine serum

(FBS), 5000U/mL of penicillin, 5000μg/mL of streptomycin and 200mM of L-glutamine. Baby hamster

kidney (BHK-21) fibroblasts were maintained in Glasgow Minimum Essential Medium (supplemented

GMEM) supplemented as above plus 10% of tryptose phosphate broth (Sigma).

3.6 Transformation of competent bacteria

Competent E. coli DH10B harboring a bacterial artificial chromosome (BAC) (Collins et al.,

2009) were transformed using the heat shock method. An aliquot of cells was gently thawed on ice and

0.5μL of plasmid DNA (BamHI-G shuttle) with a concentration between 250 and 450 ng/μL was added.

After a gentle swirl each aliquot was incubated for 30 min on ice, subsequently heat-pulsed for 90 sec

in a 42°C water bath and left for 2 min on ice again. 500 μL of Super Optimal Broth + glucose (SOC)

(2% w/v tryptone; 0.5% w/v Yeast extract; 10mM NaCl; 2.5mM KCl; 10mM MgCl2 and 20mM glucose)

was added and cells were incubated for 1h at 30°C with an agitation of 220rpm. Finally cells were spread

onto LB agar plates containing 17 μg/mL of chloramphenicol and 30 μg/mL of kanamycin and incubated

at 30°C.

3.7 Control Viruses

The WT MHV-68 (v-WT) has been described by others (18). The chimeric MHV-68 in which

mLANA DNA was replaced by the full length kLANA DNA an 5’ UTR (v-kLANA) was constructed by

Marta Miranda (Miranda and Simas unpublished results).

3.8 Generation and characterization of recombinant viruses

The MHV-68 recombinant viruses were generated by a mutagenesis procedure in E. coli DH10B

according to the Two-Step-Replacement Strategy (O’ Connor et al., 1989).

15

The BamHI-G shuttle harbouring the kLANA with the desired mutations was transformed by

heat shock into competent E. coli DH10B that contains a BAC with the entire WT MHV-68 genome

cloned. Bacteria were plated in plates that contained the selection marker of the BamHI-G shuttle

plasmid (kanamycin) and the selection marker of the BAC (chloramphenicol) for 1-2 days at 30°C

because the shuttle plasmid as a temperature-sensitive origin of replication and still needs to replicate.

A homologous recombination event between the shuttle BamHI-G and the BAC occurred, mediated by

the bacterial protein RecA which is within the shuttle plasmid (Adler et al., 2003) and it is responsible

for the complete integration of the BamHI-G shuttle plasmid into the BAC genome forming the co-

integrates. For selection of the co-integrates the bacteria were incubated overnight at 43°C in the

presence of kanamycin/chloramphenicol. Next the clones were plated only on LB agar with

chloramphenicol and incubated 1-2 days at 30°C. With this the co-integrates can resolve themselves

spontaneously by homologous recombination to either the initial condition or to the expected BAC

mutant state. In order to isolate the resolved clones with the BAC mutants the bacteria were plated on

LB agar with chloramphenicol and 5% of sucrose, which is a counter selection against SacB gene

encoded by the BamHI-G shuttle plasmid, and incubated 1-2 days at 30°C. Colonies were picked once

and plated in parallel in kanamycin LB agar and in chloramphenicol LB agar and incubated overnight at

37°C. The resistance to sucrose and the sensitivity to kanamycin are two independent indications that

the co-integrates are well resolved. To identify BAC recombinants containing kLANA coding sequences,

a colony PCR was performed using primers that amplify the C-terminal region, namely 1019 F (forward)

(5’-GTCCCTTACAGACAGATAGATGATTG-3’) and 1150 R (reverse) (5’-

AAAAAGCTTTTATTCCCCTGGCTGGGTTAATG-3’). kLANA PCR positive clones were further

characterized by restriction profile analysis using EcoRI and BamHI to assess the integrity of the

genome and confirm kLANA deletions.

.

3.9 BAC DNA Preps

To obtain BAC DNA minipreps, colonies that were positive in the colony PCR were inoculated

in 10mL of LB broth supplemented with chloramphenicol and grown, overnight at 37°C with shaking.

The bacterial cells were pelleted by centrifugation for 10 min at 4000rpm at 4°C. The pellet was first

resuspended in 200μL of buffer S1 (50mM Tris-HCl, 10mM EDTA, 100μg/mL RNase A, pH 8.0). Next

the cells were lysed by adding 200μL of buffer S2 (200mM NaOH, 1% SDS). The cell suspension was

mixed carefully and left 5 min at RT. 200μL of pre-cooled buffer S3 (2.8M KAc, pH 5.1) was added and

the suspension was mixed by inversion and incubated for 15 min on ice. In order to separate the cell

debris from the BAC DNA the mixture was centrifuged for 15 min at 13000rpm at 4°C. The supernatant

was passed to other tube, mixed with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1,

Applichem A0944, 0250), inverted for mixing and incubated for 5 min at RT. The mixture was centrifuged

for 10 min at 13000rpm at 4°C and the aqueous phase was transferred to a fresh Eppendorf. 0.7 volumes

of isopropanol were added and the mixture was inverted and incubated for 5 min at RT. To pellet the

BAC DNA the tubes were centrifuged for 20 min at 13000rpm at 4°C. Next the supernatant was

16

discarded and the pellet was washed with 200μL of 70% EtOH and centrifuged for 10 min at 13000rpm

at 4°C. Finally the supernatant was discarded and the pellet was dried and resuspended in 60μL of

nuclease-free water at stored at 4°C. To obtain large amounts of pure BAC DNA, for transfections, DNA

maxipreps were produced from 250mL cultures with the NucleoBond® BAC 100 kit (Macherey-Nagel),

according to manufacturer’s instructions. Due to the size of the BAC DNA, cut tips were used when

needed in order to avoid shearing the DNA

3.10 Reconstitution of MHV-68 virus on BHK-21 cells

BHK-21 cells were splitted one day before in order to have 106cells/6cm2dish. 1μg of BAC DNA

maxiprep was diluted in 500μL of unsupplemented GMEM with a cut tip. 2μL of X-tremeGENE HP DNA

Transfection Reagent (Roche Applied Science) was added into the media. The mixture was mixed gently

and left incubating for 20 min at RT. The transfection complexes were added to the dishes and incubated

at 37°C. After a few days a cytophatic effect (CPE) of approximately 50% was visible and the cells were

scrapped onto media and stored at -80°C (BAC+ virus). To remove completely the BAC cassette, BAC+

viral stocks were passaged trough NIH-3T3- Cre cells. 1,75x105 cells were seeded in 6 well plates and

the loxP-flanked BAC cassette was removed by cell passage coupled with limiting dilutions in order to

loose gradually the GFP signal stemming from the GFP expression cassette present in the BAC

sequences. Resulting virus stock is termed BAC- virus and was used to produce working stocks.

3.11 Production of viral stocks

Working virus stocks were prepared by infecting BHK-21 cells in 175cm2 flasks with a low MOI

(0.002 pfu/cell). Cells stayed incubating for 4 days at 37°C. After the incubation cells are scrapped into

the media and centrifuged for 5 min at 1500rpm at 4°C. Cell pellet was resuspended in 2mL of fresh

supplemented GMEM, aliquoted and stored at -80°C. These are the cell working stocks (CWS) that can

be used to produce subsequent viral stocks. The supernatant was transferred to 30mL bottles and ultra-

centrifuged for 2h at 12000rpm at 4°C to pellet virus. The supernatant was discarded and the pellet was

first softened in 2mL of fresh supplemented GMEM for 1h on ice and before resuspended in that same

volume. Virus were aliquoted and stored at -80°C. These stocks are termed working stock media (WSM)

and were used for all infectivity assays.

3.12 Plaque assay (virus titration using suspension assay)

1mL of 2,5x105 BHK-21 cells were incubated in a rotating table with viral 10-fold serial dilutions

(10-3 to 10-8) for 1h at RT in 15mL falcon tubes. After the incubation 2mL of fresh supplemented GMEM

was added to the tubes, they were inverted for mixing and the mixture was poured to 6 well plates and

17

incubated for 4 days at 37°C. After the incubation the media covering the cells was removed and the

cells were fixed with formaldehyde 4% in PBS for 10 min at RT. The fixative was removed and the cells

were covered with toluidine blue 0.1% for a minimum of 5 min. After that time the plates were washed

in tap water and dried. Titrations were performed in duplicate. Viral plaques were counted in a

magnifying glass and the viral titers were calculated from the number of viral plaques, the corresponded

dilution and the initial inoculum, according to the formula:

nº of plaques x 1

dilution x

1

inoculum

3.13 Multi-step growth curves

To perform this assay 5x104 BHK-21 cells/well were seeded in 24 well plates. The media was

removed and the cells were infected with a low MOI (0.01pfu/cell) with controls (v-WT and v-kLANA)

and the mutant viruses (v-kLANAΔ465-929 and v-kLANAΔ332-929) in 200μL of fresh supplemented

GMEM. Cells were incubated for 1h at 37°C. After the virus adsorbed to the cells, the inoculum was

removed, and cells washed 2x with 500μL of PBS. 1mL of fresh supplemented GMEM was added per

well and cells were incubated at 37°C. The multistep growth curve consists in recovering the cells and

media overtime (0, 24, 48, 72, 96 and 120 h.p.i), freeze-thawing and determine virus titers by plaque

assay. Titers were determined by plaque assay in duplicate.

3.14 Immunoblotting

Immunoblotting was performed to detect kLANA proteins in infected BHK-21 cells.

5x105cells/well in a 12 well plate were infected at a MOI of 3 pfu/cell in a total volume of 500μL. As a

control, a well with non-infected cells (mock) was analysed in parallel. The viral inoculum was added to

the wells and cells were incubated for 2h at 37°C with a gentle rock every 30 min. The inoculum was

removed and cells were washed gently with 1mL of pre-warmed supplemented GMEM. 1mL of media

was added to each well and cells were left to incubate another 4h at 37°C. After that time cells were

washed twice with ice cold PBS. 110μL of lysis buffer (150mM NaCL, 10mM Tris-HCl pH 7.4, 1mM

Na3VO4, 1mM NaF, 1% Triton™ X-100, complete protease inhibitors (Roche), millieQ H2O) were added

to each well and cells were harvested into a Eppendorf to perform a quick vortex and left for 10 min on

ice. Lysates were centrifuged for 10 min at 13000rpm at 4°C and 100μL of cleared supernatant was

recovered. 100μL of 2x Laemmli’s Buffer (100mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 10% β-

mercaptoethanol, 0.1% bromophenol blue, millieQ H2O) was added and the samples were heated at

100°C for 10 min. 50μL of each sample was loaded into a gel to be resolved by sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE). The resolving gel was prepared with 30%

Acrylamide/Bis Solution, 37.5:1 from Bio-Rad, resolving gel buffer from Bio-Rad (1.5M Tris-HCl buffer,

pH 8.8), 10% SDS, 10% ammonium persulfate and TEMED (Tetramethylethylenediamine). The stacking

18

gel was prepared with 30% Acrylamide/Bis Solution, 37.5:1 from Bio-Rad, stacking gel buffer from Bio-

Rad (0.5M Tris-HCl buffer, pH 6.8), 10% SDS, 10% ammonium persulfate and TEMED. The

electrophoresis occurred in a Bio-Rad tray in TGS buffer 1X (25mM Tris-base, 192 mM glycine and

0.1% (w/v) SDS, pH 8.3) at 180V. After the electrophoresis, proteins were transferred from the gel to

nitrocellulose membranes by the wet-transfer method. After the transfer, nitrocellulose membranes were

incubated with Ponceau dye for 2 min to confirm the transfer of proteins. The membrane was blocked

with a blocking solution (PBS+0.05% Tween-20 (PBS-T) + 2.5g of non-fat powder milk) in a rotating

platform for 30 min at RT. The membrane was washed with PBS-T and incubated with blocking solution

containing each of the primary antibodies: rat α-kLANA LN53 1:1000 (Advanced Biotechnologies),

mouse α-kLANA 4C11 1:1000 (Novus Biologicals), mouse α-mLANA mAb 6A3 1:20 (Pires de Miranda

et al., 2013), rabbit α-M3 1:3000 (Jensen et al., 2003) and rabbit α-Actin 1:1000 (Sigma) in a sealed bag

in a rotating table for 2h at RT. The membrane is washed 3x, 5 min each with PBS-T and incubated with

blocking solution containing horseradish peroxidase conjugated secondary antibody (Jackson Immuno

Research) specific to the species of the primary antibodies. Incubation was for 30 min at RT. The

membrane was washed 3x, 5 min each with PBS-T. Detection of bound antibodies was performed by

chemiluminescence using SuperSignal® West Pico Chemiluminescence Substrate (Thermo Scientific)

according to the manufacturer’s instructions.

3.15 Transcription analysis of kLANA mutants

Expression of kLANA WT and kLANA mutant viral transcripts was analysed by real-time PCR.

BHK-21 (6x105 cells/well in a 12 well plate) were infected with virus at a MOI of 5 pfu/cell in 500μL of

complete media for 8h. Total intracellular RNA was extracted using TRIzol® (Invitrogen) according to

the reagent supplier’s protocol. To avoid contamination with genomic DNA, samples were treated with

DNAse from the TURBO DNA-free™ (Ambion) according to the manufacturer’s instructions. Each

sample was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System

(Invitrogen) following the manufacturer’s instructions. 20μL reactions containing 500ng of DNAse treated

RNA, 50μM Oligo (dT) primer, 10mM dNTP mix, 10x RT buffer (200mM Tris HCl, pH 8.4 and 500mM

KCl), 25mM MgCl2, 0,1M DTT, 40 U/μL of RNase OUT and 200 U/μL of SuperScript III reverse

transcriptase were incubated in a thermocycler at 50°C for 50 min, followed by 85°C for 5min. Samples

were then treated with 2 U/μL of RNase H (Invitrogen). Real-time qPCR was performed on a Rotor Gene

6000 thermocycler from Corbett Research using the the DyNAmo Flash SYBR Green qPCR Kit

(ThermoFisher Scientific) and primers specific for the C-terminal coding region of kLANA (kLANA RT2-

F: 5’-TGGTTGTTGGTATCTTGG-3’ and kLANA RT2-R: 5’-CCTGGAGTTCGTATGAGG-3’). As a control

for viral infection, ORF50 viral transcripts were detected in parallel reactions using also specific primers

(ORF50-RT2_F: 5’-CAACATGGTCTTTTCAGG-3’ and ORF50-RT2_R: 5’-TAATGGGTGAAAATGGCC-

3’). The mix for the RT-qPCR contained 2μL of cDNA (diluted 1:10), 2x master mix (provided in the kit)

and 0,5 μM of each forward and reverse primer. The samples were incubated in a 72-well rotor at 95°C

for 10 min, followed by 40 cycles of 95°C for 10 sec, 55°C for 15 sec and 72°C for 30 sec, followed by

19

a melting analysis step from 50°C to 95°C at 0,1°C/sec. The samples were run in triplicate, the control

plasmids (psp72 PCR1_5 for kLANA full length and ORF50) were run in duplicate. Relative mRNA

values, normalized to ORF50, were calculated by the Pfaffl method (22):

Briefly this method determines a relative quantification of a gene of interest comparing it to a reference

gene. The relative expression ratio or fold difference is calculated taking into account the efficiency of

the PCR reaction (E depicted in the formula) and the Ct (in the equation is depicted as crossing points

or CP) deviation of an unknown sample against a control (v-kLANA). The target in the formula is the

samples, containing the gene of interest, which are unknown, the reference gene, or normalizer is the

ORF50, the control is the full length kLANA and the samples are the deletion mutants. The threshold

cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.

The results are normalized to a ORF50. PCR results were analysed with Rotor-Gene™ 6000 real-time

rotary analyser Software version 1.7. (Corbett Life Science).

3.16 In vivo Assays

3.16.1 Infection of mice

Six to eight week old mice were anaesthetized with isoflurane and inoculated intranasally with

104 pfu of virus in 20μL of PBS. At 14 days post-infection, mice were sacrificed by CO2 inhalation and

the spleens were surgically removed to a falcon containing 5mL of PBS with 2% FBS.

3.16.2 Single cell suspensions.

Spleens were mechanically disrupted and filtered through a 100 μm cell strainer in order to

remove stromal debris. Cells were then pelleted by centrifugation at 1200 rpm for 5 min at 4°C.

Supernatants were discarded and cells were resuspended in 1 mL of Red Blood Cells Lysis Buffer (154

mM ammonium chloride, 14 mM sodium hydrogen carbonate, 1 mM EDTA pH 7.3) and incubated on

ice for 5 min. Cells were washed with PBS/2% FBS and resuspended in 5mL of PBS/2% FBS These

splenocyte suspensions were used to assess viral latency by infectious centre assay/reactivation assay

(ICA) and determination of the frequency of viral DNA positive cells.

20

3.16.3 Infectious Center Assay (ex vivo explant co-culture assay)

Latent virus titers were determined by a reactivation assay. In this assay, co-culture of single-

cell suspension of splenocytes with permissive fibroblasts, in this case, BHK-21, leads to reactivation of

latent virus and formation of viral plaques. Briefly, 2-fold and 10-fold serial dilutions of splenocyte

suspension were prepared and plated, in duplicate, in 6 cm2 plates containing 5x105 BHK-21 cells.

Spleens were also analysed for the presence of pre-formed infectious viruses, by performing plaque

assay in freeze-thawed splenocyte suspensions. Plates were incubated for 5 days at 37°C, 5% CO2, for

the reactivation assay and 4 days for the plaque assay. Cells were fixed with 1% formaldehyde in PBS

and stained with 0.1% toluidine blue. Viral plaques were counted with the help of a magnifier lenses and

infectious centers (pfu/spleen) were determined.

3.16.4 Sorting of GC B cells

Splenocyte suspension were prepared as described above from a pool of 5 spleens for each

infection group. To block Fc receptors, cells were incubated with purified Rat anti-mouse CD16/CD32

(mouse BD Fc Block™) (1:100 in PBS with 2% FBS) for 15 min on ice. Cells were washed with PBS

with 2% FBS and pelleted by centrifugation. Splenocytes were surfaced stained by a 25 min on dark

and on ice incubation with the following antibodies: APC-H7 Rat anti-mouse CD19 from BD Biosciences

(1:400), Anti-Human/Mouse GL7 eFluor® 660 from eBiosciences (1:200) and PE conjugated hamster

anti-mouse CD95 (FAS) Mab from BD Biosciences (1:800). Cells were then washed 2x with PBS with

2% FBS, the pellets resuspended and transferred to FACS tubes. For each infection group, 1x106 GC

B cells (CD19+CD95hiGL7hi) were purified by a BD FACSAria IIu (BD Biosciences) and a BD FACSAria

III (BD Biosciences) cell sorters at the IMM flow cytometry unit. The purity of sorted cells was always

>95%, as analysed by flow cytometry.

3.16.5 Limiting dilution assay and Real-time PCR of viral DNA positive cells

Total splenocytes from a pool of 5 individual mice (starting at 2x106 cells) or sorted GC B cells

(starting at 1x106 cells) were serially two-fold diluted and eight replicates of each dilution added to PCR

tubes. Lysis buffer (10 mM Tris-HCl pH 8.3, 3mM MgCl2, 50mM KCl, 0,45% NP-40, 0,45% Tween-20

and 0,5mg/mL of proteinase K) was added to each tube and left overnight at 37°C. The next day the

proteinase K was inactivated at 95°C for 5 min in a thermocycler and the samples were analysed by

real time PCR, as previously described (Pires de Miranda et al., 2008), on a Rotor Gene 6000

thermocycler from Corbett Research using fluorescent Taqman methodology and primers and probe

sets specific for the MHV-68 M9 gene: M9-F (forward) (5’- GCCACGGTGGCCCTCTA -3’), M9-R

(reverse) (5’ – CAGGCCTCCCTCCCTTTG -3’) and M9T probe (5’- 6-FAM-CTTCTGTTGATCTTCC-

MGB-3’). PCR reactions had a final volume of 25μL containing 2,5μL of cell suspension lysate prepared

21

previously, 200μM of each primer, 300μM of probe, 1x Platinum Quantitative PCR SuperMix-UDG

(Invitrogen), 5mM of MgCl2 and 1x of Rox reference dye. The cycling program consists of a melting

temperature of 95°C for 10 min followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. In each PCR

run, a dilution series of pGBT9-M9, a plasmid that contains the M9 gene, was included as a positive

control. PCR results were analysed with Rotor-Gene™ 6000 real-time rotary analyser Software version

1.7. (Corbett Life Science). For all dilutions tested each replicate eas scored positive or negative based

on comparison with negative (water sample) and positive (plasmid containing M9 gene) controls. The

frequency of cells with viral DNA was calculated according to the single-hit Poisson model (SHPM) by

maximum likelihood (Bonnefoix et al., 2001). This model assumes that only one cell is necessary and

sufficient for generating a positive response. The method consists in modelling the data from the limiting

dilution assay according to the linear log-log regression model fitting the SHPM.

3.17 Statistical analysis

Data comparisons between groups was performed by an ordinary one-way ANOVA and Mann-

Whitney test, when applied. Statistics were calculated with GraphPad Prism Software. For limiting

dilution analysis 95% confidence intervals were determined as described (Marques, S. et al., 2003).

22

4. Results

4.1 Generation and characterization of MHV-68 recombinant viruses

Previous work from our laboratory shows that the chimeric kLANA MHV-68 virus (v-kLANA),

where the endogenous ORF73 (mLANA) was replaced by KSHV ORF73 (kLANA), is a viable model to

study KSHV pathogenesis in mice (Pires de Miranda and Simas, unpublished results). This model allows

to address the relevance of the different kLANA domains in an in vivo infection, by generating viral

recombinants with deletions in these domains. The internal acidic repeat region is the least

characterized domain of kLANA. Previous in vitro studies with two kLANA internal repeat deletion

mutants (kLANAΔ465-929 and kLANAΔ332-929) are expressed, in a KSHV background, have shown

that both these mutant proteins have a defect in viral episome maintenance (De León Vazquez and

Kaye, 2013).To test the importance of this internal repeat region in our in vivo chimeric model, the same

two recombinant proteins were introduced in a MHV-68 background: kLANAΔ465-929 harbours a

deletion encompassing aminoacids 465 to 929, which removes part of the glutamine region (Q), the full

leucine zipper (LZ) and the glutamate and glutamine region (EQE); kLANAΔ332-929 has a deletion in

aminoacids 332 to 929, disrupting the totality of the internal repeat region of the protein (Figure 7).

The construction of this viruses was made via a mutagenesis procedure in E. coli DH10B

according to the Two-Step-Replacement Strategy (O’ Connor et al., 1989) using a MHV-68 BAC plasmid

with the entire MHV-68 genome cloned (Collins et al., 2009). This technique allows the maintenance of

viral genome as a plasmid in E. coli, where a homologous recombination process occurs with a

subsequent reconstitution of the viral progeny by transfection of the BAC plasmid into eukaryotic cells

(Adler et al., 2003). The full procedure is described in Materials and Methods. To identify the BAC’s

harbouring the mutant kLANA sequences, a PCR with primers specific for the C-terminal region of

Figure 7: Diagram of KSHV LANA and LANA deletion mutant proteins. The numbers indicate aminoacid residues and the different domains of the protein are also indicated, namely the proline-rich (P), the aspartate and glutamate (DE), the glutamine (Q), the glutamate and glutamine (EQE), the leucine zipper (LZ) region and the DNA binding domain (DBD). The fold deficiency in episome maintenance in vitro is also shown and it was determined by comparison with the kLANA WT (De Leon Vazquez et al., 2013).

23

kLANA was performed (see Material and Methods - Generation and characterization of recombinant

viruses). Furthermore, the integrity of the viral genome was analysed by restriction digestion using two

different restriction enzymes, EcoRI and BamHI (Figure 8). No unexpected changes in the restriction

profile were detected. The blue boxes show the bands specific of the v-kLANAΔ465-929, the red boxes

show the bands specific to the v-kLANAΔ332-929. Two independent clones were engineered for each

mutant (i).

The mutant BAC’s were then transfected into BHK-21 fibroblasts to reconstitute the viruses in

cells. To remove completely the BAC cassette, the reconstituted viruses were sub-cultivated with NIH-

3T3-Cre cells that through the Cre-lox system, removed the BAC sequence that is flanked by lox-P sites.

4.2 The chimeric recombinant viruses display normal in vitro growth

To assess if the mutant viruses had a normal growth kinetics, a multistep growth curve was

performed. Permissive BHK-21 fibroblasts were infected at low MOI (0.01pfu/cell) and during five

Figure 8: Assessing the integrity of the mutant BAC plasmids by restriction enzyme profile analysis using EcoRI, BamHI. The blue boxes show the bands specific of the v-kLANAΔ465-929, the red boxes show the bands specific to the v-kLANAΔ332-929. Each mutant has two independent clones.

24

days cells were scrapped and the titrations were performed by plaque assay. Two different viruses

were used as controls: the WT MHV-68 that harbours the mLANA (v-WT) and the chimeric MHV-68

where mLANA was replaced by the full length kLANA (v-kLANA). It is known that the ORF73 from

the MHV-68 is not essential for in vitro growth (Fowler et al., 2003). There was no significant

difference between infection groups (one-way ANOVA) and the v-kLANA virus growth was similar

to the growth previously observed in the lab (Pires de Miranda and Simas, unpublished) (Figure 9).

This indicates that neither the substitution of the mLANA for kLANA nor the deletions in the kLANA

gene affect the lytic replication.

Figure 9: Multistep growth curve by infection of BHK-21 cells at 0.01 pfu per cell. Samples were harvested, freeze-

thawed and titers were determined by plaque assay on monolayers of BHK-21 cells.

4.3 Expression of kLANA mutant proteins

In order to investigate if all the mutants expressed the different kLANA proteins correctly, an

immunoblot was performed using antibodies against kLANA and other cellular and viral proteins.

BHK-21 cells were infected with a MOI of 3pfu/cell during 6h. LN53 is a commercial antibody that

recognizes the EQE repeats within the internal repeat region of kLANA. This is a very sensitive

antibody that recognizes multiple epitopes in the full length kLANA that are absent in the deletion

mutants. Because both mutants used lack that specific part of the protein, another commercial

antibody (4C11), that recognizes a different epitope (a.a.122-329), was ordered but it was not

sensitive enough to detect kLANA proteins under these conditions (Figure 10). kLANAΔ465-929

should have been detected with a molecular weight of approximately 140kDa and the kLANAΔ332-

929 with one of approximately 100kDa. Even the chimeric virus with the full length kLANA add a

poor recognition when compared with α-kLANA LN53. The infection levels were similar between

samples as demonstrated by the level of M3 protein and the loading control for cell infection (α-

actin) was also similar in all samples. mLANA was also detected only in the MHV-68 WT sample,

T im e p o s t- in fe c t io n (h )

Lo

g p

fu/m

L

0 2 4 4 8 7 2 9 6 1 2 0

0

2

4

6

8

v -k L A N A 3 3 2 -9 2 9 1 3

v -k L A N A 3 3 2 -9 2 9 i

v -k L A N A 4 6 5 -9 2 9 1 0

v -k L A N A 4 6 5 -9 2 9 i

v -k L A N A

v -W T

25

as expected. Clearly the problem resides in the lack of a good antibody. Serum from a patient with

Kaposi’s sarcoma, that contains polyclonal antibodies (gift from Professor Kenneth Kaye) was also

used to try to detect kLANA mutants but was also not effective in the detection (data not showed).

Figure 10: Detection of the expression of WT and mutant kLANA proteins. BHK-21 cells were infected with a MOI

of 3pfu/cell during 6h. The proteins were detected with the antibodies indicated on the right. Molecular weight (in

kDa) is indicated on the left.

4.4 Transcription analysis of kLANA mutants

Since it was impossible to detect the kLANA mutant proteins by immunoblotting, the levels of mRNA

expression were quantified instead. Because kLANA proteins affect their own transcription, these

analyses could also reveal if the lack kLANA internal regions would affect expression. BHK-21 cells

were infected with virus at a MOI of 5 pfu/cell during 8h. Total intracellular RNA was extracted, reverse

transcribed and real-time qPCR was performed using primers specific for kLANA and ORF50 viral

transcripts (see Materials and Methods - Transcription analysis of kLANA mutants). Relative kLANA

mRNA values were normalized to ORF50, a lytic gene that is transcribed during the first 8h after

infection. Ratios were calculated using the Pfaff method (Pfaff, 2001), and plotted as fold difference of

kLANA mRNA levels relative to the chimeric virus v-kLANA (Figure 11).

Analysing the data it is clear that both v-kLANAΔ465-929 independent mutants express kLANA at

the same level as v-kLANA, while v-kLANAΔ332-929 independent mutants present a deficit in kLANA

expression levels. One possible explanation for this deficit is that the aspartate and glutamate internal

26

acidic and part of the glutamine region may interact with cellular proteins with functional activity in

transcription as already discussed by others (De Leon & Kaye, 2011).

Figure 11: Quantification of kLANA transcripts. Total RNA extracted from BHK-21 infected with the corresponded

virus at an MOI of 5 pfu/cell until 8 h.p.i. Poly-A mRNA was reverse transcribed and cDNA was obtained. The

controls with no reverse transcriptase (-RT) and with mock cells were performed and both are not detectable, as

expected (results not shown). Relative mRNA values, normalized to ORF50 gene, were calculated by the Pfaffl

method taking into account the efficiency of the PCR reaction. Data are presented as fold difference in gene

expression compared to v-kLANA. Each sample was run in triplicate, and the error bars represent the standard

deviation.

4.5 The internal acidic repeat region has impact in the establishment of latency in the

spleen

It is known that the mLANA is essential for latency in the spleen (Fowler et al., 2003) and

homology between mLANA and kLANA exists (Russo et al., 1996; Virgin et al., 1997; Grundhoff

and Ganem., 2003). Furthermore, the chimeric v-kLANA is able to establish splenic latency following

intranasal inoculation (Pires de Miranda and Simas, unpublished results). It is thus essential to

discover if the kLANA mutants lacking parts of the acidic internal region can establish latency in the

spleen. An infectious centre assay was performed to quantify the levels of latent viruses in the

spleen of mice infected with the recombinant viruses (Figure 12). C57BL/6 mice were inoculated

intranasally with 104 pfu of virus and, at 14 days post-infection the mice were sacrificed, spleens

were surgically removed and processed as described in Materials and Methods – Infection of mice.

The latent load in the spleen was examined by quantification of ex vivo reactivation-competent

viruses when cultured with permissive fibroblasts. The presence of latent viruses in the splenocytes

results in the formation of viral plaques within the cell monolayer that can then be quantified (Figure

12, closed circles). To make sure that no lytic infectious viruses were present in the splenocyte

population, aliquots of splenocytes were freeze-thawed first and then co-cultured with the

kL

AN

A m

RN

A l

ev

els

re

lati

ve

to

co

ntr

ol

(fo

ld d

iffe

re

nc

e)

Co

ntr

ol (v

-kL

AN

A)

v-k

LA

NA465-9

29

v-k

LA

NA465-9

29 i

v-k

LA

NA332-9

29

v-k

LA

NA332-9

29 i

0 .0

0 .5

1 .0

1 .5

27

permissive cells. No pre-formed infectious viruses could be detected at any time point for any of the

analysed viruses (Figure 12, open circles).

As previously observed, v-kLANA established latency in the spleen albeit at lower levels when

compared with v-WT (Pires de Miranda and Simas unpublished).

In the v-kLANAΔ465-929 mutants, latency is not affected when compared to the v-kLANA.

Regarding the v-kLANAΔ332-929 mutant, however, the ability to establish latency in the spleen is

abrogated (bellow the limit of detection of this assay). This indicates that the aspartate and

glutamate internal acidic (a.a. 330-463) has influence in the ability to establish latency in the spleen.

This phenotype is unlikely to reflect inadvertent mutations introduced during the mutagenesis

process since equivalent phenotypes were observed in both independently generated mutant

viruses.

Figure 12: Quantification of latent infection in spleen 14 d.p.i. by explant co-culture plaque assay (closed circles).

Titres of infectious virus were determined in freeze/thawed splenocyte suspensions (open circles). Each circle

represents the titre of an individual mouse. Data were combined from two independent experiments each with five

mice per infection group. The dashed line represents the limit of detection of the assay. Horizontal bars indicate

arithmetic means. The comparison between v-kLANA and both v-kLANAΔ465-929 independent mutants was not

significant. ***(p<0.001) and **(p<0.01).

4.6 Quantification of the frequency of viral DNA-positive total splenocytes and GC B-

cells

To confirm the phenotype observed in the infectious centre assay, a limiting dilution assay for

quantification of DNA positive cells was performed with the total splenocyte population and with

sorted purified GC B-cells (Figures 13 and Table 1). Quantification of viral infection in GC B-cells is

highly relevant since mLANA is selectively expressed in proliferating GC B-cells (Marques et al.,

2003) and mLANA was replaced by kLANA. The viral DNA was detected by real-time PCR with

primers and probe sets specific for the MHV-68 M9 gene (see Materials and Methods - Limiting

dilution assay and Real-time PCR of viral DNA positive cells). Analysing the graphics it is clear that

1 4 d .p .i.

pfu

/sp

lee

n

v-W

T

v-k

LA

NA

vkL

AN

A465-9

29

vkL

AN

A465-9

29 i

vkL

AN

A332-9

29

vkL

AN

A332-9

29 i

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

1 4 d .p .i.

pfu

/sp

lee

n

v-W

T

v-k

LA

NA

vkL

AN

A465-9

29

vkL

AN

A465-9

29 i

vkL

AN

A332-9

29

vkL

AN

A332-9

29 i

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

***

**

**

28

the phenotype observed in the infectious centre assay is maintained. The v-WT virus has the

expected frequencies of viral DNA+ cells both in the total splenocyte population and in the GC B-

cells (Correia et al., 2013) as well as the v-kLANA despite being approximately 1log lower than the

vWT as previously observed in the lab (Pires de Miranda and Simas, unpublished).

There are no differences between v-kLANAΔ465-929 and v-kLANA and the v-kLANAΔ332-929

mutant has a frequency of viral DNA+ cells that it is not detectable both in total splenocytes (Figure

13A) and in the GC B-cells (Figure 13B). This data shows that the aspartate and glutamate internal

acidic (DE) and part of the glutamine (Q) region is crucial for the infection of GC B-cells and for

establishment of latency in the spleens in this chimeric model.

Figure 13: (A) Reciprocal frequencies of viral DNA-positive cells in total splenocytes or GC B-cells (CD19+CD95hiGL7hi) (B) were determined by limiting dilution and real-time PCR. Data were obtained from pools of five spleens per group. N.D. Not detectable. Bars represent the frequency of viral DNA-positive cells with 95% confidence intervals.

a

Data were obtained from pools of 5 spleens. b

Frequencies were calculated by limiting dilution analysis with 95% confidence intervals (numbers in parentheses). c

The purity of sorted cells was determined by fluorescence-activated cell sorting (FACS) analysis and it was always greater than 95%.

A B

29

5. Discussion

In this work, two kLANA-MHV68 chimeric mutant viruses were generated, v-kLANAΔ465-929,

lacking part of the Q, the LZ and the EQE internal repeat region and v-kLANAΔ332-929, lacking the

entire internal repeat region. They were created to assess the impact of kLANA internal repeat deletions

in the chimeric in vivo model. Results show that the DE internal repeat region and part of the Q region

(a.a. 330-463) are essential for the viral latency in the spleen.

Recombinant viruses displayed an in vitro growth similar to the v-WT and v-kLANA viruses

(Figure 3). This was expected since it has been reported that mLANA was not essential for in vitro

growth (Fowler et al., 2003). This indicates that neither the substitution of the mLANA for kLANA, nor

the internal repeat deletions in the kLANA gene dramatically affected the lytic replication.

In vivo analysis by infectious centre assay show that the v-kLANAΔ465-929 has a latency peak

very similar to vkLANA, in both independent clones and the v-kLANAΔ332-929 mutant has a completely

different result, with a phenotype emerging (Figure 12). For the latter, the ability to establish latency in

the spleen is below the limit of detection of the assay. This result indicates that the DE internal repeat

region, encompassed in a.a. 330-463, influences the ability of v-kLANA to establish latency in the

spleen. In agreement is the detection of the frequency of viral DNA positive cells, where no differences

between v-kLANAΔ465-929 and v-kLANA are observed, and the v-kLANAΔ332-929 mutant present a

frequency of viral DNA positive cells that it is not detectable both in total splenocytes and in the GC B

cells (Figures 13A and 13B and Table 1). The correlation between this two independent assays helps

to confirm the phenotype observed in the v-kLANAΔ332-929 mutant, where the lack of establishment of

latency in the spleen assessed by infectious centre assay is supported by the non detection of viral DNA

positive cells in total splenocytes and in GC B cells.

The results obtained in this work are in line with studies made by others, where in vitro, either

in transient transfected cells or in stable cell lines expressing similar LANA deletion mutants, kLANA

without the complete internal repeat region had deficits in maintaining viral episomes during latency

(Alkharsah and Schulz in 2011; De León Vasquez and Kaye, 2011; De León Vasquez and Kaye, 2013).

Some proteins, important for the correct replication and/or segregation of the episomes, might

interact with this internal region and the lack of that region impairs the persistence of viral genomes in

the spleen. Protein conformation itself might be altered by the lack of this structural parts, making it

impossible to function correctly. There also might be additional functions other than DNA segregation

and replication exerted by the internal repeat region that are essential for persistency of episome in the

spleen.

It was also important to detect the proteins by western blot to check if the lack of latency of the

v-kLANAΔ332-929 was due to low protein levels or lower stability of the protein. Given the fact that v-

kLANAΔ465-929 establishing latency in the spleen, with levels similar to v-kLANA, it is likely that the

protein is being expressed. The most common used antibody to detect kLANA is LN53, which is an

antibody that detects several epitopes in the EQE repeat region and is very sensitive. Because both v-

30

kLANAΔ465-929 and v-kLANAΔ332-929 lack that specific part, another antibody was used and it was

not successful in the detection (Figure 11). Serum from a KS patient was also used to try to detect the

proteins but is was unsuccessful as well. In the future efforts should be done to detect these mutant

proteins, like the construction of viruses with higher affinity epitope tags (HA tag for example), to be able

to detect these proteins with common commercial antibodies. The use of more cells or higher MOI to

intensify the detection and the analysis at different time-points after infection can also be done to

improve the detection levels in a western blot

The levels of kLANA poly-A transcripts in both mutant viruses were also assessed in infected

BHK-21 cells and compared to v-kLANA. The v-kLANA and the v-kLANAΔ465-929 presented very

similar levels of transcripts whereas the v-kLANAΔ332-929 have a slight deficit in the kLANA expression

levels (Figure 11). This might be due to the fact that the aspartate and glutamate internal acidic and part

of the glutamine region may interact with cellular proteins with functional activity in transcription (De

Leon & Kaye, 2011). Again, with the lack of such structural parts, the protein conformation might be

altered in such a way that blocks the access of the transcription machinery, making it difficult for the

correct transcription to occur. Overall these results confirm expression of the mutant kLANA transcripts

in infected cells and suggest that deletion of the internal regions does not affect significantly its own

expression.

Many proteins interact with different parts of LANA. Some were identified and mapped to overlap

with the internal repeat region. These proteins fall in the transcription category, in chromosome structure

or modification and in cell growth regulation (De León Vasquez and Kaye, 2011). Protein-protein

interaction assays should be performed to identify even more interactions and, more importantly, map

the location of those interactions. It is possible that most interactions occur within the internal repeat

region since it is the biggest region (Figure 5). By doing this kind of assays it might be possible to unveil

more functions of this internal structural part.

31

6. Conclusion

This work took advantage of the creation of an in vivo model to study the pathogenesis

associated with Kaposi’s sarcoma-associated herpesvirus infection. Two chimeric recombinant viruses

in a MHV-68 background were created, where the MHV-68 ORF73 (mLANA) was substituted by KSHV

ORF73 (kLANA): v-ΔkLANA465-929, which retains the DE region and part of the Q region and v-

ΔkLANA332-929, lacking the entire internal repeat region. The fact that these parts were missing helped

to unveil their importance in this in vivo model.

v-ΔkLANA465-929 had the same ability to establish latency in the spleen as the v-kLANA, while

the v-ΔkLANA332-929 has a big deficit in establish latency. This leads to conclude that the aspartate

and glutamate and part of the glutamine internal regions (a.a. 330-464) are somehow crucial for the

latency in the spleen. These results have similarities with in vitro results obtained by other authors where

it was seen that the lack of some internal parts of kLANA led to a deficit in the maintenance of the viral

episomes during latency.

In the future, a deletion mutant encompassing precisely aminoacids 330-463 should be

constructed, in order to prove that this is really a crucial part of the protein for infection and establishment

of latency. Furthermore, with this mutant virus, the EQE repeats are maintained, meaning that the

epitopes for the LN53 antibody are maintained and it is thus possible to detect the protein.

Future work on identification of more proteins that interact with this internal part is also needed to

elucidate better the mechanisms underlying LANA full function.

By having more knowledge about this protein, that is crucial for latency, might shed light into a possible

therapeutic target for fighting the KSHV infection.

32

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