Journal of Medical Virology 70:S79–S81 (2003)

Use of a Rodent Model to Show That Varicella-ZosterVirus ORF61 Is Dispensable for Establishmentof Latency

Hitoshi Sato, Lesley Pesnicak, and Jeffrey I. Cohen*

Medical Virology Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, Maryland

Varicella-zoster virus (VZV) results in a latentinfection in humans after primary infection.Latency has also been established in guinea pigsand rats after inoculation with the virus. It wasfound that infection of cotton rats with the Okavaccine strain of VZV results in a latent infection.To begin to identify which genes are requiredfor latency, we infected cotton rats with VZVstrain Oka that is deleted for ORF61. ORF61 pro-tein transactivates certain VZV promoters andenhances the infectivity of viral DNA in transienttransfections. Deletion of ORF61 results in abnor-mal syncytia and impairs the growth of VZVin vitro. Inoculation of cotton rats with ORF61-deleted Oka virus resulted in latent VZV infectionin the nervous system similar to that seen foranimals infected with parental virus. Thus, thecotton rat can be used to study the ability ofmutants in the Oka vaccine strain of VZV to esta-blish latent infection. J. Med. Virol. 70:S79–S81, 2003. � 2003 Wiley-Liss, Inc.{

KEY WORDS: varicella-zoster; latent infec-tion; ORF61; cotton rat; Okavaccine

INTRODUCTION

VZV and herpes simplex virus (HSV) establish life-long latent infections in sensory ganglia in humans.During HSV latency, only the latency associatedtranscripts (LATs) are expressed. In contrast, multipleviral transcripts have been detected during VZV latencyin humans [Meier et al., 1993; Cohrs et al., 1996, 2000;Lungu et al., 1998; Kennedy et al., 1999, 2000]. Whileextensive studies have evaluated whether HSV genesare dispensable for latency, it is unknown whetherspecific VZV genes are required for latent infection.Several studies have shown that guinea pigs and ratsinoculated with VZV develop latent infection with viralDNA present in trigeminal [Lowry et al., 1993; Brunellet al., 1999] or dorsal root [Debrus et al., 1995; Sadzot-

Delvaux et al., 1995; Annunziato et al., 1998] ganglia.Viral RNA transcripts have also been detected in ro-dents, similar to those seen in humans.

Most studies of VZV latency in humans have eval-uated infections due to non-vaccine strains of virus.Similarly, the majority of animal studies have usedlow passage clinical isolates of the VZV. While theOka vaccine strain of VZV is currently being used inseveral countries to prevent chickenpox, vaccinationwith this virus results in a latent infection. We havebeen interested in determining which viral genes areimportant for the establishment of latent infection. Alarge number of mutants have been constructed in theOka vaccine strain of VZV [Cohen and Straus, 2001].

Recently, we constructed a mutant in VZV ORF61.ORF61encodesaphosphoprotein[Stevensonetal.,1992;Ng et al., 1994] that transactivates VZV immediate-early and putative early promoters in transient expres-sion experiments [Moriuchi et al., 1993]. ORF61 proteincan enhance or repress the activation of VZV promotersby VZV immediate-early proteins depending on the cellline used [Nagpal and Ostrove 1991; Perera et al., 1992;Moriuchi et al., 1993]. Theamino terminus of theproteincontains a RING finger domain comprised of a specificpattern of cysteines and histidines; deletion of this por-tion of the protein results in loss of its transactivatingactivity [Moriuchi et al., 1994]. Deletion of the carboxyterminus of the protein results in a dominant negativemutant that inhibits the activity of the full-lengthprotein.

ORF61 is the homolog of herpes simplex virus ICP0and the VZV protein can complement an HSV ICP0mu-tant [Moriuchi et al., 1992]. Like the HSV ICP0mutant,VZV deleted for ORF61 is impaired for virus replication

*Correspondence to: Dr. Jeffrey I. Cohen, Laboratory of ClinicalInvestigation, National Institutes of Health, Bldg. 10, Rm.11N228, 10 Center Drive, Bethesda, Maryland, 20892-1888.E-mail: jcohen@niaid.nih.gov

Accepted 13 September 2002

DOI 10.1002/jmv.10326

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2003 WILEY-LISS, INC. {This article is a USgovernment work and, as such, is in the publicdomain in the United States of America.

and can be complemented by certain neuroblastomaand osteosarcoma cells lines [Yao and Schaeffer, 1995;Cohen and Nguyen, 1998]. Here we show that deletionof ORF 61 does not impair the ability of the virus toestablish a latent infection.

MATERIALS AND METHODS

Viruses

Recombinant Oka strain VZV (ROka) was derivedfrom four cosmids by transfection of Mewo (humanmelanoma)cells.ROka61Disdeleted foraminoacids1 to452 of the 467 amino acid ORF61 protein [Cohen andNguyen, 1998]. VZV strain Molly is an early passageisolate. Virus stocks were propagated and titered inmelanoma cells.

Animal Experiments and Extractionof Nucleic Acid

Female cotton rats 6 to 8 weeks of age were anesthe-tized and inoculated intramuscularly at multiple sitesalong both sides of the lumbar and thoracic spine.The animals received 5� 105 PFU of VZV-infectedmelanoma cells at each site. Some animals were notinoculated or received uninfected melanoma cells as anegative control. Onemonth after infection, the animalswere sacrificed and the lumbar and thoracic dorsal rootganglia were isolated and pooled for each animal. DNAwas obtained from the ganglia and PCR was performedusing primers specific for VZV ORF21 as previouslydescribed [Brunell et al., 1999]. Southern blotting wasperformed with a radiolabeled ORF21 probe.

RESULTS AND DISCUSSION

Cotton rats have been used for studies of pathogene-sis of a number of human viruses that grow poorly inother rodents, including respiratory syncytial virus,adenovirus, and human immunodeficiency virus [Faithet al., 1997; Langley et al., 1998]. Therefore, while priorstudies of VZV latency have been performed using adultandneonatal rats [Annunziatoetal., 1998;Brunell etal.,1999; Kennedy et al., 2001] or guinea pigs [Lowry et al.,1993], we inoculated cotton rats with VZV.

In the first set of experiments, cotton rats wereinoculated in the paraspinal region with melanomacells containing ROka61D, ROka, or Molly strain VZV.Two negative controls were performed. Animals werenot inoculated with any cells or were inoculated withuninfected cells. All animals were housed in the sameroom and none showed overt signs of infection. Onemonth after inoculation, animals were sacrificed andDNAwas obtained from the dorsal root ganglia adjacentto the inoculation sites. PCR and Southern blotting forVZVDNAshowed thatnone of theuninoculatedanimalsor those receiving uninfected cells had viral DNA intheir dorsal root ganglia (Fig. 1). Three of 6 animalsreceiving VZV ROka61D and 4 of 5 animals receivingROka hadVZVDNA in their ganglia. Three of 6 animalsreceiving a low passage clinical isolate of VZV (strain

Molly) had viral DNA in their ganglia. Prior experi-ments showed that viral DNA could not be detected inganglia from animals that received melanoma cells con-taining VZV ROka that had been heated to inactivatethe virus [Sato et al., 2002]. Therefore, infectious virus isrequired for establishment of latent infection.

The second series of experiments yielded similar re-sults (Table I). In summary, the majority of animalsreceiving VZV ROka or Molly had latent viral infection,and about half of those receiving ORF61 had VZV DNAin the ganglia. In other experiments, we have shownthat transcripts for a latency-associated gene, ORF63,are expressed in cotton rat ganglia one month afterinfectionwithVZV,whileORF40 transcripts (a gene notassociated with latency) are rarely expressed at thistime [Sato et al., 2002]. Thus, these results indicate thatORF61 is not required for the virus to establish a latentinfection. Furthermore, they show that at the titer usedin these experiments, the attenuatedOka vaccine strainestablishes a latent infection at a similar rate as a lowpassage isolate of the virus.

VZV deleted for ORF61 is impaired for syncytiaformation [Cohen and Nguyen, 1998]. While cellsinfected with the deletionmutant express normal levels

Fig. 1. VZV ORF61 is not required to establish a latent infection.Ganglia DNA was amplified by PCR using primers for ORF21 andSouthern blotting was performed using an ORF21 probe. Ganglia fromanimals 3, 4, and 6 infected with VZV ROka61D, animals 1, 2, 3, and4 infected with VZV ROka, and animals 3, 4, and 5 infected withVZV strain Molly contained viral DNA. No viral DNA was detectedin animals not inoculated with any cells (Mock) or inoculated withuninfected cells (Uninfected).

TABLE I. Frequency of Latent Viral DNA in VZV-InfectedCotton Rats*

Experimentnumber

Number of animals with VZV DNA

Un/Mock Molly ROka ROka61D

1 0/7 3/6 4/5 3/62 0/8 9/9 6/10 4/9Total 0/15 12/15 10/15 7/15

*Un¼uninfected; Mock¼animals not inoculated with cells. Molly¼low passage VZV isolate.

S80 Sato et al.

of ORF62 protein, they have reduced levels of glycopro-tein E (gE). VZV deleted for ORF61 replicates to peaktiters in human melanoma cells that are about 10-foldlower than parental virus, and to titers that are appro-ximately 100-fold lower than parental virus in schwan-noma cells. In contrast, the difference in peak titersof the deletion mutant and its parent is less evidentin human neuroblastoma and osteosarcoma cells. Theobservation that VZV deleted for ORF61 had little effecton the establishment of latent infection, suggests thatthe virus is not impaired for entry into sensory nerveterminals.

VZVORF61 is the homolog ofHSV-1 ICP0.Deletion ofICP0 reduces the establishment of latent HSV infectionin mice and impairs reactivation of the virus fromlatently infected trigeminal explants and from culturesof dissociated neurons [Clements and Stowe, 1989; Leibet al., 1989; Cai et al., 1993; Halford and Schaeffer,2001]. Expression of ICP0 by an adenovirus vectorenhances reactivation of HSV-1 from latently infectedmouse trigeminal neurons [Halford et al., 2001]. HSVICP0 enhances the ability of transfected HSV DNA toinitiate replicative infection and this process has beenpostulated to be a model for reactivation of neuronallatency that occurs in the absence of other viral pro-teins [Cai and Schaeffer, 1989]. Similarly, VZV ORF61enhances the infectivity VZV DNA up to 14-fold aftertransfection [Moriuchi et al., 1993]. Thus, while VZVORF61 may not be critical for establishment of latency,itmay have an essential role for reactivation from latentinfection.

ACKNOWLEDGMENTS

We thank Paula Annunziato for advice regardinginoculation of animals. Hitoshi Sato is the recipient ofa Japan Society for the Promotion of Science (JSPS)fellowship.

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