Virus Infection and SynthesisProteins that initiate infection

Core Protein Assembly

Table of Contents• Virus Infection

• Introduction

• Steps

• Virus Entry into Host Cell

• Uncoating

• Targeting

• Gene Expression and Genome Replication

• Proteins that Intiate Infection

• Virus Synthesis

• Viral Translational Strategies

• Viral Protein Products

• Assembly of Viral Components

• Steps of Virus Synthesis

• Encapsidation/ Coating

• Virion Budding and Maturation

• Virion Exit Patterns

• Spread of Virus

• Virus infection life cycle

Virus Infection

•Virus is a nucleo-protein having RNA or DNA as a genetic material. Infect a

limited number of different plant species, few have a wide host range. Can

multiply only inside a living cell, using host machinery.

•Viruses can die quickly if outside a cell or if the cell dies. e.g., Cucumber

mosaic virus

•Viruses can retain their ability to infect for years after the infected plant part

dies. e.g., Tobacco mosaic virus.

Introduction

•The pathological phenotypes are the result of interference and/or

competition for a substantial amount of host resources, which can disrupt

host physiology to cause disease.

Continued…

Several different stages in the virus life cycle are occurring:

1) Penetration

2) Uncoating

3) Targeting

4) Gene expression

5) Genome replication

6) Virion assembly/maturation

7) Release of new infectious virus

Steps of Virus Infection

Compete with host defense mechanism

•Although lacking an immune system comparable to animals, plants

have developed a stunning array of structural, chemical, and protein-

based defenses designed to detect invading organisms and stop them

before they are able to cause extensive damage.

•Pathogens have developed countermeasures that are able to suppress

basal resistance in certain plant species.

Virus Entry into Host Cell

Wounds•Virus enters into the plant through wounds in epidermal cells or through

roots.

Fig: Virus entry through wounds

Mechanical Damage

•Virus particles directly in contact with protoplasmic membranes are

assembled in host cells by pinocytosis

•e.g.,

•Cow pea mosaic virus

Pinocyte

•Inoculum efficiency for mechanically transmissible

viruses is increased in presence of abrasive powders

on foliage

•Abrasive increases frequency of infectable wounds

•Requires104 to 105 virus particles applied to leaf

surface for each cell infected

•e.g.,

•Potato virus X

Mechanical Inoculation

•Arthropods

•Nematodes

•Fungi

Arthropods

•Aphids, whiteflies, leafhoppers, thrips, beetles, mealybugs, mirids, and mites

•The hollow, needle-like mouthparts can penetrate the plant cell wall, either by

mechanical force and/or with the help of salivary and gut enzymes.

•It does not always irreparably damage the plant cell.

Vector-Mediated

Beetle Whitefly

Thripes Leaf hopper Zoospore

Aphid

•The genome is completely released from the capsid during or after penetration.

This is known as "uncoating“

•Different experiments indicating uncoating of viral RNA

• TMV multiplication detected some hours earlier when inoculation is done by

RNA.

Uncoating

•Since almost all DNA viruses replicate in the nucleus of

infected cells, they must be targeted there

Targeting

•Viruses contains two types of genome:

•DNA genome

•RNA genome

•For DNA genome, the replication cycle can be broken down into the

following steps:

•Early gene expression

•Replication of the viral genome

• Late gene expression

Gene Expression and Genome Replication

Viral Genome Expression

I: is transcription of dsDNA usually by host-DdRP

II: is transcription of ssDNA to give ds template for I (Gemini viruses)

III: is transcription of dsRNA usually by virus coded RdRp (Reoviruses)

IV: is replication of +ve stand RNA via –ve stand template by virus coded RdRp;

the viral (+) strand is often the template for the early translation (=ve sense RNA

viruses)

V: is the transcription of -ve sense virus genome by virus coded RdRp

( Tospoviruses)

VI: is the reverse transcription of RNA stage of retro and pararetroviruses leading

to dsDNA template for mRNA transcription.

Fig Legend

Systemic viral infection requires several different steps

Proteins that initiate the infection

NSP- Nuclear shuttle protein& Mp-Movement Proteins

Fig: Genome organization of Begomoviruses

•NSP is responsible for transport of viral DNA from the

nucleus into the cytoplasm

•Movement protein coordinates the movement of viral DNA

across plasmodesmatal boundaries.

•Whereas specific point mutations in MP or NSP prevented

this migration. 

Continued…

Fig: Role of NSP and MP in movement

•MP of Abutilon mosaic virus (AbMV) translationally fused to green fluorescent

protein or glutathione-S-transferase as reporter proteins

•It has been proposed that MP serves as a membrane anchor at the plasma

membranes, thus facilitating the movement of the transport complex along these

membranes.

• Moreover, MP can shuttle between the nuclear envelope and the cellular

periphery in order to fulfill its task, because green fluorescent protein-MP was

also observed in the vicinity of nuclei in some cells

MP for movement across membranes

•NSP, interacts with the Arabidopsis (Arabidopsis thaliana)

nuclear acetyl transferase AtNSI (nuclear shuttle protein

interactor),

•This interaction and NSI expression are necessary for

cabbage leaf curl virus infection and pathogenicity. 

NSP as a Pathogenicity Inducer

Fig: Model for NSP-AtNSI interactions in the nuclei of CaLCuV-infected

cells

•Expression of the Tomato Leaf Curl New Delhi Virus NSP from the PVX

vector in N. benthamiana resulted in leaf curling that is typical of the

disease symptoms caused by ToLCNDV in this species.

NSP as Symptom Determinant

Fig: Symptoms exhibited by plants following agroinfiltraton with

PVX constructs.

CP-Coat Protein

•For efficient systemic spread

•Translation of viral RNA

•Targeting viral genome to site of replication

•Suppression of RNA silencing

TMV CP•Required for phloem loading step

SPMV CP

(Satellite panicum mosaic virus coat protein)

•Satellite panicum mosaic virus coat protein effectively protect its cognate RNA

from deleterious events

•Enhances the performance of plant virus gene vectors. e.g., in Nicotiana

benthamiana

•SPMV CP directly or indirectly promotes spread of heterologous viruses in

compatible host systems.

Fig: Potato virus X (PVX)-derived gene vectors carrying the satellite panicum mosaic virus coat protein gene (SPCP).

Fig: Representative upper non-inoculated leaves that were previously inoculated with inoculation buffer (Mock), infectious transcripts of PVX-E1S empty vector (PVX), or a PVX vector carrying the SPMV CP gene in an minus-sense (PVX-SPCP−) or coding (PVX-SPCP+) orientation.

•DNA β satellites are symptom-modulating, single-stranded

DNA that require the helper begomovirus for replication,

spread in plant tissues, and plant-to-plant transmission by

the whitefly vector of begomoviruses 

•The DNA β satellite component encodes an essential

pathogenicity protein (βC1)

DNA β satellites

•Plants infected with PVX-βC1 produced severely distorted stems and

leaves

•Indicating that βC1 protein expression, when removed from the context of

the AYVV genome, can have a marked effect on development.

•N. benthamiana plants containing a dimeric DNA β transgene produced

severe developmental abnormalities, vein-greening, and cell proliferation in

the vascular bundles.

Continued…

Fig: Expression of βC1 protein from PVX vector

Virus Synthesis

Modifications in Translation System of Virus-infected cells

Modifications of initiation factors i.e. eIF2 nad eIF4

• Covalent modifications, especially phosphorylation

• Association with regulatory partners

• Interactions with virus-encoded subunits

Viral Translational Strategies

•Scanning

•Leaky scanning

•Non-AUG initiation – CUG and GUG codon identified.

•CAP-independent initiation

•Frame shifting

•Read-through

•Shunt – Skip 5’ UTR Region and reaches initial codon

•Hopping – 50 nucleotides are bypassed, rare event.

Viral Protein Products

• Viral proteins that are components of the mature assembled virus particles

and are produced by late genes are called as viral structural proteins i.e.

• Nucleocapsid core proteins (gag proteins)

• Viral non-structural proteins are ones that are not packaged in the

mature virus particle and are only found in the infected cells and are

produced by early genes.

Continued…

• Most viruses encode a limited number of gene products

therefore, they are dependent on the cell not only for

biosynthesis of the macromolecules that constitute the virus

particles,

• But also for the pre-existing intracellular sorting mechanisms

that the virus utilizes to achieve delivery of those

macromolecules to the sites of virion assembly.

Example

Potyvirus polyprotein: Cleavage of the polyprotein by host and viral proteases

releases the mature viral proteins.

Assembly of Viral Components

Steps of Virus Synthesis• Movement of components to assembly site(s) in the nucleus

and/or cytoplasm

• Assembly of proteins

• Incorporation of the nucleic acid genome

• Intracellular movement of viral or subviral particles

• Exit from the cell

• Virion maturation (in some cases).

Encapsidation/ Coating

• Protein monomers can aggregate in solution in various ways

depending upon:

• pH

• Ionic strength, and

• Temperature

• During the replication of many viruses, hundreds to thousands

of proteins assemble around the viral nucleic acid to form a

protein shell called a capsid.

Capsid Assembly

Capsid Types• Helical: Helical capsids are usually formed from one

protein that interlocks to form a helix-like structure around the

viral genome. 

• Icosahedral: An icosahedron is a twenty-sided object, that is

almost spherical.

• Prolate: This is an icosahedron elongated along the

fivefold axis and is a common arrangement of the heads of

bacteriophages.

Continued…

• Complex: Mostly found in non-animal viruses; are divided

into two groups:

• Viruses without identifiable capsids.

• Viruses whose capsids are attached with additional

structures.

Examples

Virion Budding and Maturation

Budding Sites

• Golgi complex

• ER-Golgi Complex Compartment

• Cellular Membranes

• Plasma membranes

• Virions bud off in the form of vesicles or in the form of

capsids depending on their formulation

Virion Exit

Patterns

Spread of Virus

Process•In order to cause infection in neighboring cells, plant viruses spread from the

initially infected cells to the rest of the plant in several distinct stages.

•First, after first viral genome replication cycles have been completed, the

progeny viruses moves intracellularly from the sites of replication to

plasmodesmata (PD)

•The virus then transverses the PD to spread intercellularly (cell-to-cell

movement). Long-distance movement of virus occurs through phloem sieve

tubes.

•The processes of plant virus movement especially cell to cell movement are

controlled by specific viral movement proteins (MPs).

Spread of Virus In a Plant

Virus Infection Life Cycle

Barley Yellow Dwarf Virus (BYDV) Life Cycle

BYDV Life cycle (In Detail)