Viral Replication

1.         Viral Growth curve

a.            Experiment setup:

i.          all cells in a culture are infected simultaneously, by using a high multiplicity of infection.

ii.          the increase in infectious virus over time is followed by sequential sampling and titration.

b.         The amount of virus produced is plotted on a logarithmic scale as a function of time after infection.

c.         Eclipse period:

i.          shortly after infection, the virus disappears; infectious particles cannot be demonstrated, even intracellularly.

ii.          the time during which no virus is found inside the cell is known as the eclipse period.

d.            Although the virus particle, as such, is no longer present, the viral nucleic acid continues to function and begins to accumulate within the cell.

e.         The eclipse period ends with the appearance of the virus.

f.          The latent period is defined as the time from the onset of infection to the appearance of virus extracellularly.

g.         The infection begins with one virus particle and ends with several hundred virus particles having been produced.

2.            Description of Growth cycle

a.         Early events:

i.            attachment

ii.            penetration

iii.            uncoating

b.         Middle events:

i.          gene expression

ii.          genome replication

c.         Late events:

i.            assembly of virus genome and protein coat

ii.          release of virus particle

3.         Key steps in Viral replication

a.            Following attachment, the virion is taken up by the host cell and is partially uncoated to expose the viral genome.

b.         Certain early viral genes are transcribed into RNA which may then be processed in a number of ways, including splicing.

c.         The early gene products translated from this mRNA are of three main types:

i.          proteins that shut down cellular nucleic acid and protein synthesis.

ii.          proteins that regulate the expression of the viral genome.

iii.            enzymes required for the replication of viral nucleic acid.

d.            Following viral nucleic acid replication, late viral genes are transcribed.

e.         The late proteins are principally viral structural proteins for assembly into new virions; some of these are subject to posttranslational modifications.

f.            Genome replication:

i.          for most families of DNA viruses, transcription and DNA replication take place in the cell nucleus.

ii.          some viruses use the cellular RNA polymerase II and other cellular enzymes, but most have their own genes for a range of other enzymes.

iii.         in addition, some carry ‘transforming genes’ which induce cellular DNA synthesis, to increase the concentration of cellular enzymes and deoxynucleotides.

iv.         RNA viruses have the advantage that ribonucleoside triphosphates are available throughout the cell cycle.

v.            however, these enzymes must encode their own RNA polymerase, since cells lack the capacity to copy RNA from an RNA template.

vi.         most RNA viruses replicate in the cytoplasm.

4.            Attachment

a.         To cause infection, virus particles must be able to bind to cells.

b.         The proteins on the surface of the virion attach to specific receptor proteins on the cell surface through weak, noncovalent bonding.

c.         The specificity of attachment determines the host range of the virus:

i.          some viruses have a narrow range: poliovirus can enter cells of only humans and other primates.

ii.          others have quite a broad range: rabies virus can enter all mammalian cells.

d.         The organ specificity of viruses is governed by receptor interection as well.

e.            Receptors for Viruses:

i.            members of the immunoglobulin superfamily, such as the integrin ICAM-1, which is the major receptor for most rhinoviruses.

ii.          CD4, the receptor for HIV.

iii.            fibroblast growth factor receptor, the receptor for herpes simplex virus type 1.

iv.         rabies virus to the acetylcholine receptor.

v.            hormone receptors and permeases.

5.            Penetration

a.            Following attachment, virions can enter cells by one of two main mechanisms, endocytosis or fusion.

b.            Endocytosis:

i.          many enveloped and nonenveloped viruses utilize receptor-mediated endocytosis to initiate infection.

ii.            attachment to receptors, which cluster at clathrin-coated pits, is followed by endocytosis into clathrin-coated vesicles that enter the cytoplasm.

iii.         the vesicle then fuses with endosomes.

iv.            acidification within the vesicle triggers changes in the capside protein VP4 of poliovirus, for example, leading to release of RNA from the virion into the cytosol.

c.         Fusion with plasma membrane

i.          the F (fusion) glycoprotein of paramyxoviruses causes the envelope of these viruses to fuse directly with the plasma membrane of the cell, even at pH 7.

ii.          this may allow the nucleocapsid to be released into the cytoplasm.

d.         Special penetrating mechanism of bacteriophages:

i.          some of the T group of bacteriophages infect E.coli by attaching several tail fibers to the cell surface and then using lysozyme from the tail to degrade a portion of the cell wall.

ii.          at this point, the tail sheath contracts, driving the tip of the core through the cell wall.

iii.         the viral DNA then enters the cell through the cell core, while the capsid proteins remain outside.

6.            Uncoating

a.         For viral genes to become available for transcription it is necessary that virions be at least partially uncoated.

b.         In the case of enveloped RNA viruses that enter by fusion of their envelope with either the plasma membrane or an endosomal membrane, the nucleocapsid is discharged directly into the cytoplasm.

c.         With the nonenveloped icosahedral reoviruses only certain capsid proteins are removed, and the viral genome expresses all its functions without ever being released from the core.

d.         For most other viruses, however, uncoating proceeds to completion.

e.         For some viruses that replicate in the nucleus the later stages of uncoating occur there, rather in the cytoplasm.

7.         Gene expression and Genome replication

a.         DNA viruses replicate in the nucleus and use the host cell DNA-dependent RNA polymerase to synthesize their mRNA.

b.         The poxviruses are the exception because they replicate in the cytoplasm, where they do not have access to the host cell RNA polymerase; they therefore carry their own polymerase within the virus particle.

c.         The genome of all DNA viruses consists of double-stranded DNA, except for the parvoviruses, which have a single-stranded DNA genome.

d.         RNA viruses, with one exception, replicate in the cytoplasm.

e.         The exception is influenza virus which undergoes a major step of its replication in the nucleus – it synthesizes its progeny RNA genomes there and completes its replication in the cytoplasm.

f.            Different strategies employed by RNA viruses for mRNA synthesis:

8.            Assembly and Release

a.         The progeny particles are assembled by packaging the viral nucleic acid within the capsid proteins.

b.            Nonenveloped viruses:

i.          all nonenveloped viruses have an icosahedral structure.

ii.          the structural proteins of simple icosahedral viruses associate spontaneously to form capsomers, which undergo self-assembly to form capsids into which viral nucleic acid is packaged.

iii.            completion of the virion often involves proteolytic cleavage of one or more species of capsid protein.

iv.         most nonenveloped viruses accumulate within the cytoplasm or nucleus and are released only when the cell eventually lyses.

c.            Budding:

i.          most enveloped viruses bud from the plasma membrane.

ii.          the budding process begins when virus-specific glycoproteins insert into the lipid bilayer at specific sites.

iii.         the viral nucleocapsid then interacts with the specific membrane site mediated by the matrix protein.

iv.         the cell membrane evaginates at that site, and an enveloped particle buds off from the membrane.

v.         budding frequently does not damage the cell, and in certain instances the cell survives while producing large numbers of budding virus particles.

d.            Exocytosis:

i.            flaviviruses, coronaviruses, and bunyaviruses mature by budding through membranes of the Golgi complex or rER.

ii.          vesicles containing the virus then migrate to the plasma membrane with which they fuse, thereby releasing the virions by exocytosis.

iii.            uniquely, the envelope of the herpesviruses is acquired by budding through the inner lamella of the nuclear membrane; the enveloped virions then pass directly to the exterior of the cell via the rER.

9.            Lysogeny

a.         Some viruses use an alternative pathway, called the lysogenic cycle, in which the viral DNA becomes integrated into the host cell chromosome and no progeny virus particles are produced at that time.

b.         The viral nucleic acid continues to function in the integrated state in a variety of ways.

c.         Of particular medical significance is the synthesis of several exotoxins in bacteria, such as diphtheria and botulium toxins, coded for by the genes of the integrated bacteriophage.

d.            Lysogenic conversion: new properties that a bacterium acquire as a result of expression of the integrated prophage (the integrated viral DNA) gene.

e.            Because the integrated viral DNA is replicated along with the cell DNA, each daughter cell inherits a copy.

f.          The prophage can be induced to resume its replicative cycle by the action of UV light and certain chemicals that damage DNA.

g.         The virus then completes its replicative cycle, leading to the production of progeny virus and lysis of the cell.