Virology lecture 2

Question 1

how do -ve ssRNA and dsRNA viruses replicate?

Answer 1

1 - trascribed by a virally packaged RNA-dependent RNA polymerase to produces +ve ssRNA
2 - this acts as mRNA and can be translated to produce capsid proteins and more RNA pol, or can be used as a template to generate more -ve ssRNA
3 - most replicate in the cytoplasm but the exception is influenza, replicating in the nucleus requiring host RNA pol2 as part of the transcription mech.
4 - the original purified RNA on its own is not infectious.

Question 2

how do +ve ssRNA viruses replicate?

Answer 2

1 - translated to make the viral proteins inc RNA dep RNA pol which replicates the input genome via a -ve ssRNA. ie +ve to -ve to +ve.
2 - the new +ve ssRNA can then be translated or packaged.
3 - this purified +ve ssRNA is infectious

Question 3

how do retroviruses replicate?

Answer 3

1 - they’re +ve ssRNA but are copied by reverse transcriptase (a RNA dep DNA pol packed in the virion) into dsDNA intermediate which is incorporated into the host genome (a provirus).
2 - provirus now transcribed by the host DNA dep RNA pol2. the resultant RNA is either packaged or transcribed (and sometimes spliced first).

Question 4

how do dsDNA viruses replicate?

Answer 4

1 - most replicate in the nucleus using host machinery inc host DNA dep RNA pol.
2 - as viral proteins are generated in the cytoplasm, some are transported back into the nucleus to assemble the new virions ie capsid proteins and DNA pol to replicate the initial DNA genome.
3 - as no nucleic acid pol is in the virion, the purified DNA alone is sufficient for infection.
4 - the exception are poxviruses which are dsDNA but replicate in the cytosol so have to package all the required enzymes (DNA dep RNA pol, capping and polyadenylating enzymes) hence the purified dsDNA is not infectious.

Question 5

temporal control of viral replication?

Answer 5

1 - early genes for nucleic acid replication and host modification
2 - late for structural proteins of virion. no need to produce these until late so that all nucleic acid can be used for replication and isnt packaged and then idle.
3 - both herpesviruses and poxviruses has temporally distinct gene classes, expression of one class dependent on proteins of the previous class.

Question 6

viral polyprotein processing vs eukaryotes

Answer 6

1 - essentially all eukaryotic mRNAs are monocistronic (code for one polypeptide) as ribosomes stop translating once they reach a stop codon. many viruses get multiple proteins from one mRNA by producing a large polypeptide which is post-translationally cleaved into the individual proteins. this is an important feature of RNA viruses. proteases usually specific to the virus and are potential chemo targets. an alternative strategy for RNA viruses is to have segmented genomes.

Question 7

viral use of splicing

Answer 7

used to place coding region of some of their proteins at 5’ end so that theyre encountered first on the mRNA by ribosomes. HIV envelope gp160 does this, it is then cleaved to produce gp120 and gp41.

Question 8

assembly of viruses

Answer 8

poorly understood. some dont require any catalytic process and may be spontaneous, others require cleavage of capsid proteins to mature forms to be infective ie HIV.

Question 9

release of viral progeny

Answer 9

1 - some via cell lysis ie some bacteriophages encode lytic proteins
2 - many bud out over prolonged periods to acquire an envelope. assciate with internal domain of viral glycoproteins inserted into the host cell membrane.

Question 10

what’s a latent infection?

Answer 10

1 - the virus infects the cell but then exists in a quiescent state.
2 - retroviruses do this. the provirus isnt trasncribed and hence isnt detected as infected by the immune system. if this occurs in the germ line it allows vertical transmission to host progeny. 8%of the human genome is retroviruses and retrotransposons.
3 - herpes virus is another example. the viral DNA enters the nucleus and is quiescent until reactivation, not integrated but as an extrachromosomal circular episome.

Question 11

how may a virus modify a host cell? 8 ways

Answer 11

1 - turn off host protein synthesis
2 - stimulate cell biochemistry to make it more virally productive
3 - increase nucleic acid synthesis
4 - modify the cell membrane ie inserting viral proteins
5 - cytopathic effect
6 -suppression of host signalling pathways for innate immunity
7 - some cause cell lysis some dont.
8 - cell transformation into a cell with many proporties associated with malignant cells.

Question 12

viruses shut off host protein synthesis

Answer 12

1 - ie poliovirus
2 - only viral proteins made.
3 - poliovirus protease destroys 5’cap binding tibosome complex so it cant bind host mRNA. poliovirus mRNA has an internal ribosome entry site (IRES) near its 5’ end that can be recognised in the absence of the 5’cap. cap-independent. IRES formed by RNA folding to recruit the ribosome.
4 - other mechs inc destruction of host DNA and increased rates of mRNA turnover.

Question 13

viruses stimulate host biochemical pathways

Answer 13

1- DNA viruses evidently need high levels of deoxyribonucleotides (dNTPs), resting cells only have low levels.
2 - some viruses synthesise factors that stimulate the cell into cycle and are made early in infection. eg simian virus 40 T antigen. (papovaviruses, adenoviruses and herpesviruses)
3 - poxviruses use a different mech
4 - the vaccinia virus (used to eradicate smallpox) expresses EGF which stimulates neighbouring cells to divide, making ideal infection targets.

Question 14

how do viruses increase nucleotide production

Answer 14

1 - initiate cell cycle
2 - express own enzymes for dNTPs in larger viruses - poxviruses and herpesviruses ie thymidine kinase, thymidylate kinase and ribonucleotide reductase.
3 - genes for these enzymes are non essential unlike those for capsid proteins but only in cells actively growing/replicating, not resting cells.

Question 15

how do viruses modify cell membranes?

Answer 15

1 - may insert viral proteins esp for viruses with a capsule.
2 - may alter cell interaction with neighbours ie measles virus causes fusion with uninfected cells, infects cells whilst avoiding neutralising antibodies. Called 'cell-associated viruses'.
3 - in contrast, small nonenveloped viruses such as poliovirus and FMDV are liberated in large numbers to target distant cells.
4 - HIV down regulates CD4, influenza removes sialic acid. prevents reinfection to improve viral dissemination.
5 - may downregulate MHC class 1 to evade CD8 T cells.

Question 16

viral cytopathic effect

Answer 16

1 - cytopathic effect = morphological changes in infected cells.
2 - vaccinia virus plaque formation
3 - virus-host specific c.p.e. so can be used diagnostically.
4 - often include cytoskeleton alterations to facilitate intracellular viral movement.
5 - can be rounded, more refractile or motile
6 - HMCV causes owl eye morphology
7 - pox viruses cause cytoplasmic eosinophilic infection bodies.

Question 17

viral suppression of innate immune system

Answer 17

1 - cell detects PAMPs with PRRs. causes host responses such as interferon release and apoptosis. viruses evolve to block these responses.
2 - large DNA viruses (herpesviruses and poxviruses) encode many proteins to block the innate immune response.

Question 18

viral infections - lytic or non, general rules

Answer 18

1 - most commonly lytic, killing host cell. approximate rules:
2 - DNA viruses cause lysis
3 - non enveloped RNA viruses are lytic
4 - host protein synthesis suppressors are lytic
5 - some enveloped RNA viruses inc retroviruses are non-lytic and release viral particles over a prolonged period.

Question 19

virally caused cell transformation

Answer 19

Means the cell exhibits uncontrolled growth, failure of contact inhibition, many properties associate with malignant cells in vivo. 2 groups of mechs:
1 - By DNA viruses - to create a suitable environment for replication. ie papilloma virus inducing proliferation and hence wart formation. Sometimes the replication fails but cell stimulation continues causing malignancy. notably strains 16 and 18 of HPV causing cervical carcinoma.

2 - By Retroviruses - reverse transcriptase and integration of a provirus, production and budding. not necessarily lethal. these viruses genetically recombine frequently and can pick up host genes, if one is important in cell growth then the virus may cause transformation due to this overexpression ie the Rous sarcoma virus. This virus is derived from an avian retrovirus (which does NOT cause transformation) but acquired the src gene encoding a tyrosine kinase. This gene is expressed at low levels and is highly regulated but if from the virus it is expressed at high levels causing abnormal growth of the cell and rapidly develops sarcomas in chickens.
3 - NB this transformation is a rare event as it requires an abortive infection (DNA virus) or activation of a KEY host gene (retroviruses). Hence neoplasia are associated with persistent infections.

Question 20

retrovirus nomenclature - gag, pol, env. helper virus?

Answer 20

gag = group specific antigen

• pol = polymerase ie reverse transcriptase including an integrase
• env = envelope glycoprotein
• any captured transforming host genes = oncogenes.
• host genes often acquired with loss of viral gene. rous sarcoma virus has gag and pol genes of avian retrovirus but lost the env for the src gene. this gene loss often disables the virus (cant now synthesise the envelope) so requires coinfection by a helper virus to make the envelope.