Midterm #3: Viral Genome

Question 1

Baltimore’s viral genome classification

Answer 1

• 7 classes of viral genomes
• Must produce mRNA’s for translation of viral proteins by host ribosomes
• Must produce copies of viral genome for packaging into newly assembled virions
• Viruses often encode a nucleic acid polymerase, which is needed for genomic replication and for protein synthesis (mRNA)

Question 2

Baltimore Viral Classification Chart

Answer 2

Question 3

2 essential viral functions for replication

Answer 3

• Must produce mRNA’s for translation of viral proteins by host ribosomes
• Must produce copies of viral genome for packaging into newly assembled virion

Question 4

Poliovirus Replication in General and Overview

Answer 4

• (+) sense, single-stranded RNA
• Exluisve to humans
• small, non-enveloped member of genus Enterovirus
• Infects GI tract but can travel to CNS
• Highly contagious even when assymptomatic
• 30 nm capsid. (very tiny)

Question 5

Poliovirus Replication in Detail

Answer 5

• (+) sense genome is equivalent to mRNA and can be directly translated
• Virally encoded polymerase makes (-) sense complementary strand from (+) strand and makes new (+) strands from the (-) template
• No DNA intermediate
• Nucleic acid replication carried out by an RNA-dependent RNA polymerase (RdRp)
  
  • ​RNA template that makes complementary RNA from that
  • Virally encoded
• Eclipse phase is very short becuase there are not many steps before producing protein

Question 6

Poliovirus Replication: Figure

Answer 6

Question 7

Virally Encoded RNA dependent RNA Polymerase (RdRp)

Answer 7

• Responsible for synthesizing complementary nucleic acid strands
• Target for many antivirals e.g. nucleoside and nucleotide analog anti-retrovirals, non-nucleoside reverse transcriptase inhibitors, anti-herpesvirus drugs, ribavirin, experimental drugs against ebola (brincidofavir, favipiravir)
• No proof-reading like in DNA polymerase, hence high error rate
  
  • May limit size of genome and complexity (# of genes) in virus
• Often tightly membrane-associated and prone to oligomerization
  
  • multiple copies link up with each other
• Structural homology among most RNA viral polymerases
  
  • similar structure, similar function

Question 8

RNA virus replication factories

Answer 8

• Compartments were RNA synthesis takes place
• localization of RdRp
• Spherule compartments whose formation is induced by viral non- structural proteins, in some cases the polymerases themselves.
• Often linked to cytoplasm through pores/necks, allowing exchange of nucleotides, synthesized genomes. Efficient RNA synthesis.
• Sometimes linked via membranous structures and cytoskeletal components to ribosome-abundant sites where mRNA translation to viral proteins take place.
• Efficient transport of newly synthesized viral genomes to assembly sites

Question 9

RNA capping and translation initiation sites

Answer 9

• mRNA that is to be actively translated in eukaryotic cells is modified at the 5’ end with a “cap”
• Some viruses include caps in their genomic RNA, others such as poliovirus do not. It has covalently attached proteins (VPg), which it uses for specific packaging of the vRNA
  
  • Instead of a cap, poliovirus (a type of picornavirus) has an Internal Ribsome Entry Site (IRES), which is located some ways into the genome, where the ribosome initiates translation

Question 10

Strategies for handling a “polycistronic” genome

Answer 10

• polycistronic: a single transcript encoding information for multiple protiens
  
  • at least 2 genes: capsid and polymerase. Usually have several more though
• Options to make multiple proteins from one mRNA

1. RNA is translated as a single, long polyprotein that is chopped up by a protease
2. Segmented genomes with multiple “monocistronic” mRNA’s
3. Some produce a long mRNA that can be processed by host splicing machinery

Question 11

Polycistronic genomes and polyproteins

Answer 11

• RNA is translated as a single, long polyprotein
• Virus encodes a protease enzyme that cuts the polyprotein at specific sites to produce functional protein components

Question 12

Polio Genome

Answer 12

Question 13

Summary of picornavirus (e.g. poliovirus) infectious cycle

Answer 13

• Endocytosis
• Structural changes that trigger uncoating to free RNA genome?
• IRES driven traslation
• Polyprotein processing
  
  • Proapoptotic effects
  • Anti-apaptotic effects
  • Shut off host cell (cap-dependent) translation
  • Shut off host cell transcription
  • Viral replication complex
• Negative strand synthesis and positive strand (genome) synthesis
• dsRNA (latency)
• Packaging
• Cell lysis, viral egress

Question 14

Picornavirus Infection Cycle: Figure

Answer 14

Question 15

Viral Nucleic Acid Polymerase

Answer 15

• Virus often encode a nucleic acid polymerase, which is needed for genome replication and for protein synthesis (mRNA)
• (-) RNA: not readily translatable to mRNA, need to produce (+) sense strand first

Question 16

(-) sense, single-stranded RNA virus genomes

Answer 16

• (-) sense genomes used as template to generate complementary (+) strand that function as mRNA, needed for translation
• No DNA intermediate
• No cellular enzyme that makes RNA from viral RNA template
• Nucleic acid replication carried out by an RNA-dependent **RNA polymerase (RdRp). ** But if the (-) gene cannot be translated, where does the polymerase come from that can make the (+) RNA?
  *

Question 17

(-) sense, single-stranded RNA virus genomes: Figure

Answer 17

Question 18

Segmented Genomes

Answer 18

• Another strategy of getting around translation initation challenges
• Ex: influenza (orthomyxovirus) has it’s genome broken into 8 segments. Each segment encodes a protein, but it is (-) sense so the (+) strands need to be made first
• Influenza’s polymerase actually “steals” mRNA caps from the host mRNA and attaches them at the 5’ end of each RNA segment
• To get around the polycistronic problem

Question 19

RNA Cap Stealing

Answer 19

• Snatching
• Influenza RdRp (PB1/PB2/PA) clips 10-20 nt from the 5’ end of host mRNA and uses it as the starting point to produce (+) sense complementary viral mRNA strand
• The resulting viral mRNA is translation-ready
• Function of mRNA cap:
  
  1. ​trafficking RNA out of nucleus
  2. Protection from exonucleases
  3. Promote translation

Question 20

Reassortment

Answer 20

• Segmented genomes are prone to reassortment
• Reassortment can occur when a cell is infected with 2 strains of the virus
• Can give rise to antigenic shifts and pandemics
  
  • Changing the surface protiens changes how our bodies recognize the antigen
• Also exploited under controlled cirumstances for generating recombinant viruses for vaccines
  
  • Use virus that propogate well in tissue culture then alter surface feature for that year

Question 21

(+) sense, single stranded RNA virus with DNA intermediate

Answer 21

• retrovirus
• virally encoded reverse transcriptase (RT) enzyme converts (+) ssRNA genome into (-) ssDNA
• RT also then generates the complementary (+) DNA strand to produce dsDNA
• HIV integrase enzyme integrates the dsDNA copy of the viral genome into the host DNA
• RT has RNA-digestion (ribonuclease) activity as well and digests the original (+) ssRNA

Question 22

HIV virion

Answer 22

• encapsulates 2 copies of (+) ssRNA tightly associated with nucleocapsid protein (NP) and contained within the cone-shaped capsid (CA)
• HIV brings along a number of virally encoded protein necessary for infection:
  
  • Reverse Transcriptase (RT)
  • Integrase
  • Protease
  • Vif, Vpr, Nef, Tat