Midterm #3: Viral Genome Flashcards Preview

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Flashcards in Midterm #3: Viral Genome Deck (22):
1

Baltimore's viral genome classification

  • 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)

2

Baltimore Viral Classification Chart

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2 essential viral functions for replication 

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

4

Poliovirus Replication in General and Overview

  • (+) 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)

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Poliovirus Replication in Detail

  • (+) 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

6

Poliovirus Replication: Figure

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7

Virally Encoded RNA dependent RNA Polymerase (RdRp)

  • 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 

8

RNA virus replication factories

  • 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 

9

RNA capping and translation initiation sites 

  • 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 

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10

Strategies for handling a "polycistronic" genome 

  • 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 

11

Polycistronic genomes and polyproteins 

  • 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

12

Polio Genome

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13

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

  • 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

14

Picornavirus Infection Cycle: Figure

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15

Viral Nucleic Acid Polymerase

  • 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

16

(-) sense, single-stranded RNA virus genomes 

  • (-) 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?
  •  

17

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

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18

Segmented Genomes

  • 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 

19

RNA Cap Stealing

  • 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

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20

Reassortment 

  • 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

21

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

  • 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

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22

HIV virion

  • 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