Lecture 12: Retroviruses

Question 1

Retroviruses

Answer 1

Enveloped, single-stranded (+) RNA viruses that replicate through a DNA intermediate using reverse transcriptase

Question 2

Retrovirus genome organization in virion

Answer 2

-Retrovirus particles contain 2 copies of genomic (+) strand RNA
-“Pseudodeploid” because even though there are 2 viral RNA copies, the produce only one dsDNA copy
-2 copies are connected by non-covalent interactions near the 5’ end
-Includes 2 molecules of a specific tRNA primer

Question 2

Reverse transcription

Answer 2

-RNA dependent DNA polymerase activity copies genetic information from an RNA template to a DNA copy. This is called cDNA
-RT’s cannot initiate polymerization de novo, but require a specific primer

Question 3

Role of tRNA’s in reverse transcription

Answer 3

-Act as a primer for the initiation of reverse transcription
-Primer tRNA’s initially unwound
-Base paired near the 5’ end of each RNA genome at the primer binding site

Question 4

Four distinct activities of reverse transcriptase

Answer 4

-RNA-directed DNA polymerase
-DNA-directed DNA polymerase
-Helicase
-Hydrolyiss of RNA in RNA-DNA heteroduplexes (RNaseH)

Question 5

Reverse transcriptase

Answer 5

-Complex molecular machine with moving parts and many activities

Question 6

Catalytic properties of RT

Answer 6

-Slow: 1-1.5 nt/sec & 1/10 the rate of DNA polymerase
-Low fidelity: Lacks editing and prone to disincorporation, dissociation, and slippage

Question 7

RNaseH

Answer 7

-Degrades RNA portion of RNA-DNA duplex
-Produces 5’ phosphate and 3’ OH ends. The latter can be used as primers for extension by RT

Question 7

Recombination during reverse transcription

Answer 7

-Diploid nature of retroviral genomes allows for high levels of recombination during replication
-A mechanism for mutation and rapid revolution

Question 8

Two mechanisms of recombination during reverse transcription

Answer 8

1. Copy choice: During (-) strand synthesis
2. Strand assimilation: During (+) strand synthesis

Question 9

Integration

Answer 9

-Process of retroviruses integrating their cDNA genomes into the host genome
-Underlying reason for the persistence of these viruses
-Mediated by integrase protein
-Changes host cell genome
-Oncogenic
-Germline mutation

Question 10

Characteristic features of retroviral integration

Answer 10

-LTRs of viral cDNA targeted to specific sequences/structures in host genome
-Viral cDNA is integrated into host DNA to produce proviral DNA
-Integration mediated by integrase (Requires ssDNA cleavage of host DNA)
-Sequence changes (Host target sequence duplicated and proviral DNA loses 2 bp from each end)
-Proviral genome serves as template for transcription of new viral RNA genomes
-Transcription mediated by host cell RNA polymerase II