Lecture 21

Question 1

RSV

Answer 1

viruses that cause cancer

knew nothing about the genome, just knew it would cause tumors in birds

even to this day, only one human cancer is caused by this class of viruses
major disease associated is AIDS

Question 2

Retroviruses-virion structure

Answer 2

Simple vs complex: number of genes, amount of splicing, etc.
more genes in HIV than there are in simple retroviruses
genome is +ss RNA
8-10 kb… avg size mrna virus

most notable features about retroviruses: genome s diploid (2 copies of ss RNA shown in red)
2 pieces of RNA intertwined, associated (using some kind of base pairing)
2 rna molecules facilitate evolution of these viruses through copy choice recombination
how would copy choice recomb work during the process of rev transcription?
despite having + RNA genome, not translated instantly like poliovirus- why not?
rna coated with nucleocapsid protein
genome coated from one end to the other
most important enzyme is reverse transcriptase
RT not associated with RNA, there is a lot of it (about 50)
IN mechanism distinct from lambda. uses hydrolysis instead of covalent bonds
3rd protein is protease (PR)
importance of transcriptase within virion is that it’s required early in NEXT infection
3 proteins: which important for next infection, and which are just leftover from previous? one leftover
enveloped virus- budding
w/in envelope: TM and SU
SU = virion attachment protein
capsid: icosahedral structure
encloses 3 proteins (all enzymes)
each tRNA for each what? post RNA? ??

Question 3

genome organization of a simple retrovirus

moloney murine leukemia virus (MLV)

Answer 3

codes for all three enzymes
polyprotein: basically enzyme region
SU:TM - cleaved, but two subunits stay together

Question 4

R

Answer 4

Short sequence: called R for repeat
also find on right hand end
terminally redundant

Question 5

U5

Answer 5

Unique sequence on 5’ end

primer binding site. also short

Question 6

Both R and U length

Answer 6

100-200 nt long

Question 7

PBS

Answer 7

18 nt long

Question 8

poly purine track

Answer 8

also 18 nt long

18 purines in a row

Question 9

sd

Answer 9

splice donor, to the left of gag

Question 10

sa

Answer 10

splice acceptor

before coding region for envelope, after pol genes

Question 11

what does the splice event do?

Answer 11

leaves aug for envelope coding region as first aug
creates monocistronic mRNA, allowing ribosomes to translate envelope region of mRNA
sub genomic mRNA not packaged b/c it lacks the packaging signal

Question 12

How does mlv enter the host cell?

Answer 12

enters cell by direct fusion- releases nucleocapsid containing genome into cytoplasm
some retroviruses enter by receptor mediated endocytosis
next step: process of reverse transcription

Question 13

Replication cycle

Answer 13

what is reverse transcription? DNA pol makes ds DNA copy of retroviral RNA
only one of these pieces of RNA in the diploid genome is really required to do this
really req one, but two could do it

ds dna linear, has all segments needed. next: ds dna must integrate into chromosome of infected cell. needs to be able to access the chromosomes
needs to be mitotic event for ds dna to access chromosomes, then it can integrate into the cell
BUT HIV can actually integrate in non dividing cell

Question 14

Replication cycle: once integrated

Answer 14

referred to as pro virus
proviral dna transcribed to produce mRNAs which are translated to produce proteins
genome RNA combined with these, assembly occurs
maturation requires proteolysis

Question 15

what other kinds of systems require proteolysis to get from immature virion to mature virion

Answer 15

???

Question 16

Cycle:

Answer 16

cell enters
reverse transcription to get ds dna
access chromosomes (requires mitosis)
makes two mRNAs
only longer one packaged (other one env)
mrna translated
gag
gag pol
and env
transcription also makes genome rna 
all go into assembly
then maturation (proteolysis)

Question 17

Reverse transcriptase properties:

Answer 17

1) a dna pol that can use either rna or dna as a template
built in helicase activity, but doesn’t use atp like helicase does
2) dna pol can carry out extensive displacement synthesis
3) in addition to a dna polymerase activity, possesses an RNas H activity
4) RNase H degrades RNA that is hybridized to dna
degrades by hydrolysis (like endonuclease)
rna strand small pieces will dissociate from dna strand
5) in most cases, the enzyme is a heterodimer (i.e. composed of two related but structurally distinct polypeptides)
6) can catalyze most, if not all, the steps of reverse transcription

Question 18

The dna pol and rnase H activities are found in different domains of monomeric MLV reverse transcriptase protein

Answer 18

N term domain has dna pol
C term domain has rnase H
HIV rev transcriptase is a heterodimer
P66 (polymerase + rnase H domains)
P51 (polymerase domain only
pol activity in P51 not active
just provides structure

Question 19

side view of hiv 1 reverse transcriptase

Answer 19

active sites far away- 2 separate domains

Question 20

overview of reverse transcription

Answer 20

would think you’d get ds dna that would be collinear
but that’s not correct. instead, produce ds dna that’s longer on both ends
has to be this way bc if above were correct, no guarantee that it would be next to a promoter
dna copy encodes own promoter
derived from U3 sequence up there
MLV: simple retrovirus
distinguish features of simple retrovirus
pbs = primer binding site
ppt = poly purine track
prod of rev transcription is ds dna, not nuc by nuc copy of rna
instead, has extra seq on both ends
ds dna prod of rev transcription also terminally redundant, but diff redundancy
reason the product ds dna is longer than viral genome: carries own promoter, derived from U3 region of rna (where promoter is located); after int of dna, virus has provided its own promoter where transcription should begin
if this wasn’t the case, no way to transcribe once integrated
begins at R and completes transcription
if exact collinear copy, wouldn’t have a promoter and couldn’t transcribe

Question 21

Reverse Transcription

Answer 21

rev transcriptase: has dna pol activity that can utilize either rna or dna as a template
has displacement activity. important for end of process
has second enzymatic activity. important for end of process
this is RNase Activity
genome itself has same seq as mrna, referred to as +
dna w/ same seq and polarity, also +
complementary strand is -
after - strand synthesis, turns over and synth the + strand

Question 22

what is the second enzymatic activity?

Answer 22

RNase activity
degrades rna bp with dna
h stands for hybrid; rnase that deg hybrids

Question 23

tRNA use in reverse transcription

Answer 23

has a tRNA bp’ed with primer binding site over an 18 nt stretch
18 bp ds rna where tuna came from host cell
3’ end on left. this tRNA used as a primer: this 3’ end extended and the product is dna
utilization of that 3’ end of tRNA is primer, 5’ genome is template
short piece - dna made, then runs out of template. call this - strong stop dna b/c it stops at this point
b/c of pause in synthesis, can detect this product in a lab
this is all going on inside capsid structure in cytoplasm of the cell
Rnase H takes DNA/RNA hybrid and degrades RNA part of hybrid
would not degrade rna where bp’ed
get ss r’ seq in dna

Question 24

Where does the first template switch happen

Answer 24

1st template switch: run out of template here after RNase first degrades rna bp’d to - strong stop dna and now have more template to extend dna
r’ bp to r and extends
makes circular intermediate

Question 25

these viruses have diploid genome. so?

Answer 25

alternate way: this r’ bp with r seq on other copy of genome in diploid structure
can and does happen
does not really matter, end result is the same

Question 26

continuation rev transcription:

Answer 26

continuing to synthesize: dna strand creates a very long stretch of dna hybrid (a substrate of rnase H)
RNase H degrades all RNA except region which corresponds to Ppt (resistant to RNase H)
piece of RNA becomes the primer for + DNA synthesis
dna not completely finished; as dna is being made, rna is being degraded behind the advancing dna polymerase
rnase H domain periodically clips RNA
PPT left behind; important bc 3’ end serves as primer to begin synth + dna
referred to as + strand primer
extension of primer is made; left to right
continues on to copy part of the tuna as a template
3’ end of a + strand fragment actually has pbs seq (B/c copies the first part of tRNA, which was originally bp’ed to PBS seq)

Question 27

why does dna pol stop 18 nuc in?

Answer 27

b/c there’s a methylated base which stops it
it’s there b/c cell tRNAs are methylated. other structural alterations, but methylated base stops from copying the rest of tRNA

Question 28

What happens the second time RNase H degrades?

Answer 28

degrades and releases tRNA
PPT removed
still resistant, but cleavage event that creates the 3’ end of PPT primer is a very specific cleavage by RNase H activity
same that cleaves between RNA and DNA at PPT
ppt left intact

Question 29

Use of displacement synthesis in reverse transcription

Answer 29

PBS and PBS’ base pair
first step: displacement synthesis
3’ end here carries out displacement synthesis using + dna strand here as template
reverse transcriptase has displacement activity allowing it to do this
these two steps probably happen simultaneously
have extension of this 3’ end using - end as a template
end up with linear ds dna molecule

Question 30

overview of retroviral replication

Answer 30

U3 is “promoter” in transcription
U3 has elements to constitute a promoter later during transcription
once completed the replication process

Question 31

Overview of integration

Answer 31

Important features of integration:
1) 2 bp lost from each end of unintegrated viral DNA
2) 4-6 bp of host DNA duplicated at target site
3) all reactions catalyzed by viral IN protein (integrase)
substrate for integration is ds dna
molecule produced during reverse transcription
needs to gain access to chromosomes
mlv- cell must go through mitosis so membrane breaks down
doesn’t have to be a dividing cell in the case of HIV

Question 32

Mechanism of integration

Answer 32

two ends have same seq of four nucleotides
this is the seq that integrase initially recognizes and utilizes in integration
2 bp lost (at base pairs from each end)
this happens by end of process, not just clipped off
4-6 bp at target site duplicated
ds target site where int will occur
say there are four nt duplicated at target site
once viral dna integrated, same seq of four nt will be replicated on both ends
target seq ends up flanking integrated pro viral dna
hydrolysis
of phosphodiester bond on both ends of the DNA
occurs two nucleotides in from 3’ end in bottom strand
integrase carries out this reaction
in lambda: never hydrolysis of phos bond. instead, worked like topoisomerase and became covalently bonded
different here: retroviral integrase uses simple hydrolysis reaction of phos bond
releases tt dinucleotide
this is the first step
will be duplication of 4 nuc that flank the end of the dna
2nd reaction: transesterification
3’ hydroxyl acts as nuc to attack phos bond
int occurs at random with respect to seq, N can be any nuc
hydrolysis attacking phos bonds
int catalyzes transesterification reaction to break and create phosphodiester bonds
these are the two reactions that retroviral integrase
two a’s probably have to be removed and filled in
cellular enzymes do ending steps
recent evidence that hiv and maybe other complex ones integrase is more specific
seeks out chromatin which is transcriptionally active

Question 33

Chemistry of run catalyzed by IN

Answer 33

3’ hydroxyl is nucleophile
this oxygen acts as nucleophile on this phosphodiester bond to create phosphodiester linkage
same arrow for hydrolysis reaction from oxygen on water to attack phos bond
all occurs within active site in integrase
both nucleophilic reactions but nucleophile is slightly different
ds dna prod contains elements that serve as promoter
end product is mrna with 5’ cap and poly a tail
translated to produce two diff proteins
some of it is spliced to prod envelope mrna, translated to produce membrane and surface proteins
initially a polypeptide; cleaved
splicing event occurs to the left of packaging signal
eliminates packaging signal from subgeneric rna here; explains why envelope mrna not packaged
transcription produces this rna which is translated

Question 34

Gene expression

Answer 34

ds dna prod contains elements that serve as promoter
end product is mrna with 5’ cap and poly a tail
translated to produce two diff proteins
some of it is spliced to prod envelope mrna, translated to produce membrane and surface proteins
initially a polypeptide; cleaved
splicing event occurs to the left of packaging signal
eliminates packaging signal from subgeneric rna here; explains why envelope mrna not packaged
transcription produces this rna which is translated
termination codon after gag gene
Termination codon after gag gene where translation would terminate between gag and pol coding region
5’ splice site
splice donor
translation begins at 5’ cap
produces gag precursor polyprotein
proteolysis by protease ultimately produces matrix p12 capsid and nucleocapsid proteins
don’t know what p12 does but know the structure of other various elements
does not happen until after virus particles released from the cell
does not occur until maturation step after release
read through of UAG codon (RSV and HIV 1 use a low frequency -1 frameshift within gag to avoid UAG codon)
about 5% of the time fails to stop here
produces gag pol precursor instead
also cleaved by proteolysis to produce all below proteins
and protease and integrase
works diff from hiv, frame shifting event

Question 35

significance of only 5% read through?

Answer 35

way more gag proteins than gag pol
reg relative abundance of enzymes vs structural proteins
need more structural

Question 36

Note about cleavage:

Answer 36

since neither of these are cleaved until after released from cell, tells us that these two polyproteins fold up and create capsid in cytoplasm without any proteolysis
all happens while these are polyproteins; ensures that these enzymes are included within virion particles. since these polyproteins are not cleaved until after the virus is released they are definitely included
cleaved to produce surface protein and TM protein
cleaved by 3 different proteases
vsv g protein and signal peptidases
golgi protease that cleaves between SU and TM = same as influenza
virally encoded protease cleaves off p2E fragment; essentially activates fusion activity of TM protein
curious: This polyprotein is cleaved by 3 diff proteases and takes us back to things we’ve seen before