Download Overview of virus life cycle

Overview of virus life cycle cell recognition
and internalization
release from
cells
progeny virus
assembly
membrane
breaching
capsid disassembly
and genome release
replication and
translation
nucleus
Credit: Internet resources
Replication scheme
Class IIa
+DNA
Class IIb
±DNA
-DNA
Class I
Class VI
+RNA
Class IV
mRNA
-RNA
Class V
±RNA
Baltimore classification
Class III
Replication overview
Similar to host DNA – leading and lagging strand synthesis, primer required
(RNA), DNA polymerase and other host factors required
lnicole3.blogspot.com
Replication overview
Involvement of several enzymes
1) 
DNA unwinding protein
2)  RNA polymerase
3)  DNA polymerase III
4)  DNA polymerase I (+ exonuclease activity)
5)  DNA ligase
1 mistake/109 - 1010 bp replications
Replication of animal DNA viruses
Enzymes mostly of host origin, some viruses carry their own enzymes
Host machinery (enzymes etc) for generating DNA are in
the nucleus (and mitochondria).
Viruses utilizing host enzymes need to enter the nucleus.
Viruses with own enzymes can replicate in the cytosol.
Nuclear DNA viruses
I. 
Parvoviruses
II. 
Polyomaviruses
III.  Adenoviruses
IV.
Herpesviruses
V.
Hepnadviruses
May carry some enzymes
Cytoplasmic DNA viruses
I. Poxvirus
Example of class I: Polyomaviruses
Example of class I: Polyomaviruses
Small, icosahedral, ~ 40 nm in diameter
Contains circular duplex DNA
Replicates in the nucleus
DNA associates with host cell histones
Utilizes host cell machinery mostly, except some viral proteins
Viral proteins required for replication (enzymes, regulatory
proteins etc) are expressed first (early genes)
Host RNA polymerase II recognizes promoter, mRNA produced
Post-transcriptional RNA processing carried out by host enzymes
Alternative processing of primary transcript produces
small and large T antigens (Tag)
Example of class I: Polyomaviruses
Example of class I: Polyomaviruses
Replication starts in the late phase of infection
Similar to host DNA replication process
Host enzymes are used
Host DNA polymerase recognizes SV40 origin of replication in the presence of
large T antigen
Host histones bind to newly synthesized viral DNA
Example of class I: Adenovirus
Large non-enveloped icosahedral virus (~ 70 nm diameter)
Linear, ds DNA, associated with virally coded basic proteins
(not host cell histones)
Example of class I: Adenovirus
Early gene expression (proteins required for replication) carried out with
host RNA pol and RNA modification enzymes
Encodes its own DNA polymerase for replication
Requires host proteins also
DNA replicated by a strand displacement mechanism
No Okazaki fragments, continuous synthesis
Protein TP acts as primer (attached to 5 end)
Example of class I: Adenovirus
Example of class I: Herpesvirus
Enveloped icosahedral virus, ~
200 nm in diameter
Linear, double stranded DNA
Example of class I: Herpesvirus
Utilizes host RNA polymerase and host RNA modification enzymes
A viral protein VP16 (tegument) enters the nucleus and binds to viral
genome. This is part of the transcription factor complex recognized by
host RNA polymerase.
Example of class I: Herpesvirus
Early proteins encoded - DNA polymerase, DNA binding proteins,
thymidine kinase, ribonucleotide reductase
Comparatively more viral proteins utilized (drug targets)
Precise mechanism of replication not known
Newly synthesized DNA exists as concatamers, cleaved to appropriate size
during packaging
Example of class I: Poxvirus
Large enveloped virus, diameter ~ 200 nm
Contains a double stranded DNA genome (192 kbp)
Contains ~ 250 genes
Example of class I: Poxvirus
Packages its own DNA dependent RNA polymerase
Naked poxvirus DNA is not infectious at all
Methylation, capping, polyadenylation of mRNA carried out by virally encoded
and packaged enzymes
One of the immediate early mRNAs codes for an uncoating protein
Replication occurs in the cytoplasm, in viral factories
Replication of DNA occurs by strand displacement and concatemer resolution
Five major viral proteins are used, along with some cellular proteins
Hepnadviruses – Class I/VII
Enveloped, icosahedral viruses containing a partially double stranded,
circular, but not covalently closed DNA genome
Examples - Hepatitis B virus
Replicates through an RNA intermediate
Packages its own DNA polymerase, which has reverse transcriptase activity
Hepnadviruses – Class I/VII
cccDNA
DNA
repair
RNA pol II
transcription
Reverse
transcription
cap
RC-DNA
Schematic of replication cycle
pA
pgRNA
Example of class IIa: Φx174
Parental genome
Parental RF
Rolling circle replication and formation of concatemers
Example of class IIb: Parvoviruses
Small, non-enveloped, icosahedral, 18-25 nm in diameter
Autonomous parvoviruses (MVM) use host cell enzymes
for replication, package -ve stranded DNA
Defective parvoviruses (AAV) need helper virus co-infection,
Package -ve and +ve stranded DNA (in different virions)
Contains palindromic terminal sequences which can serve
as primers
Replication proceeds by single strand displacement, no
lagging strand synthesis.
Replication strategy
Replication scheme
Class IIa
+DNA
Class IIb
±DNA
-DNA
Class I
Class VI
+RNA
Class IV
mRNA
-RNA
Class V
±RNA
Baltimore classification
Class III
Properties of RNA viruses
Genome usually smaller than that of DNA viruses
Probably because of the higher rate of error accumulation in RNA genomes
RNA dependent RNA polymerase necessary (no DNA stage)
Relatively few proteins
Properties of RNA viruses
Strategy to make multiple proteins a) Producing multiple monocistronic RNAs
b) Producing a primary transcript that is processed by host splicing
machinery
c) Producing a large polypeptide which is cleaved into individual proteins
d) Allowing ribosomes to bind internally to viral RNA
Example of Class IV – Poliovirus
Plus stranded RNA virus, functions as mRNA
Infectious genome
Replication proceeds in presence of DNA synthesis inhibitors, so no
DNA intermediate
Requires a RNA dependent RNA polymerase (translated initially)
Example of Class IV – Poliovirus
Roy, J Gen Virol, 2008
Example of Class IV – Poliovirus
~ 10 times more +ve strand RNA produced
+ve strand RNAs packaged into virions and removed as template quickly,
-ve strand RNAs remain available as templates continuously
IRES
VPg
start codon
for translation
Genomic (+) RNA
AAAAA
Polyprotein
(includes polymerase)
stop codon
for translation
Genome corresponds to mRNA, polyadenylated, no methylated cap
Contains internal ribosome binding site (IRES)
Polyprotein produced, which is cleaved to generate RNA polymerase
Example of Class IV – Poliovirus
VPg serves as primer for replication
May be cleaved off and recycled by a host protein TBP2 “unlinkase”
A lot of VPg and polymerase per cell!
Needs to replicate in vesicles
IRES
Genomic (+) RNA
VPg
start codon
for translation
Polyprotein
(includes polymerase)
stop codon
for translation
VP4
structural proteins
5’ VPg
VP2
VP3
VP1
AAAAA
non-structural proteins
2A
2B
2C
3A 3B
3C
3C
3D
3’
Replication scheme of poliovirus RNA
+
SS
5
RI
5
+
SS
3
RI
3
+
SS
5
+
+
+
+
polymerase
RF
RF
Poliovirus RNA dependent RNA polymerase
Hobson et al, EMBO J, 2001
Poliovirus RNA dependent RNA polymerase
RNA polymerase activity is highly
cooperative
Interaction via interface I –
necessary for binding RNA
Interaction via interface II –
necessary for catalysis
Hobson et al, EMBO J, 2001
Replication scheme of Qbeta RNA
~ 10 times more +ve strand RNA produced
+ve strand RNAs packaged into virions and removed
as template quickly, -ve strand RNAs remain available
as templates continuously
RNA dependent RNA polymerase consists of four host
encoded and one virus encoded polypeptide
Very specific for Qbeta RNA
Complete Qbeta polymerase
Polypeptide
function
Source
MW (KD)
Binding to + strand Ribosomal
Protein S1
70
Initiation
Elongation factor
Tu
45
Initiation
Elongation factor
Ts
35
Chain elongation
Qbeta encoded
65
Minus strand
synthesis
Ribosome
72
associated
(hexameric protein)
Poliovirus replication occurs in large cytoplasmic vesicles
Uninfected cell
Infected cell
Host factors are important
Doedens et al, J Virol, 2001
Class V RNA viruses
Minus stranded RNA virus
Non-Infectious genome
Requires a RNA dependent RNA polymerase, which is packaged in the
virus
Initial event after infection is synthesis of plus stranded RNA
Examples – orthomyxoviruses (influenza), Paramyxoviruses (measles),
Rhabdoviruses (rabies)
Example of class V - Rhabdovirus
+
+
+
+
polymerase and
+
other proteins
+
-
-
-
+
+
-
+
-
+
Virion RNA
polymerase
+
-
Example of class V - Rhabdovirus
Occurs in the cytosol
Synthesis of mRNAs can occur from partially packaged
nucleocapsids
mRNA modification etc carried out by packaged genome
5 proteins are synthesized - N, P, M, G, L
New copies of -ve strand RNA are coated with N
Example of class V – Influenza virus
Contains a fragmented genome - 8 -ve strand RNA wrapped with
nucleocapsid
mRNA synthesis and replication occurs in the nucleus with viral
polymerase
Unique mechanism for capping the mRNAs
Nascent strands immediately wrapped with nucleocapsid
Synthesis of influenza virus mRNAs
Cleavage
site
cap
A
Host mRNA
Viral exonuclease
A
A
U
Influenza virus nucleocapsid
Viral polymerase
A
U
Viral mRNA
Influenza virus RNA polymerase
Three subunits - PB1, PB2, PA
PB1 recognizes cellular mRNA caps, has exonuclease
activity
PB2 elongates the cleaved primer
PA involved in synthesis of -ve strand RNAs
Replication scheme of reoviruses
Entire cycle occurs in the cytoplasm
Reovirus partially uncoats into cores (infectious)
Example of class III - Reovirus
Double stranded RNA virus, contains 10 segments of ds RNA
Requires a RNA dependent RNA polymerase, which is packaged in the virus
First step is to make mRNA - produced by viral polymerase and modified by
virally encoded proteins in the core
mRNA extruded through channels at the 5-fold axes of symmetry of the
particles
Old (input) mRNA is conserved inside cores, serve as templates
New +ve strand RNA packaged into progeny virions,
where the complementary strand is synthesized
Replication scheme of reoviruses
+
+ -
+
assortment
of +ve RNA
Core
+ -
new
virion
proteins
mRNA
Schematic of bluetongue virus (orbivirus) core
VP3 and VP7 form the core surface
Roy, J Gen Virol, 2008
BTV core
VP7
VP3
VP1+VP4
Roy, J Gen Virol, 2008
Example of class VI - HIV
Plus stranded RNA virus, contains 2 RNA molecules with 5 cap and
3 polyA tail
Physically linked through hydrogen bonds
Not released into the cytosol, cannot function as mRNA
Non-infectious genome
Replication cannot proceed in presence of DNA synthesis inhibitors
(actinomycin D)
Example of class VI - HIV
An RNA dependent DNA polymerase (reverse transcriptase) is used to convert
the ssRNA genome into dsDNA
The existence of the enzyme first proposed by Howard Temin
Enzyme isolated by Temin and Baltimore
Mistake prone enzyme - 1 mistake/20,000 bases, contains Rnase H, Integrase
and protease activities, utilizes tRNA as primer
Enzyme and tRNA packaged into the particles along with genome
Example of class VI - HIV
Example of class VI - HIV
R
U5
Leader
Coding area
U3
R
A(n)
Cap
PBS
PPT
R - terminally redundant region
U3, U5 - unique, non-coding region, form 5 and 3 end of provirus genome,
contains promoter elements for provirus transcription
PBS - primer binding site
Leader region - non-coding, present at 5 of viral mRNAs
PPT - polypurine tract, initiates +ve strand synthesis during reverse
transcription
Example of class VI - HIV
R
U5
Leader
Coding area
U3
R
A(n)
Cap
PBS
PPT
U3
LTR
R
U5
U3
Viral dsDNA
Integrated into host genome by Integrase
LTR
R
U5
Expression of HIV mRNA
Transcription by host RNA polymerase II
Utilize splicing and ribosomal frameshifting to make proteins
Tat - transcription activator protein
Rev - Helps in the export of mRNA and genomic RNA to the cytosol