cell recognition and internalization
progeny virus assembly
release from cells
nucleus
capsid disassembly and genome release
replication and translation
membrane breaching
Overview of virus life cycle
Credit: Internet resources�
Replication scheme �
Class I �
Class III �
Class IIb �
Class IV�
Class VI �
Class IIa �
+DNA �
+RNA �
±DNA � -DNA�
±RNA �
-RNA �
mRNA �
Class V�
Baltimore classification�
Replication overview�
Similar to host DNA – leading and lagging strand synthesis, primer required�(RNA), DNA polymerase and other host factors required�
lnicole3.blogspot.com �
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 overview�
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 �
Cytoplasmic DNA viruses�
I. Poxvirus�
May carry some enzymes�
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: Polyomaviruses �
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) �
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: Adenovirus�
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: Herpesvirus �
Example of class I: Poxvirus�
Large enveloped virus, diameter ~ 200 nm ��Contains a double stranded DNA genome (192 kbp) ��Contains ~ 250 genes�
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�
Example of class I: Poxvirus�
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��
Schematic of replication cycle�
RC-DNA �
cccDNA �
pgRNA �pA �cap �
DNA �repair�
Reverse �transcription�
RNA pol II �transcription�
Hepnadviruses – Class I/VII �
Example of class IIa: Φx174 �
Rolling circle replication and formation of concatemers ��
Parental genome�
Parental RF�
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. ��������
Example of class IIb: Parvoviruses�
Replication strategy �
Replication scheme �
Class I �
Class III �
Class IIb �
Class IV�
Class VI �
Class IIa �
+DNA �
+RNA �
±DNA � -DNA�
±RNA �
-RNA �
mRNA �
Class V�
Baltimore classification�
�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���
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 �
�Properties of RNA viruses�
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) �����
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��
Example of Class IV – Poliovirus�
VPg � AAAAA �
IRES �
Genomic (+) RNA �
Polyprotein�(includes polymerase) �
start codon�for translation�
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 ��
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� �
Example of Class IV – Poliovirus�
VPg � AAAAA �
IRES �
Genomic (+) RNA �
Polyprotein�(includes polymerase) �
start codon�for translation�
stop codon�for translation�
structural proteins� non-structural proteins�5’� VP
4� VP1 �VP2 � VP3 � 2A � 2B � 2C� 3A � 3B � 3C� 3D�3’�
3C�
VPg�
Replication scheme of poliovirus RNA �
+ +
+
+
polymerase �
+ SS� RI � SS� RI � SS�
RF� RF�
+ +
5’� 5’� 3’� 3’� 5’�
Hobson et al, EMBO J, 2001 �
Poliovirus RNA dependent RNA polymerase
Hobson et al, EMBO J, 2001 �
RNA polymerase activity is highly�cooperative��Interaction via interface I –�necessary for binding RNA ��Interaction via interface II –�necessary for catalysis�
Poliovirus RNA dependent RNA polymerase
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 associated �(hexameric protein) �
72 �
Doedens et al, J Virol, 2001 �
Uninfected cell� Infected cell�
Poliovirus replication occurs in large cytoplasmic vesicles�
Host factors are important�
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) ����
+ + +
+ + +
+ +
+ +
- - -
- -
- -
+
Virion RNA �
polymerase �
polymerase and�other proteins�
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 - Rhabdovirus �
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�
Example of class V – Influenza virus�
A �
Cleavage�site �
Host mRNA �
Viral exonuclease �
Influenza virus nucleocapsid�
Viral polymerase �
Viral mRNA �
A �
A �
A �
U�
U�
cap �
Synthesis of influenza virus mRNAs�
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) ��
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���
Example of class III - Reovirus�
+ -
+
+
+ -
Core�mRNA �assortment�
of +ve RNA �
proteins�
new�virion�
Replication scheme of reoviruses�
Schematic of bluetongue virus (orbivirus) core �
VP3 and VP7 form the core surface �
Roy, J Gen Virol, 2008 �
VP7 �
VP3 �
VP1+VP4 �
BTV core �
Roy, J Gen Virol, 2008 �
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�
Coding area�Leader�
PBS �
U5 �R � U3 � R �
PPT �
A(n) �Cap �
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�
Coding area�Leader�
PBS �
U5 �R � U3 � R �
PPT �
A(n) �Cap �
Example of class VI - HIV�
U5 �R �U3 �U5 �R �U3 �LTR � LTR �
Viral dsDNA �
Integrated into host genome by Integrase �
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�