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�