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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