Positive sense RNA viruses.pdf

Positive sense RNA viruses Concepts > specific virus families Single stranded, linear RNA molecule Positive sense: Read from 5’ to 3’ allowing direct translation to protein ○ Except retroviruses - they have 2 copies ○ 8-12kb genome size Except corona...

Positive sense RNA viruses Concepts > specific virus families Single stranded, linear RNA molecule Positive sense: Read from 5’ to 3’ allowing direct translation to protein ○ Except retroviruses - they have 2 copies ○ 8-12kb genome size Except corona ○ Class 4 baltimore  Virus lifecycle How do viruses completely reprogram the cell with a single, small genome? ○ To dominantly express viral mRNA ○ To preferentially express viral protein ○ To replicate large amounts of viral genome ○ To produce large numbers of viral progeny Viruses modulate normal cellular control mechanisms to heavily favour virus replication ○ All while avoiding very specific and strong intracellular innate immune responses Each host cell produces 1000-10,000 virions Genome replication is amplification of progeny Objectives: ○ Robust amplification of genomic material to spread infection ○ Preserve fundamental genomic information Even non-coding elements ○ While preserving, retain ability to evolve Recombination, obtaining mutation RNA od coronaviridae is almost the same as our mRNA Flaviviridae do not have a polyA tail Picornaviridae do not have 5’ cap RdRp Aside from retroviruses, no other +ssRNA virus carries a polymerase in the capsid Genome is used as mRNA to make RNA-dependent RNA polymerase (RdRp / replicase) ○ RNA is the template, and RNA is the product Crystal structure of RdRp depicts hand-like structure to grasp viral RNA Replication of ssRNA Universal rules for synthesis of RNA ○ RNA synthesis initiates and terminates at specific sites on the template ○ RdRp may initiate synthesis de novo (like host DdRp) or require a primer ○ Other viral and cell proteins may be required ○ RNA is synthesised by template-directed stepwise incorporation of NTP, elongated in a 5’ to 3’ direction But the RNApol moves along the template 3’ to 5 RdRps produce complementary strands from a template ○ Template can be + or -  Complementary strand that is produced will always be the opposite ○ Transcription is always 5’ and 3’ RdRp recognises and binds to 3’ end Initially, a dsRNA is formed ○ Subsequent replication will form a complex that is partly double stranded and is known as a replicative intermediate ○ This form produces many more +RNA from the -RNA template ○ The newly generated +RNA can then be utilised for translation or be packaged into new viruses Translation vs replication/transcription in +RNA viruses ○ Ribosome/RDRP clash problem RNA is both template for translation and replication Translation moves in 5 to 3 Replication moves 3 to 5 Poliovirus transcription ○ Organised into separate regions ○ Genome is in a dynamic equilibrium and the RNA folds into specific structures facilitating replication Clover leaf at 5’ end CRE (cis-acting RNA element) Pseudoknot at the 3’ end ○ Polio uses viral and host factors to prevent the clash problem Host protein PCBP and the viral polyprotein 3CD bind to the cloverleaf-like structure of +RNA with host PolyA binding protein (PABP) bound to 3’ polyA tail Initiates cleavage of 3CD which becomes the RNApol When VPg is translated, it gets attached to the polyA tail to act as a primer The RNApol can then bind to the primer and transcribe Synthesis of -RNA begins Newly made dsRNA is then partly unwound by the chaperone activity of the host and viral factors binding to the 5’ end of the +RNA ○ Protein-protein and protein-RNA interactions facilitate binding  Flavivirus transcription ○ Genome encodes complementary sequences of 5’ and 3’ ends to promote cyclization Goes from linear to a circle Required for recognition by the replication complex, including NS5 (RdRp) NS5 also acts as methyltransferase and adds 5’ CAP to newly synthesised +RNA ○ Leads to -RNA templates For most +RNA viruses, the -RNA strand is the most used template resulting in 10-100 fold more +RNA compared to -RNA  Togaviridae replication ○ During initial translation, no structural proteins are produced, just the machinery required for transcription ○ -RNA template strand is transcribed and contains subgenomic promoter that enables transcription of +RNA encoding the structural proteins Promotor is not initially translated by the host ribosome - hidden within genome  ○ Subgenomic mRNA Produced by Togaviridae and Calciviridae from the 3’ end of genomes Overcomes problem with the genome organisation of Toga/noroviruses Structural genes (need a lot of protein product) are ○ At the 5’ end in picornaviruses ○ At the 3’ end in toga/noroviruses Structural proteins can only be transcribed/translated once replication has been established ○ Requires many -RNA strands Allows for coordination of when proteins are made ○ Toga/noroviruses aim to produce heaps of nucleic acid (via RdRp) before producing structural proteins for packaging and assembly Coronavirus replication ○ Huge genome ○ CAP and polyA tail added by viral enzymes ○ Common leader RNA is joined to one of several repeated transcription-regulating sequences (TRS) that cause it to skip bits of RNA TRS interact to cause bending of RNA to make it non linear During transcription of -RNA ○ Leads to production of “nested set” of discontinuous subgenomic RNAs Not the same as subgenomic promoters  ○ RdRp skilling occurs in cytoplasm ○ ○ SARS-CoV-2 replicase complex has many functions RdRp - error prone, needs other factors Processivity factors Permit high efficient RNA replication Helicase unwinds structured RNA for efficient replication ssRNA protective protein nsp16 involved in RNA capping for protein expression on ribosomes Proof-reading exonuclease activity to correct sequence errors Cytoplasm of a +RNA virus-infected cell ○ dsRNA (replicative intermediate) is a powerful inducer of interferons (inducers of antiviral activities) ○ Viruses often trigger the proliferation of small vesicles from intracellular membranes in which the RNA replication occurs, hidden from the cell’s interferon induction system Controlling translation Poliovirus and flavivirus ○ Simplest approach Encode one long ORF that produces a large polyprotein Polyprotein is then cleaved into the mature viral proteins via viral- and host-encoded proteases ○ May be inefficient Need 180 copies of some proteins but only need 1 RNApol 179 extra RNApols Many proteins have a double function E.g. affecting other cellular processes, blocking innate immunity ○ Polio does not have a 5’ cap Usually, initiation factors recognise this cap for binding eIF4e binds and recruits initiation elongation factors (eIF) Subsequently recruits 40S ribosome subunit Has IRES (internal ribosome entry site) instead Directly binds to cellular translation factors Does not need all of the machinery Type I and II IRES needs all eIFs except for eIF4e Hep C only needs eIF2 and eIF3 Nature of IRES can determine virulence  Completely CAP-independent, so no need to encode for a methyltransferase Methyltransferase attaches the CAP in the nucleus If translation/replication happens in the cytoplasm, a methyltransferase needs to be translated from virus eIF4E attaches to cap and eIF4G Polio 2A protease cleaves eIF4G -> stops both from binding to polio RNA Also stops the 4E-4G complex from binding to any capped host mRNA ○ Stops translation of host mRNA Destruction of cell translation machinery blocks the expression of essential cellular proteins from the m7Gppp-capped cellular mRNAs Hepacivirus (Hep C) translation ○ Chronic infection  ○ HCV has no cap structure to initiate translation ○ HCV also has an IRES at its 5’ end and highly structured 5’ and 3’ UTRs (untranslated region) to facilitate efficient replication ○ Uses host and viral proteases to cleave polyprotein ○ HCV IRES Very different RNA structure Does not destroy cellular protein cellular machinery and cellular pathology is negligible - chronic infection Does not directly kill hepatocytes Norovirus translation ○ Calciviridae ○ No CAP ○ Translation mediated by viral protein NS5 (VPg) VPg is a CAP substitute and recruits many factors requires for efficient translation ○ ○