RNA Virus Replication PDF

Learning Objectives Understand and describe the basic features common to (+)RNA, (-) ssRNA and dsRNA virus replication and gene expression, with example viruses described here Understand and describe the mechanisms of expressing genes encoded in single or overlapping gene fragments Under...

Learning Objectives Understand and describe the basic features common to (+)RNA, (-) ssRNA and dsRNA virus replication and gene expression, with example viruses described here Understand and describe the mechanisms of expressing genes encoded in single or overlapping gene fragments Understand and describe the basic features of eukaryotic mRNA, essential for translation, and methods used by viruses to bypass the need for these features, with examples The RNA viruses: Baltimore Groups III, IV and V  RNA viruses replicate in the cytoplasm Positive or plus (+)strand RNA viruses have genomes that are functional mRNAs; may be capped and polyadenylated Minus sense (-) RNA viruses must carry RdRp in capsid, to begin replication Host cell ribosomes assemble on viral genomes that have entered host cell, to synthesise viral proteins Earliest viral proteins are those needed to synthesise new genomes, and RdRp  Viruses modify host cell membranes to construct viral replication scaffolds Many viruses that replicate in the cytoplasm compartmentalise genome replication and transcription of proteins to virus replication complexes (VRCs) Escape recognition from host defences and recognition by toll-like receptors VRCs assembled by non-structural viral proteins, viral genomes, host lipids and host proteins Within the VRC the + strand genome is used as a template to synthesize full-length anti-genomes (- sense), which remain hydrogen bonded to the + strand Class IV (+) RNA virus genomes have distinct structures  Class IV (+) RNA viruses express multiple proteins from a single genome Eukaryotic mRNA encodes just one protein, whereas (+) strand RNA viruses must encode at least two proteins: a capsomer and an RdRp Immediate translation is critical for viral replication because it results in synthesis of the viral RdRp RdRp subsequently synthesizes the replicative forms and viral mRNA The Class IV viruses encode a polyprotein that is proteolytically processed to release many individual proteins including the capsomers and an RdRp  Viruses with genomes that lack 5’ cap must compensate with structure that allows binding to ribosome Poliovirus 5’ internal ribosome entry site IRES IRES-dependent translation initiation recruits translational machinery to an internal position in mRNA VPg is the protein primer for genome replication  Translation of mRNA occurs at eIF4G initiation complex eIF4G: eukaryotic initiation factor – serves as docking site for initiation factors and proteins involved in RNA translation  Cap-dependent initiation complex vs poliovirus initiation complex The viral internal ribosomal entry site (IRES) is a complex stem–loop structure in the 5ʹ UTR (A) Normal initiation of host mRNA involves the eIF4E protein binding to the 5ʹ cap and forming a complex with PABP The small subunit of the ribosome (40S) is closest to the AUG start codon (B) For initiation of the translation of the picornavirus genome, the host ITAF protein binds to the IRES and substitutes for eIF4E The terminal VPg protein was removed from the 5ʹ end of the genome by a host enzyme. Poliovirus proteolytically degrades eIF4E, thus preventing cap-dependent translation of host mRNA.  Overview of events during gene expression and genome replication in some (+) strand RNA viruses Examples: poliovirus; Hepatitis C virus After uncoating, the IRES enables the genome to be translated (1), making a polyprotein that is processed into individual proteins (2). The proteins go on to form virus replication compartments (3) in which double-stranded replicative forms are used to make mRNA and new genomes, (4) that are ultimately used to make new infectious virions (5). Coronavirus replication cycle  Baltimore Class V ss(-) RNA virus genomes may be segmented or non-segmented Orthobunyaviruses Orthomyxoviridae Paramyxoviridae Genome of minus sense (-)strand RNA viruses cannot be used directly as mRNA Viruses package RdRp within the virion https://viralzone.expasy.org/250?outline=all_by_species Mononegavirales: ss(-)RNA viruses with non-segmented genomes Measles virus  Baltimore Class III dsRNA virus genomes - Reoviridae Rotavirus Non-enveloped, icosahedral virion with a triple capsid structure – Uncoating leaves a double layered particle, DLP Segmented linear dsRNA genome with 11 segments coding for 12 proteins Each segment has 5’ cap, no polyA tail Co-infection of cells with different rotavirus strains belonging to the same serogroup A, B or C undergo mixing of the genome segments (genetic reassortment) Rotavirus genome Rotavirus viroplasm: cytoplasmic site of viral dsRNA synthesis A: during earlier phases of infection viroplasms are separate B: Older viroplasms fuse with one another  Rotavirus genome replication: early transcription of dsRNA genome by RdRp occurs inside DLPs Double layered particle, DLP VP1/VP3 flower proteins VP1, VP3 are internal polymerase complex Synthesis of rotavirus nucleic acids occurs inside DLPs – Transcribes capped (+)RNAs from each of the gene therefore they are fenestrated segments VP6 is outermost protein, VP2 is underneath (+)RNAs serve as either mRNAs for synthesis of viral 11 dsRNA segments proteins on cellular ribosomes , or as templates for Every vertex of particle is fenestrated allowing for entry synthesis of (-)RNA of NTPs and exit of newly synthesized mRNA Rotavirus mRNA compared to host mRNA, ready for translation to initiate Each RNA segment has 5’ cap, no polyA tail Viral NSP3 substitutes for PABP by binding to the conserved UGACC sequence near the 3’ end of the viral mRNA Gene expression and genome replication in rotaviruses 1. Cytoplasmic DLP catalyses primary transcription in which capped mRNA leaves each vertex of the virion 2. Translation of these proteins causes formation of viroplasm in which mRNA becomes enclosed by new DLPs 3. Inside the new DLPs copying of (+) strand templates results in formation of DLPs containing dsRNA genomes 4. The new DLPs cause an exponential increase in viral mRNA and protein 5. After a few hours the virus switches from gene expression and genome replication to the assembly phase of the replication cycle Rotavirus replication cycle  Summary: dsRNA viruses Rotaviruses have three-layered naked capsids surrounding a segmented dsRNA genome The double layered particle, DLP, is the form of rotavirus that is transcriptionally active in the cytoplasm of the host cell Many individual components of the DLP are enzymes needed for nucleic acid synthesis, and also structural proteins VP1 is an example of a rotavirus protein that is structural and has enzymatic activity The VP1/VP3 flower complex synthesizes the 5’cap structure and the mRNA using one of the dsRNA molecules as template The viral mRNA does not have a a poly(A) tail – a viral nonstructural protein binds to a conserved sequence in the 3’ end of each mRNA and thereby substitutes for the poly(A) binding protein PABP during translation initiation