RNA viruses: genome replication and mRNA production BSCI 437 Lecture 12 Mechanisms of viral RNA synthesis Switch from mRNA to genomic RNA production General comments All RNA viral genomes must be efficiently copied to provide Genomes for assembly into progeny virions mRNAs for synthesis of viral proteins. Two essential requirements common to RNA virus infectious cycles: RNA genome copied end to end without loss of sequence Production of (cellular) translation-competent mRNAs.

General strategies for replication and mRNA synthesis of RNA virus genomes. (See Fig 6.1) (-) Strand RNA viruses

General strategies for replication and mRNA synthesis of RNA virus genomes. (See Fig 6.1) (-) Strand RNA viruses

General strategies for replication and mRNA synthesis of RNA virus genomes. (See Fig 6.1) (+) Strand RNA viruses

General strategies for replication and mRNA synthesis of RNA virus genomes. (See Fig 6.1) (+) Strand RNA viruses

General strategies for replication and mRNA synthesis of RNA virus genomes. (See Fig 6.1) Ambisense RNA viruses

General strategies for replication and mRNA synthesis of RNA virus genomes. (See Fig 6.1) Double-stranded RNA viruses

RNA-dependent RNA polymerase (RDRP) Unique process, no cellular parallel Hallmark: resistant to actinomycin D, an inhibitor of DNA-directed RNA synthesis.

RNA-dependent RNA polymerase (RDRP) Universal rules: RNA synthesis initiates and terminates at specific sites in the template Catalyzed by virus-encoded polymerases Viral and sometimes host accessory can be required (most) can initiate RNA synthesis de novo (no primer requirement) Some do require a free 3’-OH group for priming Primer can be protein linked RNA usually synthesized by template directed, stepwise incorporation of rNTPs Elongation is in 5’  3’ direction Examples of non-templated viral RNA synthesis exist. Viral RNA synthesis is highly efficient. e.g. Poliovirus RNA copied to 50,000 copies in the course of an 8 hr infection

Three dimensional structure of RDRPs Described as analogous to a Right Hand with Thumb, Palm & Fingers. Active site located in the Palm subdomain (Fig. 6.3)

Secondary RNA structures First order information content is contained in the sequence of an RNA Second order information content is contained the structure Ability to form G-U base pairs, as well as more exotic non-Watson-Crick base pairs gives RNA the ability to produce a wide variety of structures. The wide variety of structural possibilities provides for specificity of interaction with other biomolecules, e.g. viral or host proteins.

Secondary RNA structures A wide variety of RNA structures. Stem regions Pseuodknots Each of these can contain un-paired sections called loops Hairpin loops Bulge loops Interior loops Multibranched loops

Roles of viral accessory proteins Used to direct RDRP to the correct intracellular site. Nucleus – e.g. Influenza Membranes – e.g. polio Can target RDRP to correct initiation site on RNA template Helicases unwind RNA secondary structures Processive: unwind along an mRNA Distributive: unwind at one particular spot See Figure 6.8 in text

Cellular proteins in viral RNA synthesis In the context of viral genome condensation, viruses have hijacked host proteins to their service. Qb: RDRP requires ribosomal protein S1, EF-Tu and EF-Ts for their RNA binding properties. Poliovirus: host-encoded poly(rC)-binding protein 2 helps target viral proteins to an RNA secondary structure that is the site of initiation for genome replication (see. Fig. 6.8) Poly-A binding protein 1 (PABP-1) used in both initiation of replication and translation. Cytoskeletal proteins: used in replication of many RNA viruses. Specific targeting thought to ensure high local concentrations of replication components. Tubulin: stimulates replication of measles and Sendai viruses. Actin: Human parainfluenza virus type 3, Respiratory syncytial virus

Initiation Mechanisms Most initiation occurs de-novo. Exceptions: Protein Priming: Poliovirus VPg covalently linked to 5’ end of genome. VPg becomes polyuridylated (polyU). Base pairs with polyA 3’ end of genome. Interaction with RDRP serves to target replicase to primed 3’ end of genome. See Fig. 6.8B Priming by capped RNA fragments: Influenza steals 7Methyl-Gppp caps (cap snatching) from cellular mRNAs by cleaving cellular mRNAs. Cleavage products used to prime viral mRNA synthesis. See fig. 6.9.

The Ribosome/RDRP clash problem In (+) RNA viruses, RNA is both template for translation and replication. Translation moves in the 5’ 3’ direction along the (+) strand Replication moves in the 3’ 5’ direction along the (+) strand Problem: at some point in the middle they will collide. These viruses must evolve around this.

Discrimination between viral and cellular mRNAs Q: How do virus RDRPs discriminate between self and non-self mRNAs? A: Through secondary RNA structures. Called cis-acting RNA elements. Often serve as the switches between translation and replication.

Synthesis of polyA tracts 3’ polyA tails are required for translation of (most) mRNAs polyA is attached to the 3’ ends of cellular mRNAs in the nucleus. RNA viruses replicate in cytoplasm  Many RNA Viruses have evolved mechanisms to acquire polyA tracts: e.g. Encode a 3’ polyA sequence on the (+) strand and/or 5’ polyU on the (-) strand Reiterative copying (“stuttering”) on short 3’ U-sequences on the (-) strand.

Switching from mRNA production to genome RNA synthesis No switch required when mRNA and gRNA are identical. However, mRNAs of RNA viruses are not complete copies of the viral RNA. A switching mechanism is required. Different polymerases for different functions e.g. alphaviruses sequentially produce 3 RDRPs, each with template specificity. The last one is specific for replication of full length genomic RNA e.g. Influenza and VSV viruses produce two RNA polymerases, only one of which can produce genomic RNA

Switching from mRNA production to genome RNA synthesis Different templates used for RNA synthesis and genome replication e.g. in dsRNA viruses replication of gRNA occurs only after packaging RNAs inside of capsids. All unpackaged viral RNAs are mRNA by default. (fig. 6.17)