Virology 2015 RNA Virus RdRP Enzymes

General Comments An RNA virus does not view replication of the genome and expression of the genome as separate events. An RNA virus is an RNA replicon in a world full of DNA replicons. For most RNA viruses, everything takes place in the same compartment. A biological arms race has shaped the evolution of RNA viruses

Issues, problems and strategies related to the production of many copies of progeny viral RNA (replication) The nature of the template (pp 170-171) secondary structure nucleocapsids The nature of the enzymes (pp 171-175) only viruses have RdRP (almost) The nature of the reactions (pp 175-182) initiation and priming elongation/unwinding balancing + and – strand synthesis

The Nature of the Template

RNA secondary structure is complex Secondary structures help regulate RNA function Shape is usually the key Replication machinery must deal with the shape

Example: CSE4 of alphaviruses Alphaviruses are members of a genus of the Togavirus family SS + RNA, T=4, envelope, wide host range Replication initiates at 3’ CSE using non-structural virus proteins and host factors Shown for salmonid alphavirus 3 (SAV3)

Example for nucleocapsids: VSV Vesicular Stomatitis Virus: vesiculovirus genus of Rhabdoviridae (best-studied rhabdovirus) Rhabdoviridae are – ssRNA with a “bullet” shape enveloped Wide host range among rhabdoviridae

Example: VSV Replication requiresor RNP nucleocapsid “Transcription” of genomic RNA produces + sense messages and viral proteins Production of full-length + or – RNA requires coated template

The Nature of the Enzymes

RdRp-only viruses have it??? Increasing understanding of function of RNA in an uninfected cell

Common domains or “motifs” RdRp Motifs: A: nucleotide recognition/binding, phosphoryl transfer: “two-metal” mechanism B: nucleotide recognition/binding C: active site, phosphoryl transfer: “two-metal” mechanism D: structure and shape of enzyme E: primer binding F: nucleotide entry tunnel (not shown above) (A, B, C, D in all pols)

Common shape Polio 3Dpol (above), HCV (below) Shaped like a right hand Key features: Palm, Thumb, Fingers Closed configuration Generic shape-no two are identical Shape changes affect biological properties

Location of Domains The conserved structural polymerase motifs. (a–c) The polymerase core of IBDV VP1 (a) compared with the equivalent cores in FMDV (b) and bacteriophage φ6 (c) RDRPs. The secondary structural elements containing the conserved motifs are colored as follows: A, red; B, green; C, yellow; D, purple; E, orange; F, blue. The N- and C-terminal domains are shown as thin ribbons. FMDV = Foot and mouth disease virus IBDV = infectious bursal disease virus

RdRp with template Below: Polio Rdrp with RNA in substrate channel, top removed Right (A) WNV Rdrp with motifs and position of substrate, template and product (B) 90 degree rotation of A

The Nature of the Reactions: Initiation and Priming

de novo Initiation Prime and realign Proposed mechanism for: arenaviridae, bunyaviridae: ss ambisense RNA segmented genome enveloped wide host range-rodents arena (Machupo) bunya (hantaviruses) De novo refers to lack of requirement for 3’ OH

Primer Dependent Initiation Protein priming VPg of picornaviruses = best example ss + RNA, AAAAA, icosahedral, many species 22 aa covalently linked Virion RNA Removed upon infection to produce viral mRNA

Polio Gene Products 3AB = membrane anchor 3B = VPg 3C or 3CD = protease 3D = polymerase

Replication machine anchored to membrane Addition of U to 3AB Coupled to cleavage of 3B from 3AB to produce 3A and 3B=VPg-pUpU Uridylylated VPg transferred to 3’ end 5’ VPg-pUpU  3’ ApApApApNNNNN 5’

Host proteins PCBP And PABP Are Important

The Nature of the Reactions: Unwinding

Helicase activity Example: Flaviviruses: Yellow Fever Virus, West Nile Virus, Hepatitis C Virus RNA helicase of flavivirus a member of helicase superfamily 2 Helicase a target for therapy? ATP dependent Mechanism? See p 180 Not all helicases function in this way Phage phi 6 is a dsRNA phage whose polymerase/helicase works differently

The Nature of the Reactions: Balancing + and - Synthesis

+/- shift not well-characterized Diverse mechanisms? Differential stability of + and -? Differential synthesis of + and -? Deterioration of cells/buildup of virus factories affects stoichiometry?