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Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5' exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7- methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that -- in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses. [ABSTRACT FROM AUTHOR]

The polyproteins of coronaviruses are cleaved by viral proteases into at least 15 nonstructural proteins (Nsps). Consisting of five domains, Nsp3 is the largest of these (180-210 kDa). Among these domains, the so-called X-domain is believed to act as ADP-ribose-1″-phosphate phosphatase or to bind poly(ADP-ribose). However, here we show that the X-domain of Infectious Bronchitis Virus (strain Beaudette), a Group-3 coronavirus, fails to bind ADP-ribose. This is explained on the basis of the crystal structure of the protein, determined at two different pH values. For comparison, we also describe the crystal structure of the homologous X-domain from Human Coronavirus 229E, a Group-1 coronavirus, which does bind ADP-ribose. [ABSTRACT FROM AUTHOR]

Despite the apparent natural grouping of “picorna-like” viruses, the taxonomical significance of this putative “supergroup” was never addressed adequately. We recently proposed to the ICTV that an order should be created and named Picornavirales, to include viruses infecting eukaryotes that share similar properties: (i) a positive-sense RNA genome, usually with a 5′-bound VPg and 3′-polyadenylated, (ii) genome translation into autoproteolytically processed polyprotein(s), (iii) capsid proteins organized in a module containing three related jelly-roll domains which form small icosahedral, non-enveloped particles with a pseudo-T = 3 symmetry, and (iv) a three-domain module containing a superfamily III helicase, a (cysteine) proteinase with a chymotrypsin-like fold and an RNA-dependent RNA polymerase. According to the above criteria, the order Picornavirales includes the families Picornaviridae, Comoviridae, Dicistroviridae, Marnaviridae, Sequiviridae and the unassigned genera Cheravirus, Iflavirus and Sadwavirus. Other taxa of “picorna-like” viruses, e.g. Potyviridae, Caliciviridae, Hypoviridae, do not conform to several of the above criteria and are more remotely related: therefore they are not being proposed as members of the new order. Newly described viruses, not yet assigned to an existing taxon by ICTV, may belong to the proposed order. [ABSTRACT FROM AUTHOR]

The poliovirus (PV) eradication campaign is conducted on the premise that this virus, because of the lack of a zoonotic reservoir, will not reemerge once eradicated. This report examines the origin of PV using theoretical and experimental approaches. Our rooted phylogenetic analysis suggests a speciation of PV from a C-cluster coxsackie A virus (C-CAV) ancestor through mutation of the capsid that caused a receptor switch from intercellular adhesion molecule-1 to CD155, leading to a change of pathogenicity. This hypothesis is supported experimentally with chimeras generated from three different pairs of PV and C-CAV. Those carrying the PV capsid and the replication proteins of C-CAVs replicated well, whereas their reciprocal counterparts were either debilitated or dead. This phenomenon of asymmetry is observed also in recombinants between PV1 and C-CAV20, selected in tissue culture cells using a previously undescribed protocol. The recombinants are generated at frequencies of 10-6 typical for PV interserotype recombination. Strikingly, they resemble genetically and phenotypically, including neurovirulence in CD155 transgenic mice, the large majority of circulating vaccine-derived PVs that have caused poliomyelitis outbreaks in different parts of the world. These data provide experimental evidence for C-CAVs being partners to PVs in generating diverse PV progeny by homologous recombination. They support speciation of a novel human pathogen (PV) from a pool of different human pathogens (C-CAVs). In a PV-free world without PV neutralizing antibodies, contemporary C-CAV, like their ancestor(s), could be fertile ground for a PV-like agent to emerge by mutation. [ABSTRACT FROM AUTHOR]

Replication of the giant RNA genome of severe acute respiratory syndrome (SARS) coronavirus (Coy) and synthesis of as many as eight subgenomic (sg) mRNAs are mediated by a viral replicase-transcriptase of outstanding complexity that includes an essential endoribonuclease activity. Here, we show that the CoV replicative machinery, unlike that of other RNA viruses, also uses an exoribonuclease (ExoN) activity, which is associated with nonstructural protein (nsp) 14. Bacterially expressed forms of SARS-CoV nsp14 were shown to act on both ssRNAs and dsRNAs in a 3'→5' direction. The activity depended on residues that are conserved in the DEDD exonuclease superfamily. The protein did not hydrolyze DNA or ribose-2'-O-methylated RNA substrates and required divalent metal ions for activity. A range of 5'-labeled 55RNA substrates were processed to final products of ~8-12 nucleotides. When part of dsRNA or in the presence of nonlabeled dsRNA, the 5'-Iabeled RNA substrates were processed to significantly smaller products, indicating that binding to dsRNA in cis or trans modulates the exonucleolytic activity of nsp14. Characterization of human CoV 229E ExoN active-site mutants revealed severe defects in viral RNA synthesis, and no viable virus could be recovered. Besides strongly reduced genome replication, specific defects in sg RNA synthesis, such as aberrant sizes of specific sg RNAs and changes in the molar ratios between individual sg RNA species, were observed. Taken together, the study identifies an RNA virus ExoN activity that is involved in the synthesis of multiple RNAs from the exceptionally large genomic RNA templates of CoVs. [ABSTRACT FROM AUTHOR]