Influenza's Cryptic Constraint Because of the well-known pandemic potential of influenza viruses, it is important to understand the range of molecular interactions between the virus and its host. Despite years of intensive research on the virus, Jagger et al. (p. 199 , published online 28 June; see the Perspective by Yewdell and Ince ) have found that the influenza A virus has been hiding a gene in its small negative-sense RNA genome. An overlapping open reading frame was found contained in the PA viral RNA polymerase gene, which is accessed by ribosomal frameshifting to produce a fusion protein containing the N-terminal messenger RNA (mRNA) endonuclease domain of PA and an alternative C-terminal X domain. The resulting polypeptide, PA-X, selectively degrades host mRNAs and, in a mouse model of infection, modulated cellular immune responses, thus limiting viral pathogenesis.

Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1, is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1 is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1 is the product of a ؊1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1 plays a role in viral neuroinvasiveness.

The arterivirus family (order Nidovirales) of single-stranded, positive-sense RNA viruses includes porcine respiratory and reproductive syndrome virus and equine arteritis virus (EAV). Their replicative enzymes are translated from their genomic RNA, while their seven structural proteins are encoded by a set of small, partially overlapping genes in the genomic 39-proximal region. The latter are expressed via synthesis of a set of subgenomic mRNAs that, in general, are functionally monocistronic (except for a bicistronic mRNA encoding the E and GP2 proteins). ORF5, which encodes the major glycoprotein GP5, has been used extensively for phylogenetic analyses. However, an in-depth computational analysis now reveals the arterivirus-wide conservation of an additional AUG-initiated ORF, here termed ORF5a, that overlaps the 59 end of ORF5. The pattern of substitutions across sequence alignments indicated that ORF5a is subject to functional constraints at the amino acid level, while an analysis of substitutions at synonymous sites in ORF5 revealed a greatly reduced frequency of substitution in the portion of ORF5 that is overlapped by ORF5a. The 43-64 aa ORF5a protein and GP5 are probably expressed from the same subgenomic mRNA, via a translation initiation mechanism involving leaky ribosomal scanning. Inactivation of ORF5a expression by reverse genetics yielded a severely crippled EAV mutant, which displayed lower titres and a tiny plaque phenotype. These defects, which could be partially complemented in ORF5a-expressing cells, indicate that the novel protein, which may be the eighth structural protein of arteriviruses, is expressed and important for arterivirus infection. INTRODUCTIONArteriviruses are enveloped viruses (approx. 60 nm diameter) with a 13-16 kb positive-sense RNA genome. The family Arteriviridae (Snijder & Meulenberg, 1998; currently comprises four members: equine arteritis virus (EAV, the family prototype), lactate dehydrogenase-elevating virus (LDV) of mice, porcine reproductive and respiratory syndrome virus (PRRSV) and simian haemorrhagic fever virus (SHFV). The consequences of arterivirus infection can range from an asymptomatic, persistent or acute infection to abortion or lethal haemorrhagic fever (Maclachlan et al., 2007). Three of the four arteriviruses (EAV, LDV and SHFV) were first isolated and characterized approximately 50 years ago.In the late 1980s, two distantly related PRRSV genotypes, European (PRRSV-EU) and North American (PRRSV-NA), sharing a genome-wide mean nucleotide identity of only~63 % (Allende et al., 1999; Suárez et al., 1996), emerged simultaneously on both sides of the Atlantic (Collins et al., 1992;Wensvoort et al., 1991). Since then, the virus has been causing worldwide epidemics of a previously unknown reproductive and respiratory disease and has become one of the most economically important swine diseases. Recently, a large outbreak of highly virulent PRRSV affected the Asian swine industry, causing considerable economic losses (Zhou & Yang, 2010).The unification of the ...

In Sindbis, Venezuelan equine encephalitis and related alphaviruses, the polymerase is translated as a fusion with other non-structural proteins via readthrough of a UGA stop codon. Surprisingly, earlier work reported that the signal for efficient readthrough comprises a single cytidine residue 3′-adjacent to the UGA. However, analysis of variability at synonymous sites revealed strikingly enhanced conservation within the ∼150 nt 3′-adjacent to the UGA, and RNA folding algorithms revealed the potential for a phylogenetically conserved stem–loop structure in the same region. Mutational analysis of the predicted structure demonstrated that the stem–loop increases readthrough by up to 10-fold. The same computational analysis indicated that similar RNA structures are likely to be relevant to readthrough in certain plant virus genera, notably Furovirus, Pomovirus, Tobravirus, Pecluvirus and Benyvirus, as well as the Drosophilia gene kelch. These results suggest that 3′ RNA stimulatory structures feature in a much larger proportion of readthrough cases than previously anticipated, and provide a new criterion for assessing the large number of cellular readthrough candidates that are currently being revealed by comparative sequence analysis.