The virus-encoded proteinase of Camberwell virus, a genogroup 2 norovirus, was synthesized in Escherichia coli. The purified proteinase had correct N and C termini and showed trans activity in cell-free assays. trans activity was also demonstrated in COS cells transfected with constructs encoding either the proteinase or a proteinase-polymerase fusion. The N-terminal protein of ORF1 was cleaved in COS cells, possibly at the site E 194 /S.

The ORF1 sequence was determined for Camberwell virus, a genogroup 2 Norwalk-like virus, completing the full genome of 7,555 nucleotides. ORF1 cDNA was cloned into a simian virus 40-based expression vector, and the viral proteins synthesized following transfection into COS cells were analyzed. By using antisera directed against the helicase, protease, or polymerase regions, eight polypeptides ranging in size from 19 to 117 kDa were detected by radioimmunoprecipitation. The cleavage sites determining the amino and carboxy termini of the 3C-like protease were identified at E1008/A and E1189/G, respectively.

The nucleotide sequences of the 3'-terminal open reading frame (ORF3) and 3' untranslated region (3'UTR) were determined for four Norwalk-like viruses (NLVs) belonging to genogroup 2. Three of the viruses, isolated in 1995 and 1996, were closely related to Mexico virus (92-93% nucleotide identity in ORF3). The fourth virus, isolated in 1984, was unique, showing only 49-58% nucleotide identity with other NLVs. The variation in sequence of the 3'-terminal ORF of NLVs was greater than that observed for other caliciviruses. This variation was partly due to repeated sequences and frameshifting. To investigate the properties of the ORF3 encoded polypeptide, a signal sequence and N-linked glycosylation sites predicted for Camberwell virus were tested for function by in vitro translation in the presence of microsomes. Membrane insertion, cleavage of an N-terminal signal sequence, or glycosylation were not detected.

No abstract

Contemporary antiviral development, whether by rational drug design or forward pharmacology, primarily strives to produce lock and key inhibitors. While the technology to identify druggable targets and create compounds to bind them has improved dramatically over the last century it has always been constrained by the finite availability of suitable binding sites that antiviral compounds can occupy. Here we present a new approach to drug design that utilises compounds devised to alter the microenvironment of the virion surface making it incompatible with virus entry and illustrate this strategy with inhibitors of influenza virus. We show that compounds that produce a proton-rich mantle above the virion surface induce a conformational change in the viral hemagglutinin (HA) rendering the virus unable to interact with cellular receptors and gain entry to the cell. The compounds show exceptional antiviral activity both in vitro and in vivo and protect against influenza illness in mice and ferrets after a single dosing, either therapeutically or prophylactically. The work presented here lays the foundation for a brand-new category of inhibitors that could be engineered to counter many different viruses and potentially other pathogens.