Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the Enterovirus genus of the Picornaviridae. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus cis-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be Poliovirus, the archetypal picornavirus, is arguably one of the best characterized of all viruses. The availability of infectious molecular clones (33) has enabled the application of reverse genetics to understand the function of the nonstructural proteins and the noncoding regions (NCR) of the virus genome (reviewed in reference 41). The 7.4-kb single-stranded positive (messenger)-sense RNA genome encodes a single polyprotein and contains the necessary signals for virus replication in the cell cytoplasm. Posttranslational cleavage of the polyprotein by virus-encoded proteases (2A pro and 3C pro ) yields the four capsid proteins (VP1 to VP4), the RNA-dependent RNA polymerase (3D pol ), and the accessory proteins required for replication. The input RNA acts as a template for the synthesis of a negative strand which, in turn, is used as a template for synthesis of genome sense RNA. The virus-encoded protein VPg (3A) is implicated as a protein primer for both positive-and negative-sense strand initiation, as are RNA sequences occupying the 5Ј NCR of the virus genome. A cloverleaf (CL) structure of 88 nucleotides (nt) at the 5Ј end of the genome interacts with the virus 3C pro D pol and either the VPg-containing precursor 3AB or the cellular poly-C binding protein type 2 (PCBP2) (2,3,11,28). This 5Ј NCR ribonucleoprotein complex is required for replication and may also be involved in the suppression of virus translation (10).RNA sequences and structures within the 3Ј NCR presumed to be involved in replication are much less well defined. Chimeric polioviruses in which the 3Ј NCR is replaced by the analogous region of other picornaviruses generally retain viability (35), even though there is little sequence or structural homology between substituted 3Ј NCR....

Small RNA viruses have evolved many mechanisms to increase the capacity of their short genomes. Here we describe the identification and characterization of a novel open reading frame (ORF4) encoded by the murine norovirus (MNV) subgenomic RNA, in an alternative reading frame overlapping the VP1 coding region. ORF4 is translated during virus infection and the resultant protein localizes predominantly to the mitochondria. Using reverse genetics we demonstrated that expression of ORF4 is not required for virus replication in tissue culture but its loss results in a fitness cost since viruses lacking the ability to express ORF4 restore expression upon repeated passage in tissue culture. Functional analysis indicated that the protein produced from ORF4 antagonizes the innate immune response to infection by delaying the upregulation of a number of cellular genes activated by the innate pathway, including IFN-Beta. Apoptosis in the RAW264.7 macrophage cell line was also increased during virus infection in the absence of ORF4 expression. In vivo analysis of the WT and mutant virus lacking the ability to express ORF4 demonstrated an important role for ORF4 expression in infection and virulence. STAT1-/- mice infected with a virus lacking the ability to express ORF4 showed a delay in the onset of clinical signs when compared to mice infected with WT virus. Quantitative PCR and histopathological analysis of samples from these infected mice demonstrated that infection with a virus not expressing ORF4 results in a delayed infection in this system. In light of these findings we propose the name virulence factor 1, VF1 for this protein. The identification of VF1 represents the first characterization of an alternative open reading frame protein for the calicivirus family. The immune regulatory function of the MNV VF1 protein provide important perspectives for future research into norovirus biology and pathogenesis.

Unlike other positive-stranded RNA viruses that use either a 5 0 -cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5 0 end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF4E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF4E by 4E-BP1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF4E and the cap structure or 4E-BP1, suggesting that VPg binds to eIF4E at a different site from both cap and 4E-BP1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA. Keywords: calicivirus; eIF4E; translation; VPg EMBO reports (2005) 6, 968-972.

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

The mechanism and role of RNA structure elements in the replication and translation of Caliciviridae remains poorly understood. Several algorithmically independent methods were used to predict secondary structures within the Norovirus, Sapovirus, Vesivirus and Lagovirus genera. All showed profound suppression of synonymous site variability (SSSV) at genomic 5′ ends and the start of the sub-genomic (sg) transcript, consistent with evolutionary constraints from underlying RNA structure. A newly developed thermodynamic scanning method predicted RNA folding mapping precisely to regions of SSSV and at the genomic 3′ end. These regions contained several evolutionarily conserved RNA secondary structures, of variable size and positions. However, all caliciviruses contained 3′ terminal hairpins, and stem–loops in the anti-genomic strand invariably six bases upstream of the sg transcript, indicating putative roles as sg promoters. Using the murine norovirus (MNV) reverse-genetics system, disruption of 5′ end stem–loops produced ∼15- to 20-fold infectivity reductions, while disruption of the RNA structure in the sg promoter region and at the 3′ end entirely destroyed replication ability. Restoration of infectivity by repair mutations in the sg promoter region confirmed a functional role for the RNA secondary structure, not the sequence. This study provides comprehensive bioinformatic resources for future functional studies of MNV and other caliciviruses.

Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3′-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3′ end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.