Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.

Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis. Chikungunya virus (CHIKV) is an emerging threat to world health. It is transmitted by Aedes species mosquitos, and has caused massive epidemics across the globe. The virus causes fever, rash, arthritis and can sometimes be fatal. The biology of CHIKV is poorly understood, to address this deficiency we aimed to identify functions of one of the viral proteins, nsP3. We focused on the central part of this protein, termed the alphavirus unique domain (AUD) because it is unique to the genus of viruses to which CHIKV belongs–the Alphaviruses–and not present in other related viruses. By making changes (mutations) in the amino acid sequence of the AUD and analysing the effects of these changes we show that it is involved in multiple stages of the virus lifecycle. These observations identify nsP3 and the AUD in particular as a potential target for antiviral therapy or rational vaccine design.

The spike (S) glycoprotein of the SARS-CoV-2 virus that emerged in 2019 contained a suboptimal furin cleavage site at the S1/S2 junction with the sequence ₆₈₁ P RR A R /S ₆₈₆ . This cleavage site is required for efficient airway replication, transmission, and pathogenicity of the virus. The B.1.617 lineage has recently emerged in India, coinciding with substantial disease burden across the country. Early evidence suggests that B.1.617.2 (a sublineage of B.1.617) is more highly transmissible than contemporary lineages. B.1.617 and its sublineages contain a constellation of S mutations including the substitution P681R predicted to further optimise this furin cleavage site. We provide experimental evidence that virus of the B.1.617 lineage has enhanced S cleavage, that enhanced processing of an expressed B.1.617 S protein in cells is due to P681R, and that this mutation enables more efficient cleavage of a peptide mimetic of the B.1.617 S1/S2 cleavage site by recombinant furin. Together, these data demonstrate viruses in this emerging lineage have enhanced S cleavage by furin which we hypothesise could be enhancing transmissibility and pathogenicity.

Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle. Hepatitis C virus infects 170 million people worldwide, causing long term liver disease. Recently new therapies comprising direct-acting antivirals (DAAs), small molecule inhibitors of virus proteins, have revolutionised treatment for infected patients. Despite this, we have a limited understanding of how the virus replicates in infected liver cells. Here we identify a previously uncharacterised function of the NS5A protein–a target for one class of DAAs. NS5A is comprised of three domains–we show that the first of these (domain I) plays a role in the production of new, infectious virus particles. Previously it was thought that domain I was only involved in replicating the virus genome. Mutations in domain I perturb dimer formation, enhanced binding to the 3’ end of the virus RNA genome and prevented NS5A from interacting with lipid droplets, cellular lipid storage organelles that are required for assembly of new viruses. We propose that domain I of NS5A plays multiple roles in virus assembly. As domain I is the putative target for one class of DAAs, our observations may have implications for the as yet undefined mode of action of these compounds.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes. IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.