Phenotypic analysis of mutations at residue 146 provides insights into the relationship between NS5A hyperphosphorylation and hepatitis C virus genome replication

The hepatitis C virus genotype 2a isolate, JFH-1, exhibits much more efficient genome replication than other isolates. Although basic replication mechanisms must be conserved, this raises the question of whether the regulation of replication might exhibit isolate- and/or genotype-specific characteristics. Exemplifying this, the phenotype of NS5A hyperphosphorylation is genotype-dependent; in genotype 1b a loss of hyperphosphorylation correlates with an enhancement of replication. In contrast, the replication of JFH-1 is not regulated by hyperphosphorylation. We previously identified a novel phosphorylation site in JFH-1 NS5A: S146. A phosphomimetic substitution (S146D) had no effect on replication but correlated with a loss of hyperphosphorylation. In genotype 1b, residue 146 is alanine and we therefore investigated whether the substitution of A146 with a phosphorylatable (S), or phosphomimetic, residue would recapitulate the JFH-1 phenotype, decoupling hyperphosphorylation from replication. This was not the case, as A146D exhibited both a loss of hyperphosphorylation and a reduction in replication, accompanied by a perinuclear restriction of replication complexes, reductions in lipid droplet and PI4P lipid accumulation, and a disruption of NS5A dimerization. In contrast, the S232I culture-adaptive mutation in the low-complexity sequence I (LCSI) also exhibited a loss of hyperphosphorylation, but was associated with an increase in replication. Taken together, these data imply that hyperphosphorylation does not directly regulate replication. In contrast, the loss of hyperphosphorylation is a consequence of perturbing genome replication and NS5A function. Furthermore, we show that mutations in either domain I or LCSI of NS5A can disrupt hyperphosphorylation, demonstrating that multiple parameters influence the phosphorylation status of NS5A

Association of Zinc Finger Antiviral Protein Binding to Viral Genomic RNA with Attenuation of Replication of Echovirus 7

Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNase L in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and interrelationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3′ untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect on replication or specific binding to ZAP by IP. However, the bases 3′ and 5′ of CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP binding, while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound noncompetitively with CpG-enriched RNA, consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP and colocalized with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAP’s association with larger cellular complexes may mediate the recruitment of OAS3/RNase L, KHNYN, and other RNA degradation pathways

Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication

Suppression of the CpG dinucleotide is widespread in RNA viruses infecting vertebrates and plants, and in the genomes of retroviruses and small mammalian DNA viruses. The functional basis for CpG suppression in the latter was investigated through the construction of mutants of the parvovirus, minute virus of mice (MVM) with increased CpG or TpA dinucleotides in the VP gene. CpG-high mutants displayed extraordinary attenuation in A9 cells compared to wild-type MVM (>six logs), while TpA elevation showed no replication effect. Attenuation was independent of Toll-like receptor 9 and STING-mediated DNA recognition pathways and unrelated to effects on translation efficiency. While translation from codon-optimized VP RNA was enhanced in a cell-free assay, MVM containing this sequence was highly attenuated. Further mutational analysis indicated that this arose through its increased numbers of CpG dinucleotides (7→70) and separately from its increased G+C content (42.3→57.4 %), which independently attenuated replication. CpG-high viruses showed impaired NS mRNA expression by qPCR and reduced NS and particularly VP protein expression detected by immunofluorescence and replication in A549 cells, effects reversed in zinc antiviral protein (ZAP) knockout cells, even though nuclear relocalization of VP remained defective. The demonstrated functional basis for CpG suppression in MVM and potentially other small DNA viruses and the observed intolerance of CpGs in coding sequences, even after codon optimization, has implications for the use of small DNA virus vectors in gene therapy and immunization

Multiple roles of the non-structural protein 3 (nsP3) alphavirus unique domain (AUD) during Chikungunya virus genome replication and transcription

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

Characterization of Live-Attenuated Powassan Virus Vaccine Candidates Identifies an Efficacious Prime-Boost Strategy for Mitigating Powassan Virus Disease in a Murine Model

Powassan virus (POWV) is an emerging tick-borne virus and cause of lethal encephalitis in humans. The lack of treatment or prevention strategies for POWV disease underscores the need for an effective POWV vaccine. Here, we took two independent approaches to develop vaccine candidates. First, we recoded the POWV genome to increase the dinucleotide frequencies of CpG and UpA to potentially attenuate the virus by raising its susceptibility to host innate immune factors, such as the zinc-finger antiviral protein (ZAP). Secondly, we took advantage of the live-attenuated yellow fever virus vaccine 17D strain (YFV-17D) as a vector to express the structural genes pre-membrane (prM) and envelope (E) of POWV. The chimeric YFV-17D-POWV vaccine candidate was further attenuated for in vivo application by removing an N-linked glycosylation site within the nonstructural protein (NS)1 of YFV-17D. This live-attenuated chimeric vaccine candidate significantly protected mice from POWV disease, conferring a 70% survival rate after lethal challenge when administered in a homologous two-dose regimen. Importantly, when given in a heterologous prime-boost vaccination scheme, in which vaccination with the initial chimeric virus was followed by a protein boost with the envelope protein domain III (EDIII), 100% of the mice were protected without showing any signs of morbidity. Combinations of this live-attenuated chimeric YFV-17D-POWV vaccine candidate with an EDIII protein boost warrant further studies for the development of an effective vaccine strategy for the prevention of POWV disease

Knowns and unknowns of TiLV-associated neuronal disease

Tilapia Lake Virus (TiLV) is associated with pathological changes in the brain of infected fish, but the mechanisms driving the virus’s neuropathogenesis remain poorly characterized. TiLV establishes a persistent infection in the brain of infected fish even when the virus is no longer detectable in the peripheral organs, rendering therapeutic interventions and disease management challenging. Moreover, the persistence of the virus in the brain may pose a risk for viral reinfection and spread and contribute to ongoing tissue damage and neuroinflammatory processes. In this review, we explore TiLV-associated neurological disease. We discuss the possible mechanism(s) used by TiLV to enter the central nervous system (CNS) and examine TiLV-induced neuroinflammation and brain immune responses. Lastly, we discuss future research questions and knowledge gaps to be addressed to significantly advance this field

Knowns and unknowns of TiLV-associated neuronal disease

Tilapia Lake Virus (TiLV) is associated with pathological changes in the brain of infected fish, but the mechanisms driving the virus's neuropathogenesis remain poorly characterized. TiLV establishes a persistent infection in the brain of infected fish even when the virus is no longer detectable in the peripheral organs, rendering therapeutic interventions and disease management challenging. Moreover, the persistence of the virus in the brain may pose a risk for viral reinfection and spread and contribute to ongoing tissue damage and neuroinflammatory processes. In this review, we explore TiLV-associated neurological disease. We discuss the possible mechanism(s) used by TiLV to enter the central nervous system (CNS) and examine TiLV-induced neuroinflammation and brain immune responses. Lastly, we discuss future research questions and knowledge gaps to be addressed to significantly advance this field

Viral emissions into the air and environment after SARS-CoV-2 human challenge: a phase 1, open label, first-in-human study

Background Effectively implementing strategies to curb SARS-CoV-2 transmission requires understanding who is contagious and when. Although viral load on upper respiratory swabs has commonly been used to infer contagiousness, measuring viral emissions might be more accurate to indicate the chance of onward transmission and identify likely routes. We aimed to correlate viral emissions, viral load in the upper respiratory tract, and symptoms, longitudinally, in participants who were experimentally infected with SARS-CoV-2. Methods In this phase 1, open label, first-in-human SARS-CoV-2 experimental infection study at quarantine unit at the Royal Free London NHS Foundation Trust, London, UK, healthy adults aged 18–30 years who were unvaccinated for SARS-CoV-2, not previously known to have been infected with SARS-CoV-2, and seronegative at screening were recruited. Participants were inoculated with 10 50% tissue culture infectious dose of pre-alpha wild-type SARS-CoV-2 (Asp614Gly) by intranasal drops and remained in individual negative pressure rooms for a minimum of 14 days. Nose and throat swabs were collected daily. Emissions were collected daily from the air (using a Coriolis μ air sampler and directly into facemasks) and the surrounding environment (via surface and hand swabs). All samples were collected by researchers, and tested by using PCR, plaque assay, or lateral flow antigen test. Symptom scores were collected using self-reported symptom diaries three times daily. The study is registered with ClinicalTrials.gov, NCT04865237. Findings Between March 6 and July 8, 2021, 36 participants (ten female and 26 male) were recruited and 18 (53%) of 34 participants became infected, resulting in protracted high viral loads in the nose and throat following a short incubation period, with mild-to-moderate symptoms. Two participants were excluded from the per-protocol analysis owing to seroconversion between screening and inoculation, identified post hoc. Viral RNA was detected in 63 (25%) of 252 Coriolis air samples from 16 participants, 109 (43%) of 252 mask samples from 17 participants, 67 (27%) of 252 hand swabs from 16 participants, and 371 (29%) of 1260 surface swabs from 18 participants. Viable SARS-CoV-2 was collected from breath captured in 16 masks and from 13 surfaces, including four small frequently touched surfaces and nine larger surfaces where airborne virus could deposit. Viral emissions correlated more strongly with viral load in nasal swabs than throat swabs. Two individuals emitted 86% of airborne virus, and the majority of airborne virus collected was released on 3 days. Individuals who reported the highest total symptom scores were not those who emitted most virus. Very few emissions occurred before the first reported symptom (7%) and hardly any before the first positive lateral flow antigen test (2%). Interpretation After controlled experimental inoculation, the timing, extent, and routes of viral emissions was heterogeneous. We observed that a minority of participants were high airborne virus emitters, giving support to the notion of superspreading individuals or events. Our data implicates the nose as the most important source of emissions. Frequent self-testing coupled with isolation upon awareness of first symptoms could reduce onward transmissions. Funding UK Vaccine Taskforce of the Department for Business, Energy and Industrial Strategy of Her Majesty's Government

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

The SARS-CoV-2 Omicron BA.1 variant emerged in 2021(1) and has multiple mutations in its spike protein(2). Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways(3) demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

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