Mapping of transcription termination within the S segment of SFTS phlebovirus facilitated the generation of NSs-deletant viruses

SFTS phlebovirus (severe fever with thrombocytopenia syndrome virus; SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral non-structural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3’ RACE we mapped the 3’ -end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3’ end of the N mRNA terminates upstream of a 5’ -GCCAGCC-3’ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nt of the NSs ORF were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate it is possible to recover viruses lacking NSs, expressing just a 12 amino acid NSs peptide or viruses encoding eGFP or a NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies

Differential Antagonism of Human Innate Immune Responses by Tick-Borne Phlebovirus Nonstructural Proteins

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Interferon-stimulated gene (ISG)-expression screening reveals the specific antibunyaviral activity of ISG20

Bunyaviruses pose a significant threat to human health, prosperity and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon stimulated genes (ISGs) whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional ribonuclease activity. Through use of an infectious VLP assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taken together, we report that ISG20 is a broad and potent antibunyaviral factor yet some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance could influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance

Chikungunya virus vaccine candidates with decreased mutational robustness are attenuated in vivo and have compromised transmissibility

Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates

Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil

Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. Methodology/Principal findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. Conclusions/Significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions

Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influ-enza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 50-m7G-cappedhost transcripts to prime viral mRNA synthesis (‘‘cap-snatching’’). We hypothesized that start codons withincap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We reportthe existence of this mechanism of gene origination, which we named ‘‘start-snatching.’’ Depending on thereading frame, start-snatching allows the translation of host and viral ‘‘untranslated regions’’ (UTRs) to createN-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show thatboth types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contributeto virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animaland plant viruses, a host-dependent mechanism allows the genesis of hybrid genes

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19

Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs Is a weak interferon antagonist

Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans