The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Molecular characterization of the 2022 Sudan virus disease outbreak in Uganda.

IMPORTANCE: Ebola disease (EBOD) is a public health threat with a high case fatality rate. Most EBOD outbreaks have occurred in remote locations, but the 2013-2016 Western Africa outbreak demonstrated how devastating EBOD can be when it reaches an urban population. Here, the 2022 Sudan virus disease (SVD) outbreak in Mubende District, Uganda, is summarized, and the genetic relatedness of the new variant is evaluated. The Mubende variant exhibited 96% amino acid similarity with historic SUDV sequences from the 1970s and a high degree of conservation throughout the outbreak, which was important for ongoing diagnostics and highly promising for future therapy development. Genetic differences between viruses identified during the Mubende SVD outbreak were linked with epidemiological data to better interpret viral spread and contact tracing chains. This methodology should be used to better integrate discrete epidemiological and sequence data for future viral outbreaks.

Characterization of humoral responses to Nipah virus infection in the Syrian Hamster model of disease.

Nipah virus (NiV) is a highly pathogenic paramyxovirus. The Syrian hamster model recapitulates key features of human NiV disease and is a critical tool for evaluating antivirals and vaccines. Here we describe longitudinal humoral immune responses in NiV-infected Syrian hamsters. Samples were obtained 1-28 days after infection and analyzed by ELISA, neutralization, and Fc-mediated effector function assays. NiV infection elicited robust antibody responses against the nucleoprotein and attachment glycoprotein. Levels of neutralizing antibodies were modest and only detectable in surviving animals. Fc-mediated effector functions were mostly observed in nucleoprotein-targeting antibodies. Antibody levels and activities positively correlated with challenge dose.

Lassa Virus Replicon Particle Vaccine Protects Strain 13/N Guinea Pigs Against Challenge With Geographically and Genetically Diverse Viral Strains.

Lassa virus (LASV) causes mild to severe hemorrhagic fever disease in humans. Strain 13/N guinea pigs are highly susceptible to infection with LASV strain Josiah (clade IV), providing a critical model system for therapeutics and vaccine development. To develop additional models of disease, we detail the clinical course in guinea pigs infected with 5 geographically and genetically diverse LASV strains. Two of the developed models (LASV clades II and III) were then used to evaluate efficacy of a virus replicon particle vaccine against heterologous LASV challenge, demonstrating complete protection against clinical disease after a single vaccination dose.

Probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (vOTU) structure and deubiquitinase activity.

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.

Fluorescent Crimean-Congo hemorrhagic fever virus illuminates tissue tropism patterns and identifies early mononuclear phagocytic cell targets in Ifnar-/- mice.

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/ß receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.

In Situ Imaging of Fluorescent Nipah Virus Respiratory and Neurological Tissue Tropism in the Syrian Hamster Model.

Using a recombinant Nipah virus expressing a fluorescent protein (ZsG), we visualized virus tropism in the Syrian hamster model. We found that anatomical localization of fluorescence correlated to clinical signs; signal was primarily visualized in the respiratory tract in animals with acute-onset terminal disease, whereas central nervous system localization was seen in animals that succumbed with delayed disease onset. While polymerase chain reaction (PCR) detection corresponded well to ZsG signal, virus was only isolated from some lung, brain, liver, and kidney samples that were ZsG and/or PCR positive, and only from animals euthanized on or before 15 days post infection.

Alterations in Blood Chemistry Levels Associated With Nipah Virus Disease in the Syrian Hamster Model.

Nipah virus (NiV; family Paramyxoviridae, genus Henipavirus) infection can cause severe respiratory and neurological disease in humans. The pathophysiology of disease is not fully understood, and it may vary by presentation and clinical course. In this study, we investigate changes in blood chemistry in NiV-infected Syrian hamsters that survived or succumbed to disease. Increased sodium and magnesium and decreased albumin and lactate levels were detected in animals euthanized with severe clinical disease compared with mock-infected controls. When subjects were grouped by clinical syndrome, additional trends were discernable, highlighting changes associated with either respiratory or neurological disease.

Inhibition of Nipah Virus by Defective Interfering Particles.

The error-prone nature of RNA-dependent RNA polymerases drives the diversity of RNA virus populations. Arising within this diversity is a subset of defective viral genomes that retain replication competency, termed defective interfering (DI) genomes. These defects are caused by aberrant viral polymerase reinitiation on the same viral RNA template (deletion DI species) or the nascent RNA strand (copyback DI species). DI genomes have previously been shown to alter the dynamics of a viral population by interfering with normal virus replication and/or by stimulating the innate immune response. In this study, we investigated the ability of artificially produced DI genomes to inhibit Nipah virus (NiV), a highly pathogenic biosafety level 4 paramyxovirus. High multiplicity of infection passaging of both NiV clinical isolates and recombinant NiV in Vero cells generated an extensive DI population from which individual DIs were identified using next-generation sequencing techniques. Assays were established to generate and purify both naturally occurring and in silico-designed DIs as fully encapsidated, infectious virus-like particles termed defective interfering particles (DIPs). We demonstrate that several of these NiV DIP candidates reduced NiV titers by up to 4 logs in vitro. These data represent a proof-of-principle that a therapeutic application of DIPs to combat NiV infections may be an alternative source of antiviral control for this disease.

Evaluation of a Single-Dose Nucleoside-Modified Messenger RNA Vaccine Encoding Hendra Virus-Soluble Glycoprotein Against Lethal Nipah virus Challenge in Syrian Hamsters.

In the absence of approved vaccines and therapeutics for use in humans, Nipah virus (NiV) continues to cause fatal outbreaks of encephalitis and respiratory disease in Bangladesh and India on a near-annual basis. We determined that a single dose of a lipid nanoparticle nucleoside-modified messenger RNA vaccine encoding the soluble Hendra virus glycoprotein protected up to 70% of Syrian hamsters from lethal NiV challenge, despite animals having suboptimally primed immune responses before challenge. These data provide a foundation from which to optimize future messenger RNA vaccination studies against NiV and other highly pathogenic viruses.

Griffithsin Inhibits Nipah Virus Entry and Fusion and Can Protect Syrian Golden Hamsters From Lethal Nipah Virus Challenge.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses, including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

Protection From Lethal Lassa Disease Can Be Achieved Both Before and After Virus Exposure by Administration of Single-Cycle Replicating Lassa Virus Replicon Particles.

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that postexposure vaccination can confer protection from lethal outcome.

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins.

UNLABELLED: To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the ß2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE: Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses.

Stress granule components G3BP1 and G3BP2 play a proviral role early in Chikungunya virus replication.

UNLABELLED: Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored. IMPORTANCE: Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

Temporal SILAC-based quantitative proteomics identifies host factors involved in chikungunya virus replication.

Chikungunya virus (CHIKV) is an arthropod-borne reemerging human pathogen that generally causes a severe persisting arthritis. Since 2005, the virus has infected millions of people during outbreaks in Africa, Indian Ocean Islands, Asia, and South/Central America. Many steps of the replication and expression of CHIKV's 12-kb RNA genome are highly dependent on cellular factors, which thus constitute potential therapeutic targets. SILAC and LC-MS/MS were used to define the temporal dynamics of the cellular response to infection. Using samples harvested at 8, 10, and 12 h postinfection, over 4700 proteins were identified and per time point 2800-3500 proteins could be quantified in both biological replicates. At 8, 10, and 12 h postinfection, 13, 38, and 106 proteins, respectively, were differentially expressed. The majority of these proteins showed decreased abundance. Most subunits of the RNA polymerase II complex were progressively degraded, which likely contributes to the transcriptional host shut-off observed during CHIKV infection. Overexpression of four proteins that were significantly downregulated (Rho family GTPase 3 (Rnd3), DEAD box helicase 56 (DDX56), polo-like kinase 1 (Plk1), and ubiquitin-conjugating enzyme E2C (UbcH10) reduced susceptibility of cells to CHIKV infection, suggesting that infection-induced downregulation of these proteins is beneficial for CHIKV replication. All MS data have been deposited in the ProteomeXchange with identifier PXD001330 (http://proteomecentral.proteomexchange.org/dataset/PXD001330).

Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response.

The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.

Inhibition of dengue and chikungunya virus infections by RIG-I-mediated type I interferon-independent stimulation of the innate antiviral response.

UNLABELLED: RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens, dengue virus (DENV) and chikungunya virus (CHIKV), for which no therapeutic options currently exist. We demonstrate that when a low, noncytotoxic dose of an optimized 5'triphosphorylated RNA (5'pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5'pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody-dependent enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5'pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this in vitro analysis of the antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV. IMPORTANCE: DENV and CHIKV are two reemerging mosquito-borne viruses for which no therapeutic options currently exist. Both viruses overlap geographically in tropical regions of the world, produce similar fever-like symptoms, and are difficult to diagnose. This study investigated the inhibitory effect of a RIG-I agonist on the replication of these two viruses. RIG-I stimulation using 5'pppRNA before or after DENV or CHIKV infection generated a protective antiviral response against both pathogens in immune and nonimmune cells; interestingly, the protective response against the viruses was largely independent of the classical type I interferon response. The antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.

An in vitro assay to study chikungunya virus RNA synthesis and the mode of action of inhibitors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay (32)P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5' end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15,000 G: . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3'dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.