Deactivation of the antiviral state by rabies virus through targeting and accumulation of persistently phosphorylated STAT1.

Antagonism of the interferon (IFN)-mediated antiviral state is critical to infection by rabies virus (RABV) and other viruses, and involves interference in the IFN induction and signaling pathways in infected cells, as well as deactivation of the antiviral state in cells previously activated by IFN. The latter is required for viral spread in the host, but the precise mechanisms involved and roles in RABV pathogenesis are poorly defined. Here, we examined the capacity of attenuated and pathogenic strains of RABV that differ only in the IFN-antagonist P protein to overcome an established antiviral state. Importantly, P protein selectively targets IFN-activated phosphorylated STAT1 (pY-STAT1), providing a molecular tool to elucidate specific roles of pY-STAT1. We find that the extended antiviral state is dependent on a low level of pY-STAT1 that appears to persist at a steady state through ongoing phosphorylation/dephosphorylation cycles, following an initial IFN-induced peak. P protein of pathogenic RABV binds and progressively accumulates pY-STAT1 in inactive cytoplasmic complexes, enabling recovery of efficient viral replication over time. Thus, P protein-pY-STAT1 interaction contributes to 'disarming' of the antiviral state. P protein of the attenuated RABV is defective in this respect, such that replication remains suppressed over extended periods in cells pre-activated by IFN. These data provide new insights into the nature of the antiviral state, indicating key roles for residual pY-STAT1 signaling. They also elucidate mechanisms of viral deactivation of antiviral responses, including specialized functions of P protein in selective targeting and accumulation of pY-STAT1.

Correction: SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

[This corrects the article DOI: 10.1371/journal.ppat.1009800.].

TRIM25 and DEAD-Box RNA Helicase DDX3X Cooperate to Regulate RIG-I-Mediated Antiviral Immunity.

The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25's catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-ß production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.

SARS-CoV-2 suppresses IFNß production mediated by NSP1, 5, 6, 15, ORF6 and ORF7b but does not suppress the effects of added interferon.

Type I Interferons (IFN-Is) are a family of cytokines which play a major role in inhibiting viral infection. Resultantly, many viruses have evolved mechanisms in which to evade the IFN-I response. Here we tested the impact of expression of 27 different SARS-CoV-2 genes in relation to their effect on IFN production and activity using three independent experimental methods. We identified six gene products; NSP6, ORF6, ORF7b, NSP1, NSP5 and NSP15, which strongly (>10-fold) blocked MAVS-induced (but not TRIF-induced) IFNß production. Expression of the first three of these SARS-CoV-2 genes specifically blocked MAVS-induced IFNß-promoter activity, whereas all six genes induced a collapse in IFNß mRNA levels, corresponding with suppressed IFNß protein secretion. Five of these six genes furthermore suppressed MAVS-induced activation of IFNλs, however with no effect on IFNα or IFNγ production. In sharp contrast, SARS-CoV-2 infected cells remained extremely sensitive to anti-viral activity exerted by added IFN-Is. None of the SARS-CoV-2 genes were able to block IFN-I signaling, as demonstrated by robust activation of Interferon Stimulated Genes (ISGs) by added interferon. This, despite the reduced levels of STAT1 and phospho-STAT1, was likely caused by broad translation inhibition mediated by NSP1. Finally, we found that a truncated ORF7b variant that has arisen from a mutant SARS-CoV-2 strain harboring a 382-nucleotide deletion associating with mild disease (Δ382 strain identified in Singapore & Taiwan in 2020) lost its ability to suppress type I and type III IFN production. In summary, our findings support a multi-gene process in which SARS-CoV-2 blocks IFN-production, with ORF7b as a major player, presumably facilitating evasion of host detection during early infection. However, SARS-CoV-2 fails to suppress IFN-I signaling thus providing an opportunity to exploit IFN-Is as potential therapeutic antiviral drugs.

RK-33 Is a Broad-Spectrum Antiviral Agent That Targets DEAD-Box RNA Helicase DDX3X.

Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for efficacious antiviral strategies. While antiviral drugs are available, in many cases, they are prone to the development of drug resistance. A way to overcome drug resistance associated with common antiviral therapies is to develop antivirals targeting host cellular co-factors critical to viral replication, such as DEAD-box helicase 3 X-linked (DDX3X), which plays key roles in RNA metabolism and the antiviral response. Here, we use biochemical/biophysical approaches and infectious assays to show for the first time that the small molecule RK-33 has broad-spectrum antiviral action by inhibiting the enzymatic activities of DDX3X. Importantly, we show that RK-33 is efficacious at low micromolar concentrations in limiting infection by human parainfluenza virus type 3 (hPIV-3), respiratory syncytial virus (RSV), dengue virus (DENV), Zika virus (ZIKV) or West Nile virus (WNV)-for all of which, no Food and Drug Administration (FDA)-approved therapeutic is widely available. These findings establish for the first time that RK-33 is a broad-spectrum antiviral agent that blocks DDX3X's catalytic activities in vitro and limits viral replication in cells.

Sustained Wolbachia-mediated blocking of dengue virus isolates following serial passage in Aedes aegypti cell culture.

Wolbachia is an intracellular endosymbiont of insects that inhibits the replication of a range of pathogens in its arthropod hosts. The release of Wolbachia into wild populations of mosquitoes is an innovative biocontrol effort to suppress the transmission of arthropod-borne viruses (arboviruses) to humans, most notably dengue virus. The success of the Wolbachia-based approach hinges upon the stable persistence of the 'pathogen blocking' effect, whose mechanistic basis is poorly understood. Evidence suggests that Wolbachia may affect viral replication via a combination of competition for host resources and activation of host immunity. The evolution of resistance against Wolbachia and pathogen blocking in the mosquito or the virus could reduce the public health impact of the symbiont releases. Here, we investigate if dengue 3 virus (DENV-3) is capable of accumulating adaptive mutations that improve its replicative capacity during serial passage in Wolbachia wMel-infected cells. During the passaging regime, viral isolates in Wolbachia-infected cells exhibited greater variation in viral loads compared to controls. The viral loads of these isolates declined rapidly during passaging due to the blocking effects of Wolbachia carriage, with several being lost all together and the remainder recovering to low but stable levels. We attempted to sequence the genomes of the surviving passaged isolates but, given their low abundance, were unable to obtain sufficient depth of coverage for evolutionary analysis. In contrast, viral loads in Wolbachia-free control cells were consistently high during passaging. The surviving isolates passaged in the presence of Wolbachia exhibited a reduced ability to replicate even in Wolbachia-free cells. These experiments demonstrate the challenge for dengue in evolving resistance to Wolbachia-mediated blocking.

Complete genome of Aedes aegypti anphevirus in the Aag2 mosquito cell line.

A novel negative-sense RNA virus, Aedes aegypti anphevirus, was recently identified in wild Aedes aegypti mosquitoes. We show that this virus is also present in the Aag2 Aedes aegypti cell line and characterize its complete genome and evolutionary history. The Aedes aegypti anphevirus genome is estimated to be 12 916 nucleotides in length, contains four genes and has a genome structure similar to that of other anpheviruses. Phylogenetically, Aedes aegypti anphevirus falls within an unclassified group of insect-specific viruses in the order Mononegavirales that form a sister-group to the chuviruses. Notably, the Aag2 cell line used here was also experimentally infected with dengue virus and naturally contained a Phasi Charoen-like virus and cell-fusing agent virus. All four viruses were at relatively high abundance, with 0.5 % of sequence reads assigned to Aedes aegypti anphevirus. The Aag2 cell line is therefore permissive to efficient co-infection with dengue virus and multiple insect-specific viruses.

Recognition by host nuclear transport proteins drives disorder-to-order transition in Hendra virus V.

Hendra virus (HeV) is a paramyxovirus that causes lethal disease in humans, for which no vaccine or antiviral agent is available. HeV V protein is central to pathogenesis through its ability to interact with cytoplasmic host proteins, playing key antiviral roles. Here we use immunoprecipitation, siRNA knockdown and confocal laser scanning microscopy to show that HeV V shuttles to and from the nucleus through specific host nuclear transporters. Spectroscopic and small angle X-ray scattering studies reveal HeV V undergoes a disorder-to-order transition upon binding to either importin α/ß1 or exportin-1/Ran-GTP, dependent on the V N-terminus. Importantly, we show that specific inhibitors of nuclear transport prevent interaction with host transporters, and reduce HeV infection. These findings emphasize the critical role of host-virus interactions in HeV infection, and potential use of compounds targeting nuclear transport, such as the FDA-approved agent ivermectin, as anti-HeV agents.

Wolbachia infection alters the relative abundance of resident bacteria in adult Aedes aegypti mosquitoes, but not larvae.

Insect-symbiont interactions are known to play key roles in host functions and fitness. The common insect endosymbiont Wolbachia can reduce the ability of several human pathogens, including arboviruses and the malaria parasite, to replicate in insect hosts. Wolbachia does not naturally infect Aedes aegypti, the primary vector of dengue virus, but transinfected Ae. aegypti have antidengue virus properties and are currently being trialled as a dengue biocontrol strategy. Here, we assess the impact of Wolbachia infection of Ae. aegypti on the microbiome of wild mosquito populations (adults and larvae) collected from release sites in Cairns, Australia, by profiling the 16S rRNA gene using next-generation sequencing. Our data indicate that Wolbachia reduces the relative abundance of a large proportion of bacterial taxa in Ae. aegypti adults, that is in accordance with the known pathogen-blocking effects of Wolbachia on a variety of bacteria and viruses. In adults, several of the most abundant bacterial genera were found to undergo significant shifts in relative abundance. However, the genera showing the greatest changes in relative abundance in Wolbachia-infected adults represented a low proportion of the total microbiome. In addition, there was little effect of Wolbachia infection on the relative abundance of bacterial taxa in larvae, or on species diversity (accounting for species richness and evenness together) detected in adults or larvae. These results offer insight into the effects of Wolbachia on the Ae. aegypti microbiome in a native setting, an important consideration for field releases of Wolbachia into the population.

The microbiome composition of Aedes aegypti is not critical for Wolbachia-mediated inhibition of dengue virus.

BACKGROUND: Dengue virus (DENV) is primarily vectored by the mosquito Aedes aegypti, and is estimated to cause 390 million human infections annually. A novel method for DENV control involves stable transinfection of Ae. aegypti with the common insect endosymbiont Wolbachia, which mediates an antiviral effect. However, the mechanism by which Wolbachia reduces the susceptibility of Ae. aegypti to DENV is not fully understood. In this study we assessed the potential of resident microbiota, which can play important roles in insect physiology and immune responses, to affect Wolbachia-mediated DENV blocking. METHODOLOGY/FINDINGS: The microbiome of Ae. aegypti stably infected with Wolbachia strain wMel was compared to that of Ae. aegypti without Wolbachia, using 16s rDNA profiling. Our results indicate that although Wolbachia affected the relative abundance of several genera, the microbiome of both the Wolbachia-infected and uninfected mosquitoes was dominated by Elizabethkingia and unclassified Enterobacteriaceae. To assess the potential of the resident microbiota to affect the Wolbachia-mediated antiviral effect, we used antibiotic treatment before infection with DENV by blood-meal. In spite of a significant shift in the microbiome composition in response to the antibiotics, we detected no effect of antibiotic treatment on DENV infection rates, or on the DENV load of infected mosquitoes. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that stable infection with Wolbachia strain wMel produces few effects on the microbiome of laboratory-reared Ae. aegypti. Moreover, our findings suggest that the microbiome can be significantly altered without affecting the fundamental DENV blocking phenotype in these mosquitoes. Since Ae. aegypti are likely to encounter diverse microbiota in the field, this is a particularly important result in the context of using Wolbachia as a method for DENV control.

Nucleocytoplasmic trafficking of Nipah virus W protein involves multiple discrete interactions with the nuclear import and export machinery.

Paramyxoviruses replicate in the cytoplasm with no obvious requirement to interact with the nucleus. Nevertheless, the W protein of the highly lethal bat-borne paramyxovirus Nipah virus (NiV) is known to undergo specific targeting to the nucleus, mediated by a single nuclear localisation signal (NLS) within the C-terminal domain. Here, we report for the first time that additional sites modulate nucleocytoplasmic localisation of W. We show that the N-terminal domain interacts with importin α1 and contributes to nuclear accumulation of W, indicative of a novel N-terminal NLS. We also find that W undergoes exportin-1 mediated nuclear export, dependent on a leucine at position 174. Together, these data enable significant revision of the generally accepted model of W trafficking, with implications for understanding of the mechanisms of NiV immune evasion.

Roles of nuclear trafficking in infection by cytoplasmic negative-strand RNA viruses: paramyxoviruses and beyond.

Genome replication and virion production by most negative-sense RNA viruses (NSVs) occurs exclusively in the cytoplasm, but many NSV-expressed proteins undergo active nucleocytoplasmic trafficking via signals that exploit cellular nuclear transport pathways. Nuclear trafficking has been reported both for NSV accessory proteins (including isoforms of the rabies virus phosphoprotein, and V, W and C proteins of paramyxoviruses) and for structural proteins. Trafficking of the former is thought to enable accessory functions in viral modulation of antiviral responses including the type I IFN system, but the intranuclear roles of structural proteins such as nucleocapsid and matrix proteins, which have critical roles in extranuclear replication and viral assembly, are less clear. Nevertheless, nuclear trafficking of matrix protein has been reported to be critical for efficient production of Nipah virus and Respiratory syncytial virus, and nuclear localization of nucleocapsid protein of several morbilliviruses has been linked to mechanisms of immune evasion. Together, these data point to the nucleus as a significant host interface for viral proteins during infection by NSVs with otherwise cytoplasmic life cycles. Importantly, several lines of evidence now suggest that nuclear trafficking of these proteins may be critical to pathogenesis and thus could provide new targets for vaccine development and antiviral therapies.

The immune evasion function of J and Beilong virus V proteins is distinct from that of other paramyxoviruses, consistent with their inclusion in the proposed genus Jeilongvirus.

IFN-antagonist function is a major determinant of pathogenicity and cross-species infection by viruses, but remains poorly defined for many potentially zoonotic viruses resident in animal species. The paramyxovirus family contains several zoonotic viruses, including highly pathogenic viruses such as Nipah virus and Hendra virus, and an increasing number of largely uncharacterized animal viruses. Here, we report the characterization of IFN antagonism by the rodent viruses J virus (JPV) and Beilong virus (BeiPV) of the proposed genus Jeilongvirus of the paramyxoviruses. Infection of cells by JPV and BeiPV was found to inhibit IFN-activated nuclear translocation of signal transducer and activator of transcription 1 (STAT1). However, in contrast to most other paramyxoviruses, the JPV and BeiPV V proteins did not interact with or inhibit signalling by STAT1 or STAT2, suggesting that JPV/BeiPV use an atypical V protein-independent strategy to target STATs, consistent with their inclusion in a separate genus. Nevertheless, the V proteins of both viruses interacted with melanoma differentiation-associated protein 5 (MDA5) and robustly inhibited MDA5-dependent activation of the IFN-ß promoter. This supports a growing body of evidence that MDA5 is a universal target of paramyxovirus V proteins, such that the V-MDA5 interaction represents a potential target for broad-spectrum antiviral approaches.

Bovine ephemeral fever rhabdovirus α1 protein has viroporin-like properties and binds importin ß1 and importin 7.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV α1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an α1-deficient BEFV strain) and in cells expressing a BEFV α1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of α1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.

Paramyxovirus evasion of innate immunity: Diverse strategies for common targets.

The paramyxoviruses are a family of > 30 viruses that variously infect humans, other mammals and fish to cause diverse outcomes, ranging from asymptomatic to lethal disease, with the zoonotic paramyxoviruses Nipah and Hendra showing up to 70% case-fatality rate in humans. The capacity to evade host immunity is central to viral infection, and paramyxoviruses have evolved multiple strategies to overcome the host interferon (IFN)-mediated innate immune response through the activity of their IFN-antagonist proteins. Although paramyxovirus IFN antagonists generally target common factors of the IFN system, including melanoma differentiation associated factor 5, retinoic acid-inducible gene-I, signal transducers and activators of transcription (STAT)1 and STAT2, and IFN regulatory factor 3, the mechanisms of antagonism show remarkable diversity between different genera and even individual members of the same genus; the reasons for this diversity, however, are not currently understood. Here, we review the IFN antagonism strategies of paramyxoviruses, highlighting mechanistic differences observed between individual species and genera. We also discuss potential sources of this diversity, including biological differences in the host and/or tissue specificity of different paramyxoviruses, and potential effects of experimental approaches that have largely relied on in vitro systems. Importantly, recent studies using recombinant virus systems and animal infection models are beginning to clarify the importance of certain mechanisms of IFN antagonism to in vivo infections, providing important indications not only of their critical importance to virulence, but also of their potential targeting for new therapeutic/vaccine approaches.