Vav proteins do not influence dengue virus replication but are associated with induction of phospho-ERK, IL-6, and viperin mRNA following DENV infection in vitro.

IMPORTANCE: Dengue disease is characterized by an inflammatory-mediated immunopathology, with elevated levels of circulating factors including TNF-α and IL-6. If the damaging inflammatory pathways could be blocked without loss of antiviral responses or exacerbating viral replication, then this would be of potential therapeutic benefit. The study here has investigated the Vav guanine exchange factors as a potential alternative signaling pathway that may drive dengue virus (DENV)-induced inflammatory responses, with a focus on Vav1 and 2. While Vav proteins were positively associated with mRNA for inflammatory cytokines, blocking Vav signaling didn't affect DENV replication but prevented DENV-induction of p-ERK and enhanced IL-6 (inflammatory) and viperin (antiviral) mRNA. These initial data suggest that Vav proteins could be a target that does not compromise control of viral replication and should be investigated further for broader impact on host inflammatory responses, in settings such as antibody-dependent enhancement of infection and in different cell types.

Identification of Key Residues in Dengue Virus NS1 Protein That Are Essential for Its Secretion.

Dengue virus (DENV) non-structural protein 1 (NS1) is involved in multiple aspects of the DENV lifecycle. Importantly, it is secreted from infected cells as a hexameric lipoparticle that mediates vascular damage that is a hallmark of severe dengue. Although the secretion of NS1 is known to be important in DENV pathogenesis, the exact molecular features of NS1 that are required for its secretion from cells are not fully understood. In this study, we employed random point mutagenesis in the context of an NS1 expression vector encoding a C-terminal HiBiT luminescent peptide tag to identify residues within NS1 that are essential for its secretion. Using this approach, we identified 10 point mutations that corresponded with impaired NS1 secretion, with in silico analyses indicating that the majority of these mutations are located within the ß-ladder domain. Additional studies on two of these mutants, V220D and A248V, revealed that they prevented viral RNA replication, while studies using a DENV NS1-NS5 viral polyprotein expression system demonstrated that these mutations resulted in a more reticular NS1 localisation pattern and failure to detect mature NS1 at its predicted molecular weight by Western blotting using a conformation-specific monoclonal antibody. Together, these studies demonstrate that the combination of a luminescent peptide tagged NS1 expression system with random point mutagenesis enables rapid identification of mutations that alter NS1 secretion. Two such mutations identified via this approach revealed residues that are essential for correct NS1 processing or maturation and viral RNA replication.

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection.

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNɛ) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, ß and λ. We show the necessity of IFNɛ for Zika Virus (ZIKV) protection by: increased susceptibility of IFNɛ-/- mice; their "rescue" by intravaginal recombinant IFNɛ treatment and blockade of protective endogenous IFNɛ by neutralising antibody. Complementary studies in human FRT cell lines showed IFNɛ had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNɛ activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNɛ expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNß or λ. Thus, the constitutive expression of IFNɛ provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy.

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

Applications of Deep Mutational Scanning in Virology.

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-ß. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

The Molecular Interactions of ZIKV and DENV with the Type-I IFN Response.

Zika Virus (ZIKV) and Dengue Virus (DENV) are related viruses of the Flavivirus genus that cause significant disease in humans. Existing control measures have been ineffective at curbing the increasing global incidence of infection for both viruses and they are therefore prime targets for new vaccination strategies. Type-I interferon (IFN) responses are important in clearing viral infection and for generating efficient adaptive immune responses towards infection and vaccination. However, ZIKV and DENV have evolved multiple molecular mechanisms to evade type-I IFN production. This review covers the molecular interactions, from detection to evasion, of these viruses with the type-I IFN response. Additionally, we discuss how this knowledge can be exploited to improve the design of new vaccine strategies.

Identification of Estrogen Receptor Modulators as Inhibitors of Flavivirus Infection.

Flaviviruses such as Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) are major global pathogens for which safe and effective antiviral therapies are not currently available. To identify antiviral small molecules with well-characterized safety and bioavailability profiles, we screened a library of 2,907 approved drugs and pharmacologically active compounds for inhibitors of ZIKV infection using a high-throughput cell-based immunofluorescence assay. Interestingly, estrogen receptor modulators raloxifene hydrochloride and quinestrol were among 15 compounds that significantly inhibited ZIKV infection in repeat screens. Subsequent validation studies revealed that these drugs effectively inhibit ZIKV, DENV, and WNV (Kunjin strain) infection at low micromolar concentrations with minimal cytotoxicity in Huh-7.5 hepatoma cells and HTR-8 placental trophoblast cells. Since these cells lack detectable expression of estrogen receptors-α and -ß (ER-α and ER-ß) and similar antiviral effects were observed in the context of subgenomic DENV and ZIKV replicons, these compounds appear to inhibit viral RNA replication in a manner that is independent of their known effects on estrogen receptor signaling. Taken together, quinestrol, raloxifene hydrochloride, and structurally related analogues warrant further investigation as potential therapeutics for treatment of flavivirus infections.

A Recombinant Hepatitis C Virus Genotype 1a E1/E2 Envelope Glycoprotein Vaccine Elicits Antibodies That Differentially Neutralize Closely Related 2a Strains through Interactions of the N-Terminal Hypervariable Region 1 of E2 with Scavenger Receptor B1.

The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between the J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization, and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 strain does not. Our data provide new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine.IMPORTANCE A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and ß-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies.IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.

Viperin is an important host restriction factor in control of Zika virus infection.

Zika virus (ZIKV) infection has emerged as a global health threat and infection of pregnant women causes intrauterine growth restriction, spontaneous abortion and microcephaly in newborns. Here we show using biologically relevant cells of neural and placental origin that following ZIKV infection, there is attenuation of the cellular innate response characterised by reduced expression of IFN-ß and associated interferon stimulated genes (ISGs). One such ISG is viperin that has well documented antiviral activity against a wide range of viruses. Expression of viperin in cultured cells resulted in significant impairment of ZIKV replication, while MEFs derived from CRISPR/Cas9 derived viperin-/- mice replicated ZIKV to higher titers compared to their WT counterparts. These results suggest that ZIKV can attenuate ISG expression to avoid the cellular antiviral innate response, thus allowing the virus to replicate unchecked. Moreover, we have identified that the ISG viperin has significant anti-ZIKV activity. Further understanding of how ZIKV perturbs the ISG response and the molecular mechanisms utilised by viperin to suppress ZIKV replication will aid in our understanding of ZIKV biology, pathogenesis and possible design of novel antiviral strategies.

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis.

Hepatic Overexpression of CD36 Improves Glycogen Homeostasis and Attenuates High-Fat Diet-Induced Hepatic Steatosis and Insulin Resistance.

The common complications in obesity and type 2 diabetes include hepatic steatosis and disruption of glucose-glycogen homeostasis, leading to hyperglycemia. Fatty acid translocase (FAT/CD36), whose expression is inducible in obesity, is known for its function in fatty acid uptake. Previous work by us and others suggested that CD36 plays an important role in hepatic lipid homeostasis, but the results have been conflicting and the mechanisms were not well understood. In this study, by using CD36-overexpressing transgenic (CD36Tg) mice, we uncovered a surprising function of CD36 in regulating glycogen homeostasis. Overexpression of CD36 promoted glycogen synthesis, and as a result, CD36Tg mice were protected from fasting hypoglycemia. When challenged with a high-fat diet (HFD), CD36Tg mice showed unexpected attenuation of hepatic steatosis, increased very low-density lipoprotein (VLDL) secretion, and improved glucose tolerance and insulin sensitivity. The HFD-fed CD36Tg mice also showed decreased levels of proinflammatory hepatic prostaglandins and 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictive and proinflammatory arachidonic acid metabolite. We propose that CD36 functions as a protective metabolic sensor in the liver under lipid overload and metabolic stress. CD36 may be explored as a valuable therapeutic target for the management of metabolic syndrome.

Calsyntenin-1 mediates hepatitis C virus replication.

The hepatitis C virus (HCV) RNA genome of 9.6 kb encodes only 10 proteins, and so is highly dependent on host hepatocyte factors to facilitate replication. We aimed to identify host factors involved in the egress of viral particles. By screening the supernatant of HCV-infected Huh7 cells using SILAC-based proteomics, we identified the transmembrane protein calsyntenin-1 as a factor specifically secreted by infected cells. Calsyntenin-1 has previously been shown to mediate transport of endosomes along microtubules in neurons, through interactions with kinesin light chain-1. Here we demonstrate for the first time, we believe, a similar role for calsyntenin-1 in Huh7 cells, mediating intracellular transport of endosomes. In HCV-infected cells we show that calsyntenin-1 contributes to the early stages of the viral replication cycle and the formation of the replication complex. Importantly, we demonstrate in our model that silencing calsyntenin-1 disrupts the viral replication cycle, confirming the reliance of HCV on this protein as a host factor. Characterizing the function of calsyntenin-1 will increase our understanding of the HCV replication cycle and pathogenesis, with potential application to other viruses sharing common pathways.

HCV RNA traffic and association with NS5A in living cells.

The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3'-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly.

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using 'APEX2'-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα.

The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry.

The interferon-induced transmembrane (IFITM) family of proteins have recently been identified as important host effector molecules of the type I interferon response against viruses. IFITM1 has been identified as a potent antiviral effector against hepatitis C virus (HCV), whereas the related family members IFITM2 and IFITM3 have been described to have antiviral effects against a broad range of RNA viruses. Here, we demonstrate that IFITM2 and IFITM3 play an integral role in the interferon response against HCV and act at the level of late entry stages of HCV infection. We have established that in hepatocytes, IFITM2 and IFITM3 localize to the late and early endosomes, respectively, as well as the lysosome. Furthermore, we have demonstrated that S-palmitoylation of all three IFITM proteins is essential for anti-HCV activity, whereas the conserved tyrosine residue in the N-terminal domain of IFITM2 and IFITM3 plays a significant role in protein localization. However, this tyrosine was found to be dispensable for anti-HCV activity, with mutation of the tyrosine resulting in an IFITM1-like phenotype with the retention of anti-HCV activity and co-localization of IFITM2 and IFITM3 with CD81. In conclusion, we propose that the IFITM proteins act in a coordinated manner to restrict HCV infection by targeting the endocytosed HCV virion for lysosomal degradation and demonstrate that the actions of the IFITM proteins are indeed virus and cell-type specific.