HCV NS5A inhibitors disrupt replication factory formation: a novel mechanism of antiviral action

EditorialNicholas S. Eyre, Michael R. Bear

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.Rosa C. Coldbeck-Shackley, Nicholas S. Eyre and Michael R. Bear

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 (IFNε) 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, β and λ. We show the necessity of IFNε for Zika Virus (ZIKV) protection by: increased susceptibility of IFNε -/- mice; their “rescue” by intravaginal recombinant IFNε treatment and blockade of protective endogenous IFNε by neutralising antibody. Complementary studies in human FRT cell lines showed IFNε had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNε 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 IFNε expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNβ or λ. Thus, the constitutive expression of IFNε 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.Rosa C. Coldbeck-Shackley, Ornella Romeo, Sarah Rosli, Linden J. Gearing, Jodee A. Gould, San S. Lim, Kylie H. Van der Hoek, Nicholas S. Eyre, Byron Shue, Sarah A. Robertson, Sonja M. Best, Michelle D. Tate, Paul J. Hertzog, Michael R. Bear

A mechanism for HCV NS5A-directed antiviral therapy

Covering the cover. Published Online: September 25, 2014Nicholas S. Eyre, Michael R. Bear

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 β-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.Brandon E. K. Tan, Michael R. Beard, and Nicholas S. Eyr

A rapid protocol for assessing sediment condition in eutrophic estuaries

The enrichment of sediments with nutrients and organic matter (eutrophication) is a key anthropogenic stressor of estuaries worldwide, impacting their sediment condition, ecology and ecosystem service provision. A key challenge for estuary managers and scientists is how to effectively quantify and monitor these changes in ecological condition in a timely and cost-effective manner. We developed a Rapid Assessment Protocol (RAP) for characterizing sediment condition based on the qualitative characteristics of sediment colour, odour and texture. We evaluated its utility for assessing sediment condition, and particularly the degree and effects of sediment enrichment (as quantified by complementary measurements of total C, organic C and total N) across 97 sites throughout a eutrophic microtidal estuary. RAP results were strongly and significantly correlated with the degree of sediment enrichment, with RAP scores correctly identifying the assigned enrichment class (low, medium, high) of 83.5% of sites. More enriched sediments exhibited poorer condition, manifested as significantly lower RAP scores for sediment colour, texture and odour, particularly (but not only) where enrichment coincided with elevated mud content. The RAP was particularly successful (<12% misclassification) at identifying sites with low levels of enrichment, indicating its promise as a first-pass survey approach for identifying potential reference or control sites to support impact assessments. RAP approaches based on qualitative sediment characteristics can provide a useful proxy for the degree and impacts of inorganic and organic enrichment, with potentially broad applicability for supporting timely, cost-effective assessment and monitoring of sediment condition in estuaries worldwide

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 amongst 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 -β (ER-α and ER-β) 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.Nicholas S. Eyre, Emily N. Kirby, Daniel R. Anfiteatro, Gustavo Bracho, Alice G. Russo, Peter A. White ... et al

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

Genome-wide CRISPR screen identifies RACK1 as a critical host factor for flavivirus replication

ABSTRACT 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.Byron Shue, Abhilash I. Chiramel, Berati Cerikan, Thu-Hien To, Sonja Frölich, Stephen M. Pederson, Emily N. Kirby, Nicholas S. Eyre, Ralf F. W. Bartenschlager, Sonja M. Best, and Michael R. Bear

A recombinant HCV 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 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 stain does not. Our data provides 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 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 indicates that 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.Janelle Johnson, Holly Freedman, Michael Logan, Jason Alexander Ji-Xhin Wong ... Michael R. Beard, Nicholas S. Eyre ... et al