Control of Innate Immune Signaling and Membrane Targeting by the Hepatitis C Virus NS3/4A Protease Are Governed by the NS3 Helix α
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Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatmentresistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.A ll positive-strand RNA viruses, including picornaviruses, such as poliovirus, rhinovirus, and hepatitis A virus, and flaviviruses, such as dengue virus and hepatitis C virus (HCV), rely heavily on cellular membranes at numerous stages of their infectious cycles. For example, RNA replication complexes must assemble on the topologically cytoplasmic surfaces of intracellular membranes. In some cases, such as poliovirus and hepatitis A virus, these RNA replication complexes are on the convex outer surfaces of discrete vesicles (1). In others, such as dengue virus, RNA replication complexes are assembled on invaginated membrane surfaces that are connected to the cytosol only via narrow openings (2, 3). For dengue virus, newly synthesized viral RNA exits the invaginated cytoplasm and interacts with core protein, which encapsidates the viral RNA and decorates the surfaces of nearby lipid droplets via the high-affinity binding of its N-terminal domain (4, 5). For HCV, a similar interaction of the core protein with lipid droplets has been described and seems to play a critical role in the assembly of viral particles (6-9). During dengue virus infection, formation of the nucleocapsid, subsequent interaction with envelope proteins, and budding into the ER lumen are likely to occur in close proximity (2). In the cis-Golgi, the virion undergoes a conformational change, and the viral prM (prematrix) protein is cleaved by the cellular furin protease into the mature M (matrix) protein and a peptide (pr) (10, 11). Upon cleavage, the pr peptide dissociates from the virion, resulting in the formation of mature progeny viruses that are highly infectious. This finely tuned interplay between cellular membrane remodeling, cellular lipid storage, and viral assembly is not only a fascinating cell biological puzzle, but also provides exciting...

Section: Cells and Virusesmentioning

confidence: 99%

Inhibition of Cellular Autophagy Deranges Dengue Virion Maturation

Mateo

Nagamine

Spagnolo

et al. 2013

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139

150

“…All nucleotide and amino acid positions refer to the JFH-1 genome (GenBank accession number AB047639). Mutations in the HCV coding region were introduced into pENTR-SJ*, a molecular clone of JFH-1 that has the sequence of JFH-1 and a T7 promoter (pENTR-SJ*) (30), by using site-directed mutagenesis (QuikChange Lightning kit; Stratagene). Following sequence verification, correct sequences were subcloned back into pENTR-SJ* as described below and verified again by DNA sequencing.…”

Section: Cell Linesmentioning

confidence: 99%

Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity

Aligeti

Roder

Horner

2015

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The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. IMPORTANCEHepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection. Hepatitis C virus (HCV), a positive-sense single-stranded RNA virus and a member of the Flaviviridae family, infects nearly 170 million people worldwide (1, 2). HCV infection is the leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (3). While the previous interferon-based therapies for treatment of hepatitis C were effective in only 40% of those infected with genotype 1 virus, the most prevalent HCV genotype in the United States (4), newly developed therapies for hepatitis C now use interferon-free direct-acting antiviral (DAA) regimens, with over 90% success rates (5). However, antiviral resistance is still a problem, and a vaccine is not yet available for prevention of HCV transmission.The HCV RNA genome is translated into a single polyprotein from an internal ribosome entry site located in the 5= untranslated region of the genome. The resu...

“…The M21T mutation lies in the N-terminal amphipathic helix ␣ 0 (Fig. 1A), which is conserved among all HCV genotypes and was reported to play important roles in anchoring NS3 and NS3/4A to the intracellular membrane and thus preventing NS3 from degradation (15,16,18). To determine whether the M21T mutation contributes to HCV infection, we engineered this mutation into the JFH1 genome.…”

Section: Resultsmentioning

confidence: 99%

“…It is known that HCV NS3/4A protease can shut down host interferon (IFN) signaling by cleaving MAVS, a mitochondrion-associated host protein essential for the antiviral interferon response (21), and helix ␣ 0 of NS3 could control its ability to suppress host innate immune signaling as well (18). To assess the possibility that the M21T mutation may enhance NS3's capability to cleave MAVS to suppress interferon production for viral production, we examined MAVS cleavage and interferon suppression by the wild-type and M21T mutant NS3.…”

Section: Resultsmentioning

confidence: 99%

A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

Boson

et al. 2017

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic ␣ helix (designated helix ␣ 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix ␣ 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix ␣ 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix ␣ 0 and aid understanding of the role of NS3 in HCV virion morphogenesis.IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix ␣ 0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix ␣ 0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.KEYWORDS HCV, NS3, core, helix ␣ 0 , assembly, hepatitis C virus