The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication

Lindenbach, Brett D.; Prágai, B.; Montserret, Roland; Beran, Rudolf K. F.; Pyle, Anna Marie; Pénin, François; Rice, Charles M.

doi:10.1128/jvi.00937-07

Cited by 106 publications

(106 citation statements)

References 73 publications

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“…Some of the sites of NS3 and NS5A involved in these molecular interactions also have links with some of the sites of NS4A, forming a subnetwork of coordinated substitutions. This subnetwork is in agreement with the results of a recent experimental study (15) that found that substitutions in NS4A lead to decreased replication and NS5A hyperphosphorylation and that compensatory mutations in NS3 and NS5A suppress these defects.…”

Section: Constraints On the Hypervariable Region 1 (Hvr1)supporting

confidence: 81%

“…Several experiments have provided strong evidence of extensive epistasis in RNA viruses, confirming that during viral evolution certain substitutions at different sites may occur in a coordinated manner (7)(8)(9)(10)(11)(12). Experimental studies into the adaptation of genetically modified HCV genomes propagated in cell culture also have shown antagonistic epistatic interactions between deleterious mutations exhibited in the form of compensatory mutations (13)(14)(15). These results suggest the importance of long-range interactions among HCV constitutive amino acids that place additional constraints on HCV heterogeneity.…”

mentioning

confidence: 75%

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Coordinated evolution of the hepatitis C virus

Campo

Dimitrova

Mitchell

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus is a genetically heterogeneous RNA virus that is a major cause of liver disease worldwide. Here, we show that, despite its extensive heterogeneity, the evolution of hepatitis C virus is primarily shaped by negative selection and that numerous coordinated substitutions in the polyprotein can be organized into a scale-free network whose degree of connections between sites follows a power-law distribution. This network shares all major properties with many complex biological and technological networks. The topological structure and hierarchical organization of this network suggest that a small number of amino acid sites exert extensive impact on hepatitis C virus evolution. Nonstructural proteins are enriched for negatively selected sites of high centrality, whereas structural proteins are enriched for positively selected sites located in the periphery of the network. The complex network of coordinated substitutions is an emergent property of genetic systems with implications for evolution, vaccine research, and drug development. In addition to such properties as polymorphism or strength of selection, the epistatic connectivity mapped in the network is important for typing individual sites, proteins, or entire genetic systems. The network topology may help devise molecular intervention strategies for disrupting viral functions or impeding compensatory changes for vaccine escape or drug resistance mutations. Also, it may be used to find new therapeutic targets, as suggested in this study for the NS4A protein, which plays an important role in the network.complex systems ͉ scale-free network ͉ covariation ͉ natural selection ͉ epistasis H epatitis C virus (HCV) is a major cause of liver disease worldwide. The global prevalence of HCV infection is estimated to be 2.2%, representing 130 million people (1). HCV causes chronic infection in 70-85% of infected adults (2). There is no vaccine against HCV and current antiviral therapy is relatively toxic, being effective in 50-60% of patients treated (3). HCV is a single-stranded RNA virus of Ϸ9.4 kb belonging to the Flaviviridae family (4). The positive-sense genome of HCV contains one large ORF that encodes a polyprotein that can undergo proteolytic cleavage into 10 mature proteins (C-E1-E2-P7-NS2-NS3-NS4A-NS4B-NS5A-NS5B). The structural proteins, the core (C) and envelope glycoproteins E1 and E2, are present in the N-terminal part of the polyprotein and presumably self-assemble to form the virion. The nonstructural (NS) proteins have various functions and form the replication complex (5).The HCV genome continually mutates during virus replication. Although a high rate of mutation significantly contributes to the enormous adaptability of RNA viruses, it also limits the size of viral genomes by causing error catastrophe (6). The small size of viral genomes imposes strong evolutionary constraints on their organization, as a result of which each genomic region may encode multiple and often conflicting functions. Such genomic organization requires a tight coor...

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Section: Constraints On the Hypervariable Region 1 (Hvr1)supporting

confidence: 81%

mentioning

confidence: 75%

Coordinated evolution of the hepatitis C virus

Campo

Dimitrova

Mitchell

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The helicase-protease interface is likely to be more extensive and functionally important than previously thought or implied by the existing crystal structure of the full-length protein (6). Recent genetic studies on viral replicons show that mutations in domain 2 of NS3 suppress deleterious mutations in the C terminus of NS4A (which is integrated within the protease domain) (38). Thus, the protease domain may form a network of functional interactions with the helicase domain.…”

Section: The Mechanism Of Ns3hel Activation By the Protease Domain-mentioning

confidence: 95%

The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate

Beran

Serebrov

Pyle

2007

Journal of Biological Chemistry

103

111

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Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV)replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA. Nonstructural protein (NS3)3 is an essential component of the hepatitis C virus (HCV) replication complex. It is a nucleic acid-stimulated ATPase and DEXH/D-box protein that is structurally similar to other DNA and RNA helicases from the phylogenetically conserved superfamily 2. Like these relatives, NS3 contains two RecA fold domains (domains 1 and 2) that comprise the ATPase site and the platform for RNA binding, which is supplemented by domain 3, which sits atop D1 and D2 (1). The three-lobed triangle comprised of D1-D3 is considered the "helicase" of NS3 (NS3hel), and it has been studied extensively in isolation (2-5). However, the full-length NS3 protein differs from other SF2 helicases in that it possesses a fourth domain at the N terminus that displays robust serine protease activity (the protease domain) (6, 7). This protease domain is located at the vertices of D1-D3, in an ideal location to influence the catalytic activities of the helicase region.NS3 serine protease activity is crucial for cleaving the viral polyprotein into individual, nonstructural (NS) proteins. It also facilitates viral pathogenicity by cleaving host proteins and down-regulating the innate immune response of the cell (7, 8). NS3 helicase activity may be required to assist the viral polymerase during replication of the HCV RNA genome. NS3 may unwind complex RNA structures within the genome, whereas the NS5B viral polymerase copies the RNA (9). There may ...

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“…1,2 This virus family includes several other medically important arboviruses, including the mosquitoborne viruses St. Louis encephalitis virus , Yellow fever virus , and Dengue virus (DENV), and the tick-borne viruses Tickborne encephalitis virus and Powassan virus . West Nile virus is naturally maintained through an enzootic transmission cycle between Culex mosquito vectors and avian reservoir hosts.…”

Section: Introductionmentioning

confidence: 99%

Requirement of Glycosylation of West Nile Virus Envelope Protein for Infection of, but Not Spread within, Culex quinquefasciatus Mosquito Vectors

Moudy¹,

Payne²,

Dodson³

et al. 2011

The American Society of Tropical Medicine and Hygiene

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Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV–vector interactions in a species-specific manner.

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The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication

Cited by 106 publications

References 73 publications

Coordinated evolution of the hepatitis C virus

Coordinated evolution of the hepatitis C virus

The Serine Protease Domain of Hepatitis C Viral NS3 Activates RNA Helicase Activity by Promoting the Binding of RNA Substrate

Requirement of Glycosylation of West Nile Virus Envelope Protein for Infection of, but Not Spread within, Culex quinquefasciatus Mosquito Vectors

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