The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.single-cycle growth ͉ CD81-dependent

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.The World Health Organization estimates that ϳ170 million people worldwide are infected with hepatitis C virus (HCV) (10), with an additional 4 million people infected every year. The majority of infected individuals progress to chronic hepatitis, which greatly increases their risk for developing cirrhosis and hepatocellular carcinoma (27). The standard treatment for HCV patients, based on alpha interferon (IFN-␣) and ribavirin, is expensive and is less efficacious for infections with genotypes 1 and 4, the most common genotypes. Moreover, the treatment is associated with numerous side effects. Therefore, new therapies are urgently needed.There is considerable evidence that neutralizing antibodies are involved in disease control. They emerge during the course of acute HCV infection in patients (25,28,35), and several studies have suggested that they might be involved in the control of viral loads during acute infection (17, 29). Also, polyclonal hyperimmune globulin can prevent or modify HCV infection in vivo when administered before exposure to the virus (11,13,20), and anti-E1 and anti-E2 polyclonal globulin have been reported to neutralize infection with HCV pseudotyped particles (HCVpp) or HCV cell culture-adapted viruses (HCVcc) in vitro (1,7,8,14,15,24,31,36). However, polyclonal hyperimmune globulin preparations are subject to contamination by blood-borne viruses that can be present in human plasma pools, a problem not encountered with monoclonal antibodies (MAbs). Moreover, MAbs are more easily standardized than polyclonal hyperimmune globulin. MAbs against E2 of human or primate origin have been used successfully to neutralize HCVpp of various genotypes and subtypes (18, 34), but anti-E2 MAb-based immunotherapy may be hampered by the very high strain-to-strain variation in the immunodominant hypervariable region of E2. On the other hand, E1 displays a relatively high degree of conservation within subtypes, such as subtype 1b (26), and might show a higher degree of intergenotypic cross-neutralization than E2, as suggested i...

The human immunodeficiency virus (HIV) transactivator protein, Tat, stimulates transcription from the viral long terminal repeats via an arginine-rich transactivating domain. Since arginines are often known to be methylated, we investigated whether HIV type 1 (HIV-1) Tat was a substrate for known protein arginine methyltransferases (PRMTs). Here we identify Tat as a substrate for the arginine methyltransferase, PRMT6. Tat is specifically associated with and methylated by PRMT6 within cells. Overexpression of wild-type PRMT6, but not a methylase-inactive PRMT6 mutant, decreased Tat transactivation of an HIV-1 long terminal repeat luciferase reporter plasmid in a dose-dependent manner. Knocking down PRMT6 consistently increased HIV-1 production in HEK293T cells and also led to increased viral infectiousness as shown in multinuclear activation of a galactosidase indicator assays. Our study demonstrates that arginine methylation of Tat negatively regulates its transactivation activity and that PRMT6 acts as a restriction factor for HIV replication.

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription. RNA helicase A (RHA)2 is a member of the DEXH-box (where X can be any amino acid) family of proteins and is also termed DHX9 (1, 2). The DEXH-box proteins, together with the DEAD-box and the Ski2 family members, are referred to as RNA helicases that are able to rearrange the structures of RNA molecules (3). RHA contains a helicase core domain consisting of seven motifs that are conserved for all RNA helicases. Within the N-terminal region of RHA there are two copies of type A double-stranded RNA binding domains. Together with an RGG-box domain located at the C terminus, double-stranded RNA binding domains regulate RNA binding as well as helicase activities of RHA (4). RHA is a nuclear protein and shuttles between the nucleus and the cytoplasm with the assistance of a bidirectional nuclear transport domain consisting of 110 amino acids at the C terminus (5). This function of the RHA nuclear transport domain is subject to regulation of arginine methylation catalyzed by PRMT1 (protein-arginine methyltransferase 1) (6). RHA is able to unwind double-stranded RNA or DNA with the energy derived from hydrolysis of NTPs by virtue of its NTPase activity (1). This property enables RHA to participate in multiple cellular processes from RNA transcription to RNA processing to RNA nuclear export (7). These multiple functions underlie the vital role of RHA in the germ line proliferation and development of Caenorhabditis elegans (8) and also account for the early embryonic lethality observed with RHA knock-out mice (9).The regulation activity of RHA in RNA transcription is implicated by its presence within the RNA polymerase II holoenzyme complex. For example, RHA has been shown to bridge the interactions between RNA polymerase II and transcription co-activators such as CREB-binding protein and BRAC1 (breast cancer-specific tumor suppressor protein 1) (10, 11). RHA also directly interacts with the p65 subunit of NF-B and stimulates NF-B-mediated reporter gene expression (12). Involvement of RHA in transcription is further indicated by the function of its homologue in Drosophila, named the maleless (MLE) gene, that increases gene expression fr...

A 14-amino-acid spacer peptide termed SP1 that separates the capsid (CA) and nucleocapsid (NC) sequences plays an active role in the assembly of human immunodeficiency virus type 1. This activity of SP1 involves its amino-terminal residues that, together with adjacent CA residues, constitute a putative ␣-helical structure spanning Gag residues from positions 359 to 371. In this study, we have determined that the virus assembly determinants within this putative ␣-helix were residues H359, K360, A361, L364, A367, and M368, of which K360 and A367 contribute to virus production to lesser extents. Notably, changes of the two basic amino acids H359 and K360 to arginine (R) impaired virus production, whereas mutations L364I and M368I, in contrast to L364A and M368A, generated near-wild-type levels of virus particles. This suggests that within Gag complexes, amino acids H359 and K360 are involved in stricter steric interactions than L364 and M368. Since L364 and M368 are separated by four residues and thus presumably located on the same side of the helical surface, they may initiate synergistic hydrophobic interactions to stabilize Gag association. Further analysis in the context of the protease-negative mutation D185H confirmed the key roles of amino acids H359, A361, L364, and M368 in virus assembly. Importantly, when transfected cells were subjected to Dounce homogenization and the cell lysates were treated by ultracentrifugation at 100,000 ؋ g, Gag molecules containing each of the H359A, A361V, L364A, and M368A mutations were found mainly in the supernatant fraction (S100), whereas approximately 80% of wild-type Gag proteins were found in the pellet. Therefore, these four mutations must have prevented Gag from generating large complexes.

The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10 −3 per site) for G∶U∕U∶G mismatches. The strong preference for G∶U∕U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naïve patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.A pproximately 170 million people worldwide are infected with hepatitis C virus (HCV) (1). Chronically infected individuals are at risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma (2). Although the infection can be cured with a combination of interferon-α and ribavirin, severe side effects and complicated treatment schedules can compromise therapy (3). Moreover, the response is particularly poor in patients infected with HCV genotype 1, which is the most prevalent genotype in North America and Europe (4).Several direct-acting antivirals (DAAs) targeting important viral and host factors are currently undergoing clinical evaluation. Drugs acting against the viral NS5B RNA-dependent RNA polymerase (RdRp) or NS3 protease are in advanced clinical trials, with the protease inhibitors (PIs) telaprevir and boceprevir recently gaining FDA approval. Combining interferon-α and ribavirin with either PI can produce increases in sustained virological response when compared with the combination of interferon-α and ribavirin without PI (5, 6). However, the emergence of resistance-conferring mutations can lead to treatment failure (7). Like other RNA viruses, HCV shows quasispecies-like characteristics (8). Accordingly, resistant variants are selected from a genetically highly diverse population. Resistance to PIs is rapidly selected in cell-based replicon systems and in vivo (9). Similar observations have been made with the various classes of nonnucleoside analogue inhibitors (NNIs) of NS5B (9). Moreover, mutations that decrease susceptibility to highly potent inhibitors of the RNA binding protein NS5A also emerge rapidly in the replicon system (10). In contrast, the barrier to the development of resistance to nucleoside analogue inhibitors (NIs) appears to be significantly higher. The 2′-C-methylated NIs were shown to select in v...

The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the ⌿ and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alternative conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the ⌿ hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA packaging, but how these diverse mutations affect the packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies ( This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.Retroviral particles package a dimeric RNA genome that is subsequently reverse transcribed into DNA by the virion-associated reverse transcriptase enzyme. The cis-acting RNA sequences and the trans-acting viral proteins that mediate RNA dimerization and RNA packaging have been studied extensively for several retroviruses, including human immunodeficiency virus type 1 (HIV-1) (9,20,35). It has proven particularly difficult to accurately map the RNA signals that execute these two processes, but most studies have indicated that these elements cluster within the untranslated leader region of the HIV-1 genome. In vitro studies have identified the dimerization initiation signal (DIS) as the primary dimerization signal, which acts through base pairing of a palindromic 6-nucleotide (nt) sequence motif in the exposed loop of the DIS hairpin (4,27,32,40). Nevertheless, studies with mutant virions have indicated that the in vivo situation is much more complex, suggesting the possibility of multiple accessory dimerization signals (6,21,38). The ⌿ hairpin has been implicated as the core element that mediates packaging, but there is ample evidence for the involvement of additional sequence elements (25,28). It has also been suggested that the process of RNA dimerization may be coupled to RNA encapsidation; such a coupling seems to be an elegant mechanism for ensuring that only dimeric RNA genomes are packaged (18).Huthoff and Berkhout demonstrated that the HIV-1 untranslated leader RNA can ado...

Accumulating evidence suggests that cellular lipoprotein components are involved in hepatitis C virus (HCV) morphogenesis, but the precise contribution of these components remains unclear. We investigated the involvement of apolipoprotein C1 (ApoC1) in HCV infection in the HCV pseudotyped particle system (HCVpp), in the recently developed cell culture infection model (HCVcc), and in authentic HCV isolated from viremic chimpanzees. Viral genomes associated with HCVcc or authentic HCV were efficiently immunoprecipitated by anti-ApoC1, demonstrating that ApoC1 was a normal component of HCV. The infectivities of HCVpp that had been mixed with ApoC1 and, more importantly, untreated HCVcc collected from lysates or media of infected Huh7.5 cells were directly neutralized by anti-ApoC1. Indeed, convalescent anti-HCV immunoglobulin G and anti-ApoC1 each neutralized over 75% of infectious HCVcc particles, indicating that many, if not all, infectious particles were recognized by both antibodies. Moreover, peptides corresponding to the C-terminal region of ApoC1 blocked infectivity of both HCVpp and HCVcc. Altogether, these results suggest that ApoC1 associates intracellularly via its C-terminal region with surface components of virions during viral morphogenesis and may play a major role in the replication cycle of HCV.Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus belonging to the Flaviviridae family. The HCV genome encodes a polyprotein that is co-and posttranslationally processed by host and viral proteases into at least 10 proteins, including 2 envelope glycoproteins, E1 and E2. The glycoproteins form heterodimers and are believed to be essential for HCV entry (32). Nevertheless, the mechanism by which HCV attaches to and enters the cells is not clear. For many viruses, entry into target cells is a multistep process that can involve the successive use of multiple attachment factors, receptors, and coreceptors (27). Several putative receptors have been proposed for HCV entry into cells: human tetraspanin CD81, tight junction component claudin 1, scavenger receptor class B type I, low-density lipoprotein (LDL) receptor, mannose binding lectins L-SIGN and DC-SIGN, asialoglycoprotein receptor, and glycosaminoglycans (GAGs) (3,7,10,13).Lipoproteins are synthesized mainly in the liver and intestines. HCV particles isolated from the plasma samples of HCV-infected patients and experimentally infected chimpanzees are associated with LDLs. LDL and HCV components are believed to form LDL-virus complexes, characterized by verylow-to-low buoyant density (1,2,23,29,30,34). In addition, assays characterizing the recently developed consensus JFH-1 molecular HCV clone (HCVcc) (19,33,35) provided evidence that the infectious particles generated in vitro display sedimentation velocity and buoyant density profiles similar to those described for HCV particles isolated from the plasma samples of HCV-infected patients (15, 16). Moreover, it has been proposed that apolipoproteins E and B, components of lipoproteins, we...