The cytoplasmic tail of the influenza A virus M2 protein is highly conserved among influenza A virus isolates.The cytoplasmic tail appears to be dispensable with respect to the ion channel activity associated with the protein but important for virus morphology and the production of infectious virus particles. Using reverse genetics and transcomplementation assays, we demonstrate that the M2 protein cytoplasmic tail is a crucial mediator of infectious virus production. Truncations of the M2 cytoplasmic tail result in a drastic decrease in infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding events, and a reduction in budding efficiency. The M1 protein binds to the M2 cytoplasmic tail, but the M1 binding site is distinct from the sequences that affect infectious virus particle formation. Influenza A virus strains A/Udorn/72 and A/WSN/33 differ in their requirements for M2 cytoplasmic tail sequences, and this requirement maps to the M1 protein. We conclude that the M2 protein is required for the formation of infectious virus particles, implicating the protein as important for influenza A virus assembly in addition to its well-documented role during virus entry and uncoating.A productive virus infection begins with the release of the viral genome into a permissive host cell. The viral genome can then be replicated, transcribed, and translated. To complete a productive life cycle, a virus must temporarily package its genome into a particle that facilitates the transport and release of the viral genetic material into another permissive cell. For most human viral pathogens, the mechanisms responsible for genome incorporation and virus assembly are yet to be clearly defined.Budding of enveloped viruses requires the action of a viral protein capable of mediating curvature of the lipid bilayer, followed by fission of the new viral membrane from the cellular membrane (43, 61). The Gag protein of human immunodeficiency virus (12,19); the matrix proteins of influenza A virus (20, 31), vesicular stomatitis virus (VSV) (33), and simian virus 5 (63); and the VP40 proteins of Ebola (36,51) and Marburg viruses (71) have been shown to mediate budding of virus-like particles (VLPs) in the absence of other viral proteins.While matrix proteins alone can drive the process of VLP formation, coexpression of other viral proteins can improve the efficiency of VLP formation. In the case of Ebola and Marburg viruses, coexpression of the viral glycoprotein with VP40 enhances the efficiency of VLP formation over that observed with VP40 alone (3,35,71). The extracellular, membrane-proximal region of the VSV G protein has been shown to increase the budding efficiency of VSV (58). The budding of a fully infectious viral particle requires the coordinated action of multiple viral proteins with the lipid bilayer, viral genome, and most likely host proteins. This concerted event increases the efficiency of particle release over that demonstrated in most VLP systems (35,58).Influenza A viruses are classified...

The M 2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M 2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M 2 proteins with truncated C termini. Deletion of 28 amino acids (M 2 Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M 2 protein in trans restored the replication of the M 2 truncated virus. Although the M 2 Stop70 virus particles were similar to wild-type virus in morphology, the M 2 Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M 2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

Specific inhibitors of hepatitis C virus (HCV) replication that target the NS3/4A protease (e.g., VX-950) or the NS5B polymerase (e.g., R1479/R1626, PSI-6130/R7128, NM107/NM283, and HCV-796) have advanced into clinical development. Treatment of patients with VX-950 or HCV-796 rapidly selected for drug-resistant variants after a 14-day monotherapy treatment period. However, no viral resistance was identified after monotherapy with R1626 (prodrug of R1479) or NM283 (prodrug of NM107) after 14 days of monotherapy. Based upon the rapid selection of resistance to the protease and nonnucleoside inhibitors during clinical trials and the lack of selection of resistance to the nucleoside inhibitors, we used the replicon system to determine whether nucleoside inhibitors demonstrate a higher genetic barrier to resistance than protease and nonnucleoside inhibitors. Treatment of replicon cells with nucleoside inhibitors at 10 and 15 times the 50% effective concentration resulted in clearance of the replicon, while treatment with a nonnucleoside or protease inhibitor selected resistant colonies. In combination, the presence of a nucleoside inhibitor reduced the frequency of colonies resistant to the other classes of inhibitors. These results indicate that the HCV replicon presents a higher barrier to the selection of resistance to nucleoside inhibitors than to nonnucleoside or protease inhibitors. Furthermore, the combination of a nonnucleoside or protease inhibitor with a nucleoside polymerase inhibitor could have a clear clinical benefit through the delay of resistance emergence.Hepatitis C virus (HCV) is a positive-strand RNA virus that is a member of the Hepacivirus genus within the Flaviridae family. There are an estimated 170 million individuals chronically infected with HCV worldwide, which amounts to almost 3% of the global population (1). In the United States, an estimated 20,000 new HCV infections occurred in 2005, adding to the approximately 4 million individuals previously infected with HCV (2, 38). Liver cirrhosis, as a result of HCV infection, is currently the leading reason justifying liver transplantation; however, reinfection occurs immediately posttransplantation and can result in graft loss (39).The current treatment of pegylated alpha interferon in combination with ribavirin results in a sustained viral response in approximately 50% of HCV patients infected with genotype (GT) 1 virus, the most prevalent GT worldwide. Therefore, a specific HCV antiviral therapy is highly desirable. Viral proteases and viral polymerases have been validated as clinically effective targets for a number of different viruses, including human immunodeficiency virus, hepatitis B virus, and herpesviruses (6,7,14,15). Two potential drug targets encoded by HCV are the NS3/4A serine protease and the NS5B RNA-dependent RNA polymerase (5). Several anti-HCV compounds that inhibit the activity of either the NS3/4A protease or the NS5B RNA-dependent RNA polymerase have resulted in decreased viral loads when administered to HCV-infected pat...

Hepatitis C virus (HCV)4 constitutes a global health problem. Current therapies are unable to effectively eliminate viral infection in a significant number of patients. The RNA-dependent RNA polymerase (RdRp) of HCV NS5B is an attractive target for the development of orally bioavailable small molecule inhibitors (1, 2). The structure of the NS5B apoenzyme and the NS5B-RNA complex reveals the characteristic right hand architecture of polymerase enzymes, comprising three distinct domains (palm, thumb, and finger) encircling the enzyme active site located in the palm domain (3-6). The structural and biochemical characterization of HCV NS5B polymerase can provide a basis for drug design efforts, and the elucidation of the mechanism of inhibition can guide the optimization of inhibitor efficiency against wild-type and resistant mutants.Among the extensively investigated non-nucleosides documented to inhibit the RdRp activity of HCV NS5B, derivatives of various benzofuran and benzothiadiazine have been reported to bind to allosteric binding sites in the palm domain of NS5B (7,8). The palm domain, whose geometry is conserved in virtually all DNA and RNA polymerases, contains catalytic aspartic acids responsible for the nucleotidyl transfer reaction. The benzofuran compound HCV-796 has been shown to have significant antiviral effects in patients chronically infected with HCV (9, 10). In addition, two series of compounds based on the thiophene and benzimidazole scaffolds have been reported to inhibit NS5B by binding to two different binding pockets in the thumb domain of NS5B (11,12). The thumb domain is connected to the palm domain by a ␤-hairpin termed the primer grip motif. The C-terminal region of the thumb protrudes toward the active site (3). The thumb binding inhibitors have been proposed to inhibit the RdRp activity of NS5B, perhaps by interfering with template/primer interaction and conformational dynamics of the protein (13,14).Despite the elucidation of a number of NNIs that bind to the thumb and palm binding sites, the mechanism by which NNIs cause inhibition of RNA synthesis is unclear. Also, our understanding of the kinetics of NNI interaction with NS5B, the role of NNI binding and kinetics for inhibition, and the inhibitor efficacy on NS5B-resistant mutations remains incomplete. The four representative palm-and thumb-binding NNIs selected in this study have been reported to effectively inhibit replication of subgenomic replicons with low toxicity. Noncompetitive inhibition of NS5B polymerase activity with respect to NTPs has been reported (2, 15, 16). Based on co-crystallization studies with NS5B, it has been proposed that allosteric inhibitors may lock the NS5B protein in an inactive formation by binding tightly to the protein (16,17). It is important to understand how the binding affinity relates to inhibition potency and resistance to HCV inhibition. Because the intrinsic potency of slowly binding compounds can be underestimated in the short time □ S The on-line version of this article (available at htt...

In vitro, telaprevir selects subtype-specific resistance pathways for hepatitis C virus GT-1a and GT-1b, as described to have occurred in patients. In GT-1a, the HCV-796 resistance mutation C316Y has low replication capacity (7%) that can be compensated for by the emergence of the mutation L392F or M414T, resulting in an increase in replication levels of >10-fold.The current standard of care for hepatitis C virus (HCV)-infected patients involves a treatment regimen of pegylated alpha interferon in combination with ribavirin, which results in a sustained viral response of approximately 50% for genotype 1 (GT-1)-infected patients (1, 11). There is a clear medical need for more efficacious therapies, and to this effect, a number of novel specific antiviral compounds are currently in preclinical and clinical development. A majority of these compounds inhibit the enzymatic activity of either the NS3/4A serine protease or the NS5B RNA-dependent RNA polymerase.One factor that may limit the clinical efficacy of specific HCV antiviral drugs is the development of resistance. HCV presents a number of features that make drug resistance likely to occur upon treatment, such as the following: (i) the NS5B polymerase lacks proofreading activity, which results in the introduction of random mutations during the replication of the genomic RNA; (ii) HCV replicates as a genetic population known as a quasispecies that allows quick adaptation of the viral population upon changes in the environment (12); (iii) HCV produces a large number of infectious particles (up to 10 12 ) per day, which means that each genetic variant made during RNA replication may be packaged into an infectious viral particle and can quickly spread (15); and (iv) the short half-life of the HCV genome, as estimated for the circulating virus (14) and calculated for the HCV replicon (3), is such that a variant present at low prevalence within the quasispecies can quickly become the dominant sequence if it offers a selective advantage. Resistance to specific HCV inhibitors in vitro has been well characterized through the use of the HCV GT-1b replicon system, and these studies have been predictive of the amino acid substitution(s) selected in HCV-infected patients upon drug treatment (4, 7-10, 13). For example, for the NS3/4A protease inhibitor telaprevir and the nonnucleoside polymerase inhibitor HCV-796, the resistance mutations identified in vitro (NS3 substitutions at residues T54 and A156 for telaprevir and an NS5B substitution at residue C316 for HCV-796) were also identified in GT-1b-treated patients (4, 5, 16).One limitation of the majority of the replicon resistance studies reported to date is that only a single HCV subtype, GT-1b, has been used. HCV subtypes can vary by up to 25% at the nucleotide level, and this variability may lead to subtypespecific differences in the resistance profiles. In fact, subtypespecific resistance profiles for HCV-infected patients treated with telaprevir have been described previously. Substitutions at NS3 residues V36 and R155...