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A patient developed reactivation of herpes zoster infection (shingles) after a routine liver biopsy. Reactivation of herpes is often related to trauma. This entity should be considered when patients report postbiopsy pain inappropriate to the procedure. If the typical rash of shingles develops, antiviral therapy should be considered.
Interferon is currently the only available treatment for chronic hepatitis C but is effective in only about 40% of patients. Most of these responding patients relapse after short courses, so that sustained long-term viral-negative responses are uncommon (10% to 20%). However, longer treatment courses have significantly reduced relapse, thereby increasing sustained response rates. Furthermore, the combination of ribavirin with interferon shows promise for reducing relapse even further. The challenge for the future is to develop antiviral agents that will be effective in the large proportion of patients who do not respond to interferon.
The Boehringer Ingelheim compound collection was screened for inhibitors of the ATPase activity of human papillomavirus E1 helicase to develop antiviral agents that inhibit human papillomavirus (HPV) DNA replication. This screen led to the discovery of (biphenyl-4-sulfonyl)acetic acid 1, which inhibits the ATPase activity of HPV type 6 E1 helicase with a low micromolar IC(50) value. A hit-to-lead exercise rapidly converted 1 into a low nanomolar lead series.
Influenza A viruses pose a serious threat to world public health, particularly the currently circulating avian H5N1 viruses. The influenza viral nucleoprotein forms the protein scaffold of the helical genomic ribonucleoprotein complexes, and has a critical role in viral RNA replication. Here we report a 3.2 A crystal structure of this nucleoprotein, the overall shape of which resembles a crescent with a head and a body domain, with a protein fold different compared with that of the rhabdovirus nucleoprotein. Oligomerization of the influenza virus nucleoprotein is mediated by a flexible tail loop that is inserted inside a neighbouring molecule. This flexibility in the tail loop enables the nucleoprotein to form loose polymers as well as rigid helices, both of which are important for nucleoprotein functions. Single residue mutations in the tail loop result in the complete loss of nucleoprotein oligomerization. An RNA-binding groove, which is found between the head and body domains at the exterior of the nucleoprotein oligomer, is lined with highly conserved basic residues widely distributed in the primary sequence. The nucleoprotein structure shows that only one of two proposed nuclear localization signals are accessible, and suggests that the body domain of nucleoprotein contains the binding site for the viral polymerase. Our results identify the tail loop binding pocket as a potential target for antiviral development.
Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.
Picornaviruses, including foot-and-mouth disease virus (FMDV), enterovirus 71 (EV71) and encephalomyocarditis virus (EMCV), are important pathogens that cause fever, herpes, and myocarditis in humans and animals. The interplay between picornaviruses and their hosts remains enigmatic. Here we perform porcine genome-wide CRISPR/Cas9 screens and identify methionine sulfoxide reductase B3 (MSRB3) as an essential factor for FMDV. MSRB3 deficiency inhibits FMDV replication. Mechanistically, MSRB3 eliminates methionine oxidation of FMDV 3D polymerase and stabilizes its expression. Further studies show that radical SAM domain-containing protein 1 (RSAD1) catalyzes methionine oxidation of FMDV 3D polymerase and promotes its aggregation and subsequent degradation through the autophagy-lysosome pathway. Importantly, RSAD1-MSRB3-mediated redox modification also affects the stability of 3D polymerases of EV71 and EMCV, and regulates their infectivity and pathogenesis both in vitro and in vivo. Collectively, this study corroborates that RSAD1-MSRB3-mediated redox cycling of 3D polymerase plays a conserved function in modulating picornavirus infection, providing insights into viral pathogenesis and broad-spectrum antiviral development.
BACKGROUND & AIMS: HCV nonstructural protein 5A (NS5A) has been implicated in regulating cell growth and interferon response. The NS5A protein contains proline-rich regions that are highly conserved among HCV genotypes and match Src homology 3 (SH3)-binding motifs (PxxP) found in various cellular signaling molecules. METHODS: We screened for HCV NS5A interacting proteins by using the yeast 2-hybrid system and studied the functional consequence of this interaction. RESULTS: Several independent clones containing SH3 domains were isolated along with Bin1, a tumor suppressor with pro-apoptotic properties, being the most frequently identified clone. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding, in vivo co-immunoprecipitation, and confocal microscopy. Deletion and mutation analyses indicated that the SH3 binding motif of HCV NS5A and SH3 domain of Bin1 are essential for interaction. Human hepatoma (HepG2) cells lacking expression of Bin1 undergo apoptosis upon infection with adeno-Bin1. Bin1-induced apoptosis was inhibited in HepG2 cells expressing wild-type NS5A but not NS5A mutant with mutations in the SH3 binding motif. Infectious HCV genome containing mutations in the SH3 binding motif was not infectious in chimpanzees. CONCLUSIONS: Our results indicate that this interaction is implicated in productive HCV infection and may contribute to the pathogenesis of hepatocellular carcinoma. In addition, the NS5A PxxP motif may represent a novel target for antiviral development.
Phosphorylation of the P proteins of nonsegmented negative-strand RNA viruses is critical for their function as transactivators of the viral RNA polymerases. Using unphosphorylated P protein of human parainfluenza virus type 3 (HPIV3) expressed in Escherichia coli, we have shown that the cellular protein kinase that phosphorylates P in vitro is biochemically and immunologically indistinguishable from cellular protein kinase C isoform zeta (PKC-zeta). Further, PKC-zeta is specifically packaged within the progeny HPIV3 virions and remains tightly associated with the ribonucleoprotein complex. The P protein seems also to be phosphorylated intracellularly by PKC-zeta, as shown by the similar protease digestion pattern of the in vitro and in vivo phosphorylated P proteins. The growth of HPIV3 in CV-1 cells is completely abrogated when a PKC-zeta-specific inhibitor pseudosubstrate peptide was delivered into cells. These data indicate that PKC-zeta plays an important role in HPIV3 gene expression by phosphorylating P protein, thus providing an opportunity to develop antiviral agents against an important human pathogen.
N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg(2+) or Mn(2+)) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or pi-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication "fitness" of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and in N-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development.
Current treatment of HIV-1-infected individuals involves the administration of several drugs, all of which target either the reverse transcriptase or the protease activity of the virus. Unfortunately, the benefits of such treatments are compromised by the emergence of viruses exhibiting resistance to the drugs. This situation warrants new approaches for interfering with virus replication. Considering the activation of protease in the virus particles, a novel strategy to inhibit HIV-1 replication was tested targeting the dimerization domain of the protease. To test this idea, we have selected four residues from the C terminus of HIV-1 protease that map to the dimer interface region of the enzyme. We have exploited Vpr to display the peptides in the virus particles. The chimeric Vpr exhibited expression and virion incorporation similar to wildtype Vpr. The virus derived from the HIV-1 proviral DNA containing chimeric Vpr sequences registered a reduced level of replication in CEM and CEM X 174 cells in comparison with viruses containing wildtype Vpr. Similar results were observed in a single-round replication assay. These results suggest that the intravirion display of peptides targeting viral proteins is a powerful approach for developing antiviral agents and for dissecting the dynamic interactions between structural proteins during virus assembly and disassembly.
Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).
Probably the most tragic examples of virus infections that have caused the deaths of many millions of people in the past century were the influenza and AIDS pandemics. These events occurred as a direct result of the introduction of animal viruses into the human population. Similarly, mass mortalities among aquatic and terrestrial mammals were caused by the introduction of viruses into species in which they had not previously been present. It seems paradoxical that at a time when we have managed to control or even eradicate major human virus infections like polio and smallpox we are increasingly confronted with new or newly emerging virus infections of humans and animals. A complex mix of social, technological and ecological changes, and the ability of certain viruses to adapt rapidly to a changing environment, seems to be at the basis of this phenomenon. Extensive diagnostic and surveillance networks, as well as novel vaccine- and antiviral development strategies should provide us with the safeguards to limit its impact.
Influenza A virus continues to cause annual epidemics. The emergence of avian viruses in the human population poses a pandemic threat, and has highlighted the need for more effective influenza vaccines and antivirals. Development of such therapeutics would be enhanced by the use of a small-animal model that is permissive for replication of human influenza virus, and for which reagents are available to dissect the host response. A model is presented of nasal and pulmonary infection in adult inbred cotton rats (Sigmodon hispidus) that does not require viral 'adaptation'. It was previously demonstrated that animals infected intranasally with 10(7) TCID50 of a recent H3N2 influenza, A/Wuhan/359/95, have increased breathing rates. In this report it is shown that this is accompanied by weight loss and decreased temperature. Virus replication peaked within 24 h in the lung, with peak titres proportional to the infecting dose, clearing by day 3. Replication was more permissive in nasal tissues, and persisted for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia, which persisted for nearly 3 weeks. Interleukin 1 alpha (IL1alpha), alpha interferon (IFN-alpha), IL6, tumour necrosis factor alpha (TNF-alpha), GROalpha and MIP-1beta mRNA were elevated soon after infection, and expression coincided with virus replication. A biphasic response was observed for RANTES, IFN-gamma, IL4, IL10 and IL12-p40, with increased mRNA levels early during virus replication followed by a later increase that coincided with pulmonary inflammation. These results indicate that cotton rats will be useful for further studies of influenza pathogenesis and immunity.
Human immunodeficiency virus-1 (HIV-1) Vif is essential for viral evasion of host antiviral factor CEM15/APOBEC3G. We report that Vif interacts with cellular proteins Cul5, elongins B and C, and Rbx1 to form an Skp1-cullin-F-box (SCF)-like complex. The ability of Vif to suppress antiviral activity of APOBEC3G was specifically dependent on Cul5-SCF function, allowing Vif to interact with APOBEC3G and induce its ubiquitination and degradation. A Vif mutant that interacted with APOBEC3G but not with Cul5-SCF was functionally inactive. The Cul5-SCF was also required for Vif function in distantly related simian immunodeficiency virus mac. These results indicate that the conserved Cul5-SCF pathway used by Vif is a potential target for antiviral development.
This study was undertaken to determine the immune response of humans to viral capsid polypeptides of hepatitis A virus (HAV) after natural infection, which is very important for vaccine development. Antiviral capsids in 73 serum samples from patients with acute and chronic HAV infections were analyzed by immunoblotting against individual HAV capsid polypeptides (VP1, VP2, VP3, and VP4) by using a cell culture-based HAV antigen. For reference, total anti-HAV immunoglobulin G (IgG) and anti-HAV IgM were also determined by radioimmunoassay. As a result, a dominant immune response against VP1 (98% IgG, 94% IgM) was found in the acute phase. However, many other sera also reacted with VP0 (88% IgG; 35% IgM) and VP3 (81% IgG and 29% IgM). In contrast to the acute phase, anti-VP1, anti-VP0, and anti-VP3, IgG antibodies against all three viral proteins (29, 29, and 73% respectively), especially those against VP3, were found years after onset of HAV disease and over long periods in the sera of hepatitis patients. These results suggest that antibodies for capsid polypeptides are present over an extended period in the sera of HAV-infected patients. They are likely of importance in maintaining long-term immunity.
The significance of the conserved cytoplasmic tail sequence of influenza A virus neuraminidase (NA) was analyzed by the recently developed reverse genetics technique (W. Luytjes, M. Krystal, M. Enami, J. D. Parvin, and P. Palese, Cell 59:1107-1113, 1989). A chimeric influenza virus A/WSN/33 NA containing the influenza B virus cytoplasmic tail rescued influenza A virus infectivity. The transfectant virus had less NA incorporated into virions than A/WSN/33, indicating that the cytoplasmic tail of influenza virus NA plays a role in incorporation of NA into virions. However, these results also suggest that the influenza A virus and influenza B virus cytoplasmic tail sequences share common features that lead to the production of infectious virus. Transfectant virus was obtained with all cytoplasmic tail mutants generated by site-directed mutagenesis of the influenza A virus tail, except for the mutant resulting from substitution of the conserved proline residue, presumably because of its contribution to the secondary structure of the tail. No virus was rescued when the cytoplasmic tail was deleted, indicating that the cytoplasmic tail is essential for production of the virus. The virulence of the transfectant viruses in mice was directly proportional to the amount of NA incorporated. The importance of the NA cytoplasmic tail in virus assembly and virulence has implications for use in developing antiviral strategies.
Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.
Arenaviruses include Lassa fever virus (LFV) and the South American hemorrhagic fever viruses. These viruses cause severe human disease, and they pose a threat as agents of bioterrorism. Arenaviruses are enveloped viruses with a bisegmented negative-strand RNA genome whose proteomic capability is limited to four polypeptides: nucleoprotein (NP); surface glycoprotein (GP), which is proteolytically processed into GP1 and GP2; polymerase (L); and a small (11-kDa) RING finger protein (Z). Our investigators have previously shown that Z has a strong inhibitory activity on RNA synthesis mediated by the polymerase of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). In this report we show that cells transduced with a replication-deficient recombinant adenovirus expressing Z (rAd-Z) are resistant to LCMV and LFV infection. Virus cell entry mediated by LCMV or LFV GP was not affected in rAd-Z-transduced cells, but both virus transcription and replication were strongly and specifically inhibited, which resulted in a dramatic reduction in production of infectious virus. These findings open new avenues for developing antiviral strategies to combat the highly pathogenic human arenaviruses, including LFV.