Dylan Harris

Identification of cellular factors associated with the 3' nontranslated region of the hepatitis C virus genome

Chronic infection by hepatitis C virus (HCV) is the leading cause of severe hepatitis that often develops into liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms underlying HCV replication and pathogenesis are poorly understood. Similarly, the role(s) of host factors in the replication of HCV remains largely undefined. Based on our knowledge of other RNA viruses, it is likely that a number of cellular factors may be involved in facilitating HCV replication. It has been demonstrated that elements within the 3′-nontranslated region (3′-NTR) of the (+) strand HCV genome are essential for initiation of (−) strand synthesis. The RNA signals within the highly conserved 3′-NTR may be the site for recruiting cellular factors that mediate virus replication/pathogenesis. However, the identities of putative cellular factors interacting with these RNA signals remain unknown. In this report, we demonstrate that an RNA affinity capture system developed in our laboratory used in conjunction with LC/MS/MS allowed us to positively identify more than 70 cellular proteins that interact with the 3′-NTR (+) of HCV. Binding of these cellular proteins was not competed out by a 10-fold excess of nonspecific competitor RNA. With few exceptions, all of the identified cellular proteins are RNA-binding proteins whose reported cellular functions provide unique insights into host cell-virus interactions and possible mechanisms influencing HCV replication and HCV-associated pathogenesis. Small interfering RNA-mediated silencing of selected 3′-NTR-binding proteins in an HCV replicon cell line reduced replicon RNA to undetectable levels, suggesting important roles for these cellular factors in HCV replication.

The FUSE Binding Protein Is a Cellular Factor Required for Efficient Replication of Hepatitis C Virus

ABSTRACT Hepatitis C virus (HCV) infection is the leading cause of liver cirrhosis and hepatocellular carcinoma and one of the primary indications for liver transplantation. The molecular mechanisms underlying the actions of host factors in HCV replication remain poorly defined. FUSE (far upstream element of the c-myc proto-oncogene) binding protein (FBP) is a cellular factor that we have identified as a binder of HCV 3′ nontranslated region (3′NTR). Mapping of the binding site showed that FBP specifically interacts with the poly(U) tract within the poly(U/UC) region of the 3′NTR. Silencing of FBP expression by small interfering RNA in cells carrying HCV subgenomic replicons severely reduced viral replication, while overexpression of FBP significantly enhanced viral replication. We confirmed these observations by an in vitro HCV replication assay in the cell-free replicative lysate, which suggested that there is a direct correlation between the cellular FBP level and HCV replication. FBP immunoprecipitation coprecipitated HCV nonstructural protein 5A (NS5A), indicating that FBP interacts with HCV NS5A, which is known to function as a link between HCV translation and replication. Although FBP is mainly localized in the nucleus, we found that in MH14 cells a significant level of this protein is colocalized with NS5A in the cytosol, a site of HCV replication. While the mechanism of FBP involvement in HCV replication is yet to be delineated, our findings suggest that it may be an important regulatory component that is essential for efficient replication of HCV.

Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides

The transactivator responsive region (TAR) present in the 5′-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA–protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNATAR) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418–428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNATAR–peptide conjugates where uptake of the PNATAR–penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC50 values in the range of 28–37 nM while IC50 for inhibition of HIV-1 replication was lowest with PNATAR–transportan-27 (0.4 μM) followed by PNATAR–tat (0.72 μM) and PNATAR–penetratin (0.8 μM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.

Insertion of a small peptide of six amino acids into the β7–β8 loop of the p51 subunit of HIV-1 reverse transcriptase perturbs the heterodimer and affects its activities

BackgroundHIV-1 RT is a heterodimeric enzyme, comprising of the p66 and p51 subunits. Earlier, we have shown that the β7-β8 loop of p51 is a key structural element for RT dimerization (Pandey et al., Biochemistry 40: 9505, 2001). Deletion or alanine substitution of four amino acid residues of this loop in the p51 subunit severely impaired DNA binding and catalytic activities of the enzyme. To further examine the role of this loop in HIV-1 RT, we have increased its size such that the six amino acids loop sequences are repeated in tandem and examined its impact on the dimerization process and catalytic function of the enzyme.ResultsThe polymerase and the RNase H activities of HIV-1 RT carrying insertion in the β7-β8 loop of both the subunits (p66INS/p51INS) were severely impaired with substantial loss of DNA binding ability. These enzymatic activities were restored when the mutant p66INS subunit was dimerized with the wild type p51. Glycerol gradient sedimentation analysis revealed that the mutant p51INS subunit was unable to form stable dimer either with the wild type p66 or mutant p66INS. Furthermore, the p66INS/p66INS mutant sedimented as a monomeric species, suggesting its inability to form stable homodimer.ConclusionThe data presented herein indicates that any perturbation in the β7-β8 loop of the p51 subunit of HIV-1 RT affects the dimerization process resulting in substantial loss of DNA binding ability and catalytic function of the enzyme.

Peptide inhibition of HIV-1: current status and future potential.

More than 2 decades of intensive research has focused on defining replication mechanisms of HIV type 1 (HIV-1), the etiologic agent of AIDS. The delineation of strategies for combating this viral infection has yielded many innovative approaches toward this end. HIV-1 is a lentivirus in the family retroviridae that is relatively small with regard to both structure and genome size, having a diploid RNA genome of approximately 9 kb, with only three major genes and several gene products resulting from alternate splicing and translational frameshifting. Most marketed drugs for treating AIDS are inhibitors of HIV-1 reverse transcriptase or protease enzymes, but new targets include the integrase enzyme, cell surface interactions that facilitate viral entry, and also virus particle maturation and assembly. The emergence of drug-resistant variants of HIV-1 has been the main impediment to successful treatment of AIDS. Thus, there is a pressing need to develop novel treatment strategies targeting multiple stages of the virus life-cycle. Research efforts aimed at developing successful means for combating HIV-1 infection have included development of peptide inhibitors of HIV-1. This article summarizes past and current endeavors in the development of peptides that inhibit replication of HIV-1 and the role of peptide inhibitors in the search for new anti-HIV drugs.