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 More than a decade ago, hepatitis C virus (HCV) 1 was discovered as the major causative agent of parenteral non-A non-B hepatitis (1, 2), and the number of HCV-infected individuals worldwide is currently estimated at 170 million. Although some infected individuals are able to clear the virus without treatment, most infections persist leading in about 50% of all cases to chronic hepatitis, which may develop into chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. The main therapeutic regimen currently in clinical use is a combination treatment consisting of high doses of interferon-␣ and the nucleoside analog ribavirin, and a large percentage of patients receiving this regimen are not responsive. These stark facts underscore the importance of expediently developing new strategies to combat this viral infection.The hepatitis C virus is a positive strand RNA virus of the flaviviridae family having a genome roughly 9.6 kb in length (3). The RNA genome contains a single ORF that encodes a polyprotein of ϳ3000 amino acids that is processed by a combination of host-and virus-encoded proteases (4) into four structural proteins (core, E1, E2, and p7) and six nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. Replication of HCV is initiated at the 3Ј-NTR of the RNA genome, although little is known about the mechanism of initiation or the factors required for this process. The NS5B protein is the RNA-dependent RNA polymerase, or HCV replicase, responsible for replication of the HCV genome whose structure and enzymatic activities have been well characterized (5, 6). Although it has been established that NS5B can initiate primer-independent RNA synthesis, the mechanistic details of this process remain the source of some controversy (7-9). Hong et al. (10) have proposed that a structural motif within NS5B positions the terminal nucleotides of the genome in such a way that de novo synthesis is initiated from the 3Ј-end of the genome. The initiation of HCV virus replication may very well involve the aid of cellular factors in...

The neamine part of the aminoglycoside antibiotic neomycin B was conjugated to a 16 mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. Attachment of the neamine core allows cellular uptake of the PNA and results in potent inhibition of HIV-1 replication. The polycationic neamine moiety imparts greater solubility to the PNA and also confers a unique RNA cleavage property to the conjugate which is specific to its target site and functional at physiological concentrations of Mg(2+). These properties suggest a potential therapeutic application for this class of compounds.

We have earlier reported that anti-TAR PNA conjugated with the membrane-transducing peptide transportan inhibits transactivation of the HIV-1 LTR resulting in decreased production of HIV-1 virions by chronically infected H9 cells (N., Kaushik, A., Basu, P., Palumbo, R.L., Myers, V.N., Pandey, 2002. Anti-TAR polyamide nucleotide analog conjugated with a membrane permeating peptide inhibits HIV-1 production. J. Virol. 76, 3881-3891). In this study, we have found that the PNA(TAR)-transportan conjugate is efficiently internalized by cells and kinetics analysis reveals a sigmoidal curve with a cooperativity index of 6, indicating very rapid cellular uptake. Additionally, analysis of uptake at varying temperatures or in the presence of phenylarsine oxide revealed that the mechanism of uptake is neither receptor-dependent nor occurs via endocytosis. We also found that the PNA(TAR)-transportan conjugate exhibits potent virucidal activity as HIV-1 virions pretreated with the conjugate were rendered noninfectious, suggesting that the conjugate may also permeate the virus envelope. The anti-HIV-1 virucidal activity of the conjugate may be useful either in topical formulations designed to block HIV-1 infection or as a prophylactic agent for inactivation of HIV-1 in the circulating plasma prior to attachment and entry.

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.