Rajeev Banerjee

Specific Interaction of Hepatitis C Virus Protease/Helicase NS3 with the 3′-Terminal Sequences of Viral Positive- and Negative-Strand RNA

ABSTRACT The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and ΔpNS3 (helicase domain only) and examined their abilities to interact with the 3′-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3′ untranslated region (UTR). The results presented here demonstrate that NS3 (and ΔpNS3) interacts efficiently and specifically with the 3′-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5′-terminal RNA sequences. The interaction of NS3 with the 3′-terminal negative strand [called 3′(−) UTR127] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3′ terminus (nucleotides 5 to 20 from the 3′ end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and ΔpNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3′-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3′(−) UTR binding, specific interaction of NS3 (or ΔpNS3) with the 3′-terminal sequences of the positive-strand RNA [3′(+) UTR] appears to require the entire 3′(+) UTR of HCV. Deletion of either the 98-nucleotide 3′-terminal conserved region or the 5′ half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3′-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

Amino-terminal region of poliovirus 2C protein is sufficient for membrane binding

The poliovirus-encoded, membrane associated polypeptide 2C is required for viral replication. We have previously established that, while the 2C protein lacks a defined membrane binding domain, the N-terminal region containing a putative amphipathic helix plays an important role in membrane binding both in vivo and in vitro. In order to determine whether the N-terminal region is sufficient for membrane binding, we have made fusion constructs between this region of 2C (amino acids 1-72 and 1-88) and a soluble protein, chloramphenicol acetyltransferase (CAT). The ability of CAT and the fusion polypeptides to bind to membranes was examined by in vitro translation in the presence of microsomal membrane. While CAT was found in the soluble fraction, both 2C/CAT fusion constructs (1-72/CAT and 1-88/CAT) were membrane associated, suggesting that the N-terminal region of 2C was sufficient to impart membrane binding. To confirm these results in vivo, CAT, 1-72/CAT, and 1-88/CAT were expressed in HeLa cells and their localization was examined using indirect immunofluorescence. Results presented here demonstrate that, while CAT is expressed throughout the cell, 1-72/CAT and 1-88/CAT constructs are capable of localizing to the endoplasmic reticulum (ER) area in transfected cells in the absence of other poliovirus proteins. These results suggest that the first 72 amino acids of 2C contain a membrane binding domain that is capable of targeting soluble proteins to the ER region of the cell.

Regulation of Poliovirus 3C Protease by the 2C Polypeptide

ABSTRACT Poliovirus-encoded nonstructural polypeptide 2C is a multifunctional protein that plays an important role in viral RNA replication. 2C interacts with both intracellular membranes and virus-specific RNAs and has ATPase and GTPase activities. Extensive computer analysis of the 2C sequence revealed that in addition to the known ATPase-, GTPase-, membrane-, and RNA-binding domains it also contains several “serpin” (serine protease inhibitor) motifs. We provide experimental evidence suggesting that 2C is indeed capable of regulating virus-encoded proteases. The purified 2C protein inhibits 3Cpro-catalyzed cleavage of cellular transcription factors at Q-G sites in vitro. It also inhibits cleavage of a viral precursor by the other viral protease, 2Apro. However, at least three cellular proteases appear not to be inhibited by 2C in vitro. The 2C-associated protease inhibitory activity can be depleted by anti-2C antibody. A physical interaction between 2C and His-tagged 3Cpro can be demonstrated in vitro by coimmunoprecipitation of 2C with anti-His antibody. Deletion analysis suggests that the 2C central and C-terminal domains that include several serpin motifs are important for 3Cpro-inhibitory activity. To examine the 2C protease inhibitory activity in vivo, stable HeLa cell lines were made that express 2C in an inducible fashion. Infection of 2C-expressing cells with poliovirus led to incomplete (or inefficient) processing of viral precursor polypeptides compared to control cell lines containing the vector alone. These results suggest that 2C can negatively regulate the viral protease 3Cpro. The possible role of the 2C protease inhibitory activity in viral RNA replication is discussed.