Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661 and E2661 lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion.IMPORTANCE This study reveals conformational and antigenic differences between the Δ123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to reduce the ability of HCV to elicit NAbs directed toward the conserved core domain. Future studies aimed at generating a three-dimensional structure for intact E2 containing HVR1, and the adjoining NAb epitope at residues 412 to 428, together with HVR2, will reveal how the variable regions modulate antigenic structure.

Amiloride derivatives are known blockers of the cellular Na . Here, we demonstrate that amiloride and EIPA strongly inhibit coxsackievirus B3 (CVB3) RNA replication and do not inhibit CVB3 release, in contrast to our previous findings on HRV2. Passaging of plasmid-derived CVB3 in the presence of amiloride generated mutant viruses with amino acid substitutions in position 299 or 372 of the CVB3 polymerase. Introduction of either of these mutations into the CVB3 plasmid produced resistance to amiloride and EIPA, suggesting that they act as inhibitors of CVB3 polymerase, a novel mechanism of antiviral activity for these compounds.The Picornaviridae are small, nonenveloped, positive-sense RNA viruses. They are currently divided into nine genera, three of which are significant causes of human disease, the Enteroviruses, Rhinoviruses, and Hepatoviruses (26). The picornaviral genome is approximately 7,500 nucleotides long and directs synthesis of a ϳ240-kDa polyprotein containing one structural (P1) and two nonstructural (P2 and P3) domains. The polyprotein is proteolytically cleaved by viral proteases 2A, 3C, and 3CD into three to four capsid proteins and a number of nonstructural proteins. Most of the nonstructural proteins, including the 3C and 3CD proteases, are used in viral RNA replication (reviewed in reference 2). The 2B, 2BC, and 2C proteins from the P2 domain are involved in intracellular membrane rearrangement and formation of replication complexes. The P3 proteins most directly involved in RNA synthesis are 3B (VPg), used as a protein primer for both positive-and negative-strand RNA synthesis, and 3D pol , an RNA-dependent RNA polymerase. Other P3 proteins (3AB, 3C, and 3CD) are also involved in RNA synthesis.Amiloride and its derivates are known blockers of the cellular Na ϩ /H ϩ exchanger and the epithelial Na ϩ channel (reviewed in reference 14). In addition, amiloride derivatives, principally 5-(N,N-hexamethylene)amiloride (HMA), have been shown to inhibit ion channels formed by the proteins of human immunodeficiency virus (HIV), hepatitis C virus, coronavirus, and dengue viruses (7,13,18,19,27). We have previously reported that amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and benzamil inhibit propagation of human rhinovirus 2 (HRV2; a Rhinovirus) in HeLa cells and that the antiviral activity is unlikely to be due to the inhibition of the cellular Na ϩ /H ϩ exchanger or the epithelial Na ϩ channel (9).Here, we demonstrate that these compounds inhibit replication of coxsackievirus B3 (CVB3; an Enterovirus) in HeLa cells to a greater extent than seen previously for HRV2 and via a significantly different mechanism. We show that amiloride and EIPA specifically inhibit CVB3 RNA replication, and mutations conferring resistance to both compounds are located close to the active center of the RNA-dependent RNA polymerase. MATERIALS AND METHODSChemicals, media, cells, and viruses. Amiloride, EIPA, HMA, benzamil, actinomycin D, and guanidine hydrocholoride (GHCl) were purchased from Sigma-Aldrich. Amiloride ...

). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D pol in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D pol by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg 2؉ . Docking analysis suggested a binding site for amiloride and EIPA in 3D pol , located in close proximity to one of the Mg 2؉ ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.The family Picornaviridae is a family of positive-sense RNA viruses which contains numerous human pathogens, causing poliomyelitis, myocarditis, meningitis, hepatitis, the common cold, and other diseases. The viral genomic RNA is ϳ7,500 nucleotides (nt) long and contains a 22-amino-acid peptide, VPg, covalently linked to the 5Ј end and a poly(A) tail at the 3Ј end. Genome replication occurs via synthesis of a complementary, negative-sense RNA strand, catalyzed by the viral RNA-dependent RNA polymerase, 3D pol , in association with a number of viral and host proteins. It is a complex process taking place in membrane-associated replication complexes in the cytoplasm of infected cells (reviewed in references 5, 7, and 23). The synthesis of both complementary and genomic RNA strands is initiated by attachment of two UMP nucleotides to a tyrosine residue of VPg, resulting in the production of VPgpUpU. VPg uridylylation requires a template. In the case of genomic strand synthesis, an internal stem-loop in the genomic RNA strand (cis-acting replication element [CRE]) is used as a template, with subsequent translocation of VPg-pUpU to the 3Ј end of the complementary strand and its elongation into a full-length genomic strand (9,15,16,26). The complementary strand synthesis does not absolutely depend on CRE-it can also be templated by the poly(A) tail of the genomic strand (9,15,16,26).VPg uridylylation and RNA elongation have been reproduced successfully in vitro by use of purified components. VPg uridylylation assays require 3D pol , VPg, CRE or poly(A), UTP, and Mg 2ϩ or Mn 2ϩ (19,20), with CRE-templated reaction stimulated by viral proteins 3CD or 3C (18, 19), whereas an elongation assay mix contains an RNA primer instead of VPg (21).Coxsackievirus B3 (CVB3) is a picornavirus responsible for 14 to 32% of human myocarditis cases (1). Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit CVB3 propagation in cell culture by inhibiting viral genome replication (11). Two amino acid substitutions in 3D pol (S299T and A372V) confer partial resistance of the virus to the compounds, suggesting that amiloride analogues may act as inhibitors of CVB3 3D pol (11). Here we show that amiloride and EIPA inhibit VPg uridylylation and RNA elongation by CVB3 3D pol in vitro, acting as competitive inhibitors with respect to...