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Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB-1), occludin (OCLN), and claudin-1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh-7.5) or CLDN6 (HuH6). Huh-7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6-dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1-specific neutralizing antibodies (Abs) fully abrogated infection of Huh-7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti-CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1-specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB-1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. Conclusion: These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1-specific therapeutic strategies. (HEPATOLOGY 2014;59:24-34) H epatitis C virus (HCV) is a highly variable, plus-strand RNA virus of the family Flaviviridae and a leading cause of liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. 1 The pronounced variability of HCV facilitates viral immune evasion and is attributable to enormous replication rate and error-prone RNA replication. Seven genotypes (GTs) and multiple subtypes are known, with genetic diversity being in the order of more than 30% between individual viral GTs. 2 Although the basic genome structure is conserved among HCV GTs, there are remarkable genotypedependent differences with regard to treatment response and pathophysiology of the infection. For instance, GTs 1 and 4 exhibit inferior response rates, when compared with GTs 2 and 3, in interferon-based therapy regimens, and GT3 virus infection shows a particularly strong association with liver steatosis. 3,4 Abbreviations: Abs, antibodies; CHC, chronic hepatitis C; CLDN, claudin; FACS, fluorescence-activated cell sorting; GTs, genotypes; HCV, hepatitis C virus; HCVcc, cell culture-derived HCV; HCVpp, HCV pseudoparticles; IgG, immunoglobulin G; miR-122, microRNA 122; MLV, murine leukemia virus; mRNA, messenger ...

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Lentiviral budding is governed by group-specific antigens (Gag proteins) and proceeds in the absence of cognate viral envelope proteins, which has been exploited to create pseudotypes incorporating envelope proteins from nonlentiviral families. Here, we report the generation of infectious lentiviral pseudoparticles incorporating human respiratory syncytial virus (hRSV) F protein alone (hRSV-Fpp) or carrying SH, G, and F proteins (hRSV-SH/G/Fpp). These particles recapitulate key infection steps of authentic hRSV particles, including utilization of glycosaminoglycans and low-pH-independent cell entry. Moreover, hRSV pseudoparticles (hRSVpp) can faithfully reproduce phenotypic resistance to a small-molecule fusion inhibitor in clinical development (BMS-433771) and a licensed therapeutic F protein-targeting antibody (palivizumab). Inoculation of several human cell lines from lung and liver revealed more than 30-fold differences in susceptibility to hRSVpp infection, suggesting differential expression of hRSV entry cofactors and/or restriction factors between these cell types. Moreover, we observed cell-type-dependent functional differences between hRSVpp carrying solely F protein or SH, G, and F proteins with regard to utilization of glycosaminoglycans. Using hRSVpp, we identified penta-O-galloyl-␤-D-glucose (PGG) as a novel hRSV cell entry inhibitor. Moreover, we show that PGG also inhibits cell entry of hRSVpp carrying F proteins resistant to BMS-433771 or palivizumab. This work sheds new light on the mechanisms of hRSV cell entry, including possible strategies for antiviral intervention. Moreover, hRSVpp should prove valuable to dissect hRSV envelope protein functions, including the interaction with cell entry factors. IMPORTANCELentiviral pseudotypes are highly useful to specifically dissect the functions of viral and host factors in cell entry, which have been exploited for numerous viruses. Here, we successfully created hRSVpp and show that they faithfully recapitulate key characteristics of parental hRSV cell entry. Importantly, hRSVpp accurately mirror hRSV resistance to small-molecule fusion inhibitors and clinically approved therapeutic antibodies. Moreover, we observed highly different susceptibilities of cell lines to hRSVpp infection and also differences between hRSVpp types (with F protein alone or with SH, G, and F proteins) in regard to cell entry. This indicates differential expression of host factors determining hRSV cell entry between these cell lines and highlights the fact that the hRSVpp system is useful to explore the functional properties of hRSV envelope protein combinations. Therefore, this system will be highly useful to study hRSV cell entry and host factor usage and to explore antiviral strategies targeting hRSV cell entry. H uman respiratory syncytial virus (hRSV) is an enveloped, nonsegmented, negative-strand RNA virus classified in the family Paramyxoviridae. It causes upper and lower respiratory tract disease in children and elderly and immunosuppressed persons (1-3). hRSV po...

Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available.

We recently uncovered a systemic error in our report describing lentiviral pseudoparticles decorated with functional respiratory syncytial virus (RSV) envelope proteins. It has been described that lengthy cytoplasmic tails of viral envelope proteins can preclude efficient lentiviral pseudotyping. Considering this, we initially created a full-length and a C-terminally truncated variant of the RSV F protein. Initial experiments comparing these constructs did not reveal gross differences in lentiviral pseudotyping between full-length and truncated RSV F proteins. Now we have uncovered an early mix-up between these constructs. Consequently, all experimental data presented in Fig. 1 to 4 were obtained with a truncated RSV F protein, which we now designate RSV F trc. Moreover, RSV F-derived constructs such as RSV F K394R (Fig. 2 and 4) and RSV F K272E (Fig. 2 and 4) were created in the context of this truncation mutant and are now designated RSV F trc K394R and RSV F trc K272E. Page 3068, Fig. 1: We have now compared lentiviral pseudotyping between fulllength RSV F and the RSV F trc constructs and summarize these novel data in Fig. 1 given below. The figure below complements the published data by providing a comparison of the functioning of RSV F trc and RSV F in lentiviral pseudotyping. Our comparison revealed a trend that RSV F trc constructs produce slightly higher titers of lentiviral pseudotypes than RSV F (Fig. 1B and C). This trend was independent of RSV F constructs being expressed alone or in combination with additional RSV envelope proteins, including small hydrophobic (SH) proteins and glycoprotein G (G) (Fig. 1B). Moreover, this trend was also seen for RSV F constructs carrying a single point mutation (K272E) conferring resistance to palivizumab, an F protein-targeting monoclonal antibody, or resistance to a membrane fusion inhibitor (K394R; bis-hydrochloride monohydrate [BMS-433771]) (Fig. 1C). Since the truncation does not affect the ectodomain of the RSV F protein, we assumed that the function of the truncation mutant RSV F trc closely phenocopies the behavior of the wild-type RSV F in terms of entry factor usage and inhibition by antibodies and small molecules. To support this, we confirmed that both proteins share comparable susceptibility to palivizumab (Fig. 1D) and the BMS-433771 membrane fusion inhibitor (Fig. 1E). Page 3070, Fig. 3A: In the displayed experiment, we used heparin at a concentration of 1 g/ml, not 5 g/ml. This error has no influence on the general conclusion derived from these data.

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