Claire L. Brimacombe*, Joe Grove*, Luke W. Meredith, Ke Hu, Andrew J. Syder, Maria
Victoria Flores, Jennifer M. Timpe, Sophie E. Krieger, Thomas F. Baumert, Timothy L.
Tellinghuisen, Flossie Wong-Staal, Peter Balfe and Jane A. McKeating (* - joint first authorship). Journal of Virology, 85:596-605 2011.
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We have previously reported that HCV strain JFH-1 could transmit via cell-free and cell-to-cell routes in vitro. In this study we demonstrate that a diverse panel of chimeric HCVcc viruses representing the seven major genotypes readily transmit in the presence of patient derived antibodies that are able to neutralize cell-free virus infectivity, demonstrating that direct cell-to-cell routes of transmission is a universal property of HCV. We extend these findings by demonstrating that disruption of HCV particle assembly or physical separation of target and producer cells ablates transmission, indicating that intact virions transfer via cell-cell contacts. HCV entry is a complex process that is dependent on host cell molecules: scavenger receptor BI (SR-BI), tetraspanin CD81 and the tight junction proteins Claudin-1 and Occludin.
In this paper we demonstrate that CD81 and both tight junction protein entry factors were required for cell-free and cell-to-cell transmission. However, antibodies and small molecule entry inhibitors targeting SR-BI preferentially inhibit cell-to-cell transmission. Furthermore, increased SR-BI expression in the target cell augments cell-to-cell transmission, suggesting that SR-BI expression levels limit cell-to-cell transmission. These findings shed new light on the strategies employed by HCV to evade the humoral immune response and have major implications for the development of targeted anti-glycoprotein immune therapies and highlight the importance of targeting virus receptors, in particular SR-BI, as a method to curtail HCV transmission and immune evasion.
Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact dependent transmission, in this study we use a novel infectious co-culture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy to escape host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody resistant cell-to-cell transmission. HCV entry is a multi-step process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight junction components Claudin-1 and Occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in virus cell-to-cell transmission, with SR-BI specific antibodies and small molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver.
The infectious co-culture assay.
(A) Summary of the infectious co-culture procedure. (B) Representative data; i) co-culture medium was titrated in a standard infectious assay allowing cell-free infectivity to be quantified as foci forming units per ml (FFU/ml), the histogram displays the infectivity of cell-free virus in the presence of 10µg/ml control or nAb (anti-E2 mAb 9/27). ii) Monitoring newly infected target cell frequency by flow cytometry allows quantification of co-culture transmission. CMFDA labeled producer cells appear to the right of the plot, whereas NS5A positive cells appear in the upper quadrants, the frequency of infected targets is annotated above the appropriate cell population (upper left quadrant). Representative plots of co-culture transmission in the presence of control or nAb are shown.
Diverse HCVcc transmission.
Huh-7.5 cells were electroporated with a panel of chimeric JFH-1 HCV RNAs, where the infecting genotype is depicted prior to the strain nomenclature. 72h post electroporation the cells were labeled with CMFDA and co-cultured with Huh-7.5 target cells in the presence of control or cross reactive patient pooled HCV+ IgG at 300µg/ml for 48h. Extracellular media was collected and the levels of infectious virus quantified (A) and the % neutralization by patient HCV+ IgG determined (B). The stacked histogram displays nAb resistant cell-to-cell (white bars) and nAb sensitive transmission (black bars) for each virus (C). All treatments were performed in duplicate and the error bars indicate the standard deviation. The data set is representative of 3 experiments.
Neutralizing antibody resistant HCV transmission requires cell contact
The standard H77/JFH co-culture assay was modified either by performing the co-culture at low (0.25x) seeding density or by seeding the target and producer cells on opposing faces of the culture well using a transwell insert. In both cases a 1:1 target:producer ratio was maintained. The frequency of infected target cells in the absence (nAb sensitive) or presence (nAb resistant) of mAb 9/27 at 4µg/ml is shown. The experiments were performed in duplicate and error bars indicate the standard deviation. The data set is representative of 4 experiments.
In summary these data show the key role of cell-cell contacts in HCV spreading infection and the dependance of these events on the HCV co-receptors. These data contrast with our previous report (click here) reflecting the improvements to the assay format which has resulted in improved sensitivity and lower rates of false positives.