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Substitution of herpes simplex virus 1 entry glycoproteins with those of saimiriine herpesvirus 1 reveals a gD-gH/gL functional interaction and a region within the gD profusion domain that is critical for fusion.

Journal of virology (2014-03-29)
Qing Fan, Richard Longnecker, Sarah A Connolly
ABSTRACT

To gain insight into the mechanism of herpesvirus entry into cells, the four glycoproteins that are necessary for herpes simplex virus (HSV) fusion were cloned from the saimiriine herpesvirus 1 (SaHV-1) genome, a primate member of the alphaherpesvirus family. Cell-cell fusion assays indicate that SaHV-1 entry glycoproteins function with the previously identified alphaherpesvirus entry receptors nectin-1 and CD155 but not with herpesvirus entry mediator (HVEM) or paired immunoglobulin-like type 2 receptor alpha (PILRα). Replacement of HSV-1 gD with the SaHV-1 gD homolog resulted in a complete loss of fusion function when coexpressed with HSV-1 gB and gH/gL. HSV-1 gD was also unable to substitute for SaHV-1 gD when coexpressed with SaHV-1 gB and gH/gL. Similarly, the gH/gL heterodimers from HSV-1 and SaHV-1 were not interchangeable. In contrast, both the HSV-1 and SaHV-1 gB homologs retained function in a heterotypic context. These results suggest that an essential interaction between homotypic gD and gH/gL occurs during both HSV-1 and SaHV-1 entry. To map the site of this homotypic interaction, we created a series of gD chimeras, focusing on the "profusion domain" (PFD) that consists of HSV-1 gD residues 261 to 305 or SaHV-1 gD residues 264 to 307. We identified a seven-amino-acid stretch (264 RTLPPPK 270) at the N terminus of the SaHV-1 gD PFD that contributes to homotypic fusion. Finally, we found that the gD receptor-binding region and PFD cannot function independently but that both can inhibit the function of wild-type gD. The herpesvirus entry machinery requires the concerted action of at least four glycoproteins; however, details of the interactions among these glycoproteins are not well understood. Like HSV-1, SaHV-1 belongs to the alphaherpesvirus subfamily. Using cell-cell fusion experiments, we found that SaHV-1 uses the entry receptors nectin-1 and CD155 but not HVEM or PILRα. By swapping the entry glycoproteins between HSV-1 and SaHV-1, we revealed a functional interaction between gD and gH/gL. To examine the homotypic interaction site on gD, we evaluated the function of a panel of HSV-1/SaHV-1 gD chimeras and identified a small region in the SaHV-1 gD profusion domain that is critical for SaHV-1 fusion. This study contributes to our understanding of the molecular mechanisms of herpesvirus entry and membrane fusion.

MATERIALS
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Product Description

Millipore
ANTI-FLAG® antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution