Glycoprotein M and ESCRT in herpes simplex virus type 1 assembly

Herpes simplex virus type 1 (HSV-1) has a large linear double-stranded DNA genome in an icosahedral capsid shell, a cell-derived lipid envelope and a proteinaceous tegument layer. There are over fifty viral proteins and many host proteins identified in HSV-1 virions. The final formation of mature virus particles requires the membrane wrapping of tegumented capsids in the cytoplasm, a process termed secondary envelopment. This process involves the coordination of numerous viral and cellular proteins and results in double-membrane structures with enveloped virions contained within cellular vesicles. Mature viruses are then released through the fusion of these virion-containing vesicles and plasma membranes. This thesis describes investigation into the functions of viral glycoprotein M (gM) and the cellular Endosomal Sorting Complexes Required for Transport (ESCRT) in secondary envelopment. Firstly, it has been reported that gH/L can be efficiently internalised and targeted to the TGN by the co-expression of gM in transfection assays. In order to examine the role of gM in guiding the localisation of viral proteins in infected cells, a HSV-1 gM deletion virus (∆gM), and its revertant virus were constructed. The major phenotype demonstrated was that the absence of gM caused the internalisation of cell surface gH/L to be inhibited and higher levels of gH/L to be observed on the cell surface. Further, lower levels of gH/L were detected in purified ∆gM virions, which was in agreement with the delayed entry kinetics, smaller plaque sizes and greater replication deficits at low multiplicity of infection observed in ∆gM infected cells. Over all the results presented in this thesis demonstrate that in infected cells the efficient incorporation of gH/L into virions relies on the function of gM in HSV-1. Secondly, during HSV-1 secondary envelopment the budding and scission of the viral envelope from the host membrane share topological similarities with the formation of intraluminal vesicle in multivesicular bodies, retrovirus budding, and abscission at the end of cytokinesis, processes that require the cellular ESCRT machinery. There are four multiprotein ESCRT complexes and many associated proteins involved in their regulation. It has been previously shown that the ESCRT-III complex and a functional ATPase VPS4 are required for HSV-1 secondary envelopment, but different from the strategy utilised by HIV-1, the recruitment of ESCRT during HSV-1 infection is independent of TSG101 and/or ALIX. Data presented in this thesis demonstrate that CHMP4A/B/C proteins of the ESCRT-III complex are specifically crucial for HSV-1 secondary envelopment. Simultaneous depletion of CHMP4A/B/C proteins significantly inhibited HSV-1 replication. Ultrastructure analysis revealed that there were virtually no extracellular virions in CHMP4A/B/C depleted samples while more free capsids were observed in the cytoplasm, although the nuclear capsids and primary envelopment events appeared to be normal. In order to identify interactions between HSV-1 and ESCRT proteins, 22 HSV-1 tegument proteins were cloned and tested against a panel of ESCRT and ESCRT-associated proteins in yeast two-hydrid assays. Analysis of positive hits from yeast two-hybrid interaction screens using GST pull-down, co-immunoprecipitation and protein co-localisation assays have validated interactions of pUL47 with CC2D1A/1B, CIN85, CHMP6 and ALIX, pUL46 and pUL49 with CC2D1A/1B and CIN85, and pUL16 with CC2D1A/1B. Furthermore, the newly identified ESCRT associated proteins CC2D1A and CC2D1B have been detected in purified virions. The role of the identified ESCRT proteins in HSV-1 replication has been investigated using siRNA depletion. Unfortunately siRNA depletions of the various ESCRT candidates individually or in combinations did not show any significant effect on HSV-1 replication. Overall these data suggest that unlike HIV and other retroviruses, HSV-1 has evolved multiple parallel pathways to hijack the ESCRT machinery to facilitate its replication, particularly, through the interactions that lead directly to the recruitment of CHMP4A/B/C proteins. Disruption of some of these pathways did not prevent HSV-1 replication in tissue culture, suggesting any one potential pathway is sufficient for ESCRT recruitment to sites of HSV-1 assembly.This work was supported by the Royal Society (UF090010) and the MRC Grant (G0700129). PhD studentship funding for Yudan Ren was offered jointly by Cambridge Overseas Trust, Department of Pathology, Robinson College, the Board of Graduate Studies and Mr. Xuefeng He