Binding of Herpes Simplex Virus 1 UL20 to GODZ (DHHC3) Affects Its Palmitoylation and Is Essential for Infectivity and Proper Targeting and Localization of UL20 and Glycoprotein K

Abstract

Herpes simplex virus 1 (HSV-1) UL20 plays a crucial role in the envelopment of the cytoplasmic virion and its egress. It is a nonglycosylated envelope protein that is regulated as a γ1 gene. Two-hybrid and pulldown assays demonstrated that UL20, but no other HSV-1 gene-encoded proteins, binds specifically to GODZ (also known as DHHC3), a cellular Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein. A catalytically inactive dominant-negative GODZ construct significantly reduced HSV-1 replication and affected the localization of UL20 and glycoprotein K (gK) and their interactions but not glycoprotein C (gC). GODZ is involved in palmitoylation, and we found that UL20 is palmitoylated by GODZ using a GODZ dominant-negative plasmid. Blocking of palmitoylation using 2-bromopalmitate (2-BP) affected the virus titer and the interaction of UL20 and gK but did not affect the levels of these proteins. In conclusion, we have shown that binding of UL20 to GODZ in the Golgi apparatus regulates trafficking of UL20 and its subsequent effects on gK localization and virus replication. We also have demonstrated that GODZ-mediated UL20 palmitoylation is critical for UL20 membrane targeting and thus gK cell surface expression, providing new mechanistic insights into how UL20 palmitoylation regulates HSV-1 infectivity. HSV-1 UL20 is a nonglycosylated essential envelope protein that is highly conserved among herpesviruses. In this study, we show that (i) HSV-1 UL20 binds to GODZ (also known as DHHC3), a Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein; (ii) a GODZ dominant-negative mutant and an inhibitor of palmitoylation reduced HSV-1 titers and altered the localization of UL20 and glycoprotein K; and (iii) UL20 is palmitoylated by GODZ, and this UL20 palmitoylation is required for HSV-1 infectivity. Thus, blocking of the interaction of UL20 with GODZ, using a GODZ dominant-negative mutant or possibly GODZ shRNA, should be considered a potential alternative therapy in not only HSV-1 but also other conditions in which GODZ processing is an integral component of pathogenesis.

The Absence of DHHC3 Affects Primary and Latent Herpes Simplex Virus 1 Infection

“…We have demonstrated previously that UL20 binds to both GODZ (6) and HSV-1 gK (23). In addition, we found that overexpression of a dominant-negative mutant of GODZ (GODZ C157S ) affected UL20 and gK expression and localization in vitro (6). As utilization of dominant-negative mutant approaches to analysis of function can be problematic, we determined the expression of UL20 and gK, and their interaction, in HSV-1-infected GODZ Ϫ/Ϫ MEFs.…”

“…UL20 has been shown to interact with HSV-1 glycoprotein K (gK) (1), and both UL20 and gK are classified as essential genes for HSV-1 infectivity (1)(2)(3)(4)(5). We found recently that UL20 binds to GODZ (Golgi-specific DHHC zinc finger protein)/DHHC3, a member of the Asp-His-His-Cys (DHHC) family of palmitoyl transferases (6). These enzymes are characterized by a conserved DHHC motif embedded within a larger conserved cysteine-rich domain (7)(8)(9).…”

“…GODZ is localized predominately within the cis-Golgi complex (12,18,19) and has been shown to be involved in the palmitoylation of several proteins, especially in neurons (18)(19)(20)(21)(22). Using a dominant-negative construct of GODZ to characterize its role in virus infectivity in vitro, we have shown that the interaction of UL20 with GODZ is required for optimal virus infectivity, that UL20 is palmitoylated by GODZ in cultured cells, and that this palmitoylation is required for virus infectivity (6).…”

“…It is well established that GODZ is a Golgi complex protein (12,18,19), and binding of GODZ with UL20 has been shown to occur within the Golgi complex (6). We therefore infected MEFs from WT and GODZ Ϫ/Ϫ mice with VC1 virus for 16 h and then fixed and permeabilized the infected cells prior to incubation with anti-GM130 (to detect the Golgi complex), as well as anti-FLAG (to detect UL20) and anti-V5 (to detect gK) antibodies ( Fig.…”

Characteristics of herpes simplex virus infection and pathogenesis suggest a strategy for vaccine development

“…Most tegument proteins form complex structures by interacting with each other and anchoring to capsids or membranes to stabilize the viral structure . Topological data suggest that the interactions between tegument proteins and cellular structure may or may not depend upon the myristyl‐ and palmityl‐base anchors produced by posttranslational modifications on the surfaces of these proteins . Interestingly, some tegument proteins play important roles through their interactions with cellular molecules, as they are involved in the viral structural network .…”