Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization

Abstract

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the -Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization. Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.

Cellular Cullin RING Ubiquitin Ligases: Druggable Host Dependency Factors of Cytomegaloviruses

“…Several other interactions of pUL35 with host proteins have been described: pUL35 interacts with PML, Sp100, and Daxx in PML bodies [137]. Another interaction of p75-UL35 with sorting nexin 5 (SNX5), affects the localization of glycoprotein B (gB) (also known as gpUL55) [138]. A proteomics study revealed that pUL35 also interacts with Usp7, DDB1, DET1 - and DDB1 -Associated 1 (DDA1), and DCAF1 to modulate the DNA repair response [119].…”

The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription

“…It has been reported that deletion of the complete UL35 ORF led to a strong defect in virus replication, assembly, and particle formation, depending on the multiplicity of infection (MOI) [18]. A recent study showed that loss of UL35a led to a comparable growth deficit as the complete deletion of the UL35 ORF [19]. In contrast, when only UL35 expression was impaired but expression of UL35a was unaffected, a moderate defect in viral replication was observed [19].…”

“…A recent study showed that loss of UL35a led to a comparable growth deficit as the complete deletion of the UL35 ORF [19]. In contrast, when only UL35 expression was impaired but expression of UL35a was unaffected, a moderate defect in viral replication was observed [19]. UL35 was shown to positively affect the activation of the viral major immediate-early promoter (MIEP) by interacting with the viral transactivator pp71, encoded by UL82 [20].…”

Human Cytomegalovirus Primary Infection and Reactivation: Insights From Virion-Carried Molecules

“…More evidence based on expression of the UL35 mutant virus in transfected cells has revealed decreased production of enveloped particles and disappearance of dense bodies (DBs), indicating a role of pUL35 in envelopment ( Schierling et al, 2005 ). Furthermore, the transport of envelope glycoprotein B is dependent on the binding of pUL35 and its interacting partner, sorting nexin 5 (SNX5) ( Maschkowitz et al, 2018 ).…”