Human cytomegalovirus final envelopment on membranes containing bothtrans-Golgi network and endosomal markers

Abstract

SummaryThe human cytomegalovirus (HCMV) has been shown to complete its final envelopment on cytoplasmic membranes prior to its secretion to the extracellular medium. However, the nature of these membranes has not been characterized. It is thought that HCMV acquires its final envelope from the trans-Golgi network (TGN), though we and others have previously reported a role for endocytic membranes. Here we studied the localization of cellular markers in HCMV-infected cells and in isolated viruses. Immunofluorescence staining indicated that HCMV induces the recruitment of TGN and endosomal markers to the virus factory. Immuno-gold labelling of isolated viral particles and electron microscopy demonstrated the incorporation of TGN46, endosomal markers early endosomal antigen 1, annexin I, transferrin receptor and CD63, and the cation-independent mannose 6-phosphate receptor, which traffics between the TGN and endosomes into the viral envelope. Virus immunoprecipitation assays demonstrated that virions containing TGN46 and CD63 were infectious. This study reconciles the apparent controversy regarding the nature of the HCMV assembly site and suggests that HCMV has the ability to generate a novel membrane compartment containing markers for both TGN and endosomes, or that the membranes that HCMV uses for its envelope may be vesicles in transit between the TGN and endosomes.

Human cytomegalovirus pUL37x1-induced calcium flux activates PKCα, inducing altered cell shape and accumulation of cytoplasmic vesicles

“…The AC is thought to be where virions acquire tegument proteins and envelopes. Early endosome antigen-1 (EEA1) is a cellular protein present in early endosomes (35) that participates in endosomal membrane fusion (36) and is localized to the AC within infected cells (37,38). Our findings confirm these characteristics, showing that EEA1 was colocalized with two markers of the AC, a Golgi component targeted by HPA lectin and the late viral protein pUL99 (Fig.…”

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

“…1G upper panel), an observation in line with the fact that other TGN markers, such as TGN-46 and p230, also localize at the AC. 6,7,11,50,54 Live cell imaging using an autofluorescent RhoB construct, mRFP-RhoB, and the recombinant UL32-EGFP-HCMV-TB40 virus, not only confirmed the observations from the experiments in fixed cells but additionally shed light into the dynamics of RhoB spatial localization in association with the pUL32 tegument protein at the late stages of HCMV infection. Apart from its marked upregulation, RhoB is also relocated during viral infection, changing from an initial cytoplasmic, relatively scattered distribution to a striking recruitment to sites where new virions are assembled, along with structural HCMV proteins, such as pUL32 ( Fig.…”

“…1G lower panel). The same pattern of subcellular association has been observed among several TGN markers, Rab5 and HCMV AC proteins 6,7,11,54 corroborating the specificity of RhoB localization in the assembly site of the virus. …”

“…This is of interest, as it has been reported that the cellular proteins, CD63, TGN46 and CD-M6PR, that also localize at the AC are downregulated in infected cells and are incorporated in new virions. 7 During the formation of the AC, extensive cytoplasmic remodeling occurs, resulting in dramatic reorientation of the secretory machinery, including the early endosomes. 6,7,10,14 Although RhoB has been reported to localize to early endosomes, 20 it is not fully spatially associated with the early endosome protein Rab5 65,66 in HCMV-infected cells (Fig.…”

“…The endosomal sorting complex required for transport (ESCRT) machinery and multivesicular bodies (MVB) play a role in virion maturation. [6][7][8][9][10][11][12][13][14] RhoB belongs to the Rho GTPase family of proteins that comprises 6 subfamilies. 15 The RhoA subfamily consists of RhoA, RhoB and RhoC, which exhibit approximately 85% amino acid sequence identity.…”