Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection

Abstract

Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an unlimited array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies that incorporate viral products including dense bodies and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). Cells were fixed and labeled with antibodies against subcellular compartment markers and imaged using confocal and super-resolution microscopy. In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in endothelial cells were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that virus-containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. Virus containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non-canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV.ImportanceHuman cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with increased risk vascular disease. HCMV infects many cells in the human and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact the outcome of infection.

The unconventional way out—Egress of HCMV through multiviral bodies

“…While investigating the provenance of these MViBs, we also found that they carry CD63, which is also present at the release events. The presence of CD63 potentially links MViBs to the endosomal/exosomal pathway, which would be in line with earlier studies, which reported multivesicular structures filled with HCMV virions (Bughio et al, 2013; Fraile‐Ramos et al, 2007, 2010; Momtaz et al, 2021; Schauflinger et al, 2011; Severi et al, 1988). However, in our hands, the MVB inhibiting drug U18666A does not inhibit EVA generation, which indicates that the properties of MViBs are different from classical cellular MVBs.…”

“…Although current data (Read et al, 2019; Schauflinger et al, 2013; Shaga Devan et al, 2021; Taisne et al, 2019) have mainly described that HCMV capsids individually bud into small vesicles in the AC (Figure 1a,b), several studies have described large vesicles filled with virus particles (Bughio et al, 2013; Fraile‐Ramos et al, 2010; Momtaz et al, 2021; Schauflinger et al, 2011; Severi et al, 1988). However, the relevance of these structures has remained unclear.…”

“…Based on the presence of the cellular markers LBPA, RAB5, CD63, ALIX, and clathrin adaptor proteins, the authors concluded that MViBs in fibroblasts originate from the classical endocytic pathway with the involvement of UL71. In contrast, MViBs in endothelial cells originate from a nonclassical MVB biosynthesis pathway with the involvement of UL135 (Momtaz et al, 2021).…”

Myeloid cell tropism enables MHC-E–restricted CD8 + T cell priming and vaccine efficacy by the RhCMV/SIV vaccine

“…What is still unclear is why RhCMV vector-mediated MHC-II-restricted CD8 + T cell priming would be restricted to this cell type, as intrinsic MHC-II processing occurs in many cell types, including professional Agpresenting cells and non-Ag-presenting cells (36). It is possible that aspects of CMV infection in endothelial cells that differ from other viral target cells contribute an essential element of this priming mechanism (43)(44)(45).…”

Intermittent Bulk Release of Human Cytomegalovirus

“…It is plausible to hypothesize that these virus populations might undergo different envelopment processes in the cell and are exocytosed with a different spatiotemporal profile (Scrivano et al, 2011). While this manuscript was in preparation, a study from the Wilson and Goodrum lab suggested that virus-containing MVBs in fibroblasts and endothelial cells are derived from different cellular membranes, which would add another potential HCMV egress pathway that could result in different virus populations; however, it is unclear if these pathways are functional in egress (Momtaz et al, 2021).…”