Three-Dimensional Structure of the Human Cytomegalovirus Cytoplasmic Virion Assembly Complex Includes a Reoriented Secretory Apparatus

Abstract

Human cytomegalovirus (HCMV) induces profound changes in infected cell morphology, including a large cytoplasmic inclusion that corresponds to the virion assembly complex (AC). In electron micrographs, the AC is a highly vacuolated part of the cytoplasm. Markers of cellular secretory organelles have been visualized at the outer edge of the AC, and we recently showed that a marker for early endosomes (i.e., early endosome antigen 1) localizes to the center of the AC. Here, we examined the relationship between the AC and components of the secretory apparatus, studied temporal aspects of the dramatic infectioninduced cytoplasmic remodeling, examined the three-dimensional structure of the AC, and considered the implications of our observations for models of HCMV virion maturation and egress. We made three major observations. First, in addition to being relocated, the expression levels of some organelle markers change markedly during the period while the AC is developing. Second, based on three-dimensional reconstructions from z-series confocal microscopic images, the observed concentric rings of vesicles derived from the several compartments (Golgi bodies, the trans-Golgi network [TGN], and early endosomes) are arranged as nested cylinders of organelle-specific vesicles. Third, the membrane protein biosynthetic and exocytic pathways from the endoplasmic reticulum to the Golgi bodies, TGN, and early endosomes are in an unusual arrangement that nonetheless allows for a conventional order of biosynthesis and transport. Our model of AC structure suggests a mechanism by which the virus can regulate the order of tegument assembly.

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

“…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 pattern of partial subcellular association between RhoB and Rab5 is in agreement with the fact that Rab5 only partially co-localizes with the viral AC protein gB 11 and does not precisely designate the viral AC. 6 Moreover, limited colocalization between Rab5 and the early endosome protein EEA1 has also been observed in HCMV-infected fibroblasts, whereas EEA1 localizes at the AC. 6 On the contrary, in HCMV infected cells RhoB shows the same distribution pattern as the TGN marker, TGN-38 ( Fig.…”

“…6 Moreover, limited colocalization between Rab5 and the early endosome protein EEA1 has also been observed in HCMV-infected fibroblasts, whereas EEA1 localizes at the AC. 6 On the contrary, in HCMV infected cells RhoB shows the same distribution pattern as the TGN marker, TGN-38 ( Fig. 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.…”

“…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. 1G lower panel).…”

“…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.…”