Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions

Abstract

Beta-herpesvirus infection completely reorganizes the membrane system of the cell. This system is maintained by the spatiotemporal arrangement of more than 3000 cellular proteins that continuously adapt the configuration of membrane organelles according to cellular needs. Beta-herpesvirus infection establishes a new configuration known as the assembly compartment (AC). The AC membranes are loaded with virus-encoded proteins during the long replication cycle and used for the final envelopment of the newly formed capsids to form infectious virions. The identity of the envelopment membranes is still largely unknown. Electron microscopy and immunofluorescence studies suggest that the envelopment occurs as a membrane wrapping around the capsids, similar to the growth of phagophores, in the area of the AC with the membrane identities of early/recycling endosomes and the trans-Golgi network. During wrapping, host cell proteins that define the identity and shape of these membranes are captured along with the capsids and incorporated into the virions as host cell signatures. In this report, we reviewed the existing information on host cell signatures in human cytomegalovirus (HCMV) virions. We analyzed the published proteomes of the HCMV virion preparations that identified a large number of host cell proteins. Virion purification methods are not yet advanced enough to separate all of the components of the rich extracellular material, including the large amounts of non-vesicular extracellular particles (NVEPs). Therefore, we used the proteomic data from large and small extracellular vesicles (lEVs and sEVs) and NVEPs to filter out the host cell proteins identified in the viral proteomes. Using these filters, we were able to narrow down the analysis of the host cell signatures within the virions and determine that envelopment likely occurs at the membranes derived from the tubular recycling endosomes. Many of these signatures were also found at the autophagosomes, suggesting that the CMV-infected cell forms membrane organelles with phagophore growth properties using early endosomal host cell machinery that coordinates endosomal recycling.

Overview of how HCMV manipulation of host cell intracellular trafficking networks can promote productive infection

“…HCMV manipulates intracellular trafficking components to facilitate VAC formation, viral assembly, and egress HCMV lytic infection of fibroblasts, endothelial cells, epithelial cells, smooth muscle cells, and potentially macrophages and dendritic cells, causes the formation of a large structure within the cell known as the viral assembly compartment (VAC) which functions as a space for viral tegument acquisition and envelopment (1,6,7,19,(24)(25)(26)(27)(28)(29)(30)(31). Infection causes extensive remodeling of nearly all secretory pathway components including the ER, Golgi complex, and the endosomal system to form the VAC, complete morphogenesis of progeny virions, and facilitate egress of the viral particle from the host cell (1,6,7,19,24,25,27,32). Due to the complexity of these events, definition of the specific HCMV-driven molecular processes involved has proven to be a challenging area of research.…”

“…In addition to these organelles, other cellular components have been found to be associated with the VAC, including members of the SNARE family, ESCRT machinery, and Rab GTPases, all of which are important factors for intracellular trafficking. ESCRT machinery regulates multi-vesicular body (MVB) cargo incorporation, thus its association with the multi-vesicular VAC indicates that ESCRT machinery may facilitate formation of the VAC compartments (3,(24)(25)(26)32).…”

“…Because the formation and function of the VAC is thought to be largely dependent on viral proteins, VAC formation and subsequent virion assembly is an active process facilitated by viral manipulation of host cell trafficking machinery. A number of specific cellular trafficking proteins have been shown to be needed for VAC formation and maintenance, the most well-studied being the small Rab GTPase Rab11, Syntaxin 5, Bicaudal D1, FIP4, BiP, and dynein (1,2,6,11,16,24,26,32,33,36) (Figure 1A). Endosomal recycling depends on proteins such as Rab11 as well as their effectors, and interference can significantly alter endosomal function and localization within the cell,…”

“…Additionally, HCMV commandeers trafficking proteins to maintain the VAC and facilitate virion maturation. Some known trafficking proteins utilized in this process are: Stx5, Bicaudal D1, BiP, Dynein, Rab11, and FIP4 (1,2,6,11,16,24,26,32,34). While exact mechanisms for HCMV-induced organelle restructuring and protein recruitment are unclear, there is evidence implicating roles for HCMV-encoded miRNAs (miR-UL112-1 and miR-US5-1), UL71, UL99, and potentially structural proteins (4,25,26).…”

“…In infected cells, Stx3 is found within the VAC, particularly at sites of viral glycoprotein gH and tegument protein UL99 accumulation, where it is shown to be important in the tegumentation and secondary envelopment process within the VAC, as well as in the production of infectious HCMV particles (Figure 1B) (6,25,36,41). Mass spectrometry analysis of progeny virion proteomes found the presence of: MHC-I, Rab12, Rab 23, Rab32, SNX2, SNX3, VAMP2, VAMP3, STX12, VTI1A, and PIK3C2A, suggesting that membrane components for virion envelopment are derived from the tubular portions of early endosomes that participate in Arf6/ Rab8-dependent cargo recycling (32). Newer data also supports that the envelopment and egress of HCMV is likely via a Rab3/ Rab27a-linked lysosome or sorting vesicle-like trafficking pathway (Figure 1B) (6).…”