Abstract
The assembly of infectious virus particles is a complex event. For human cytomegalovirus (HCMV) this process requires the coordinated expression and localization of at least 60 viral proteins that comprise the infectious virion. To gain insight into the mechanisms controlling this process, we identified protein binding partners for two viral proteins, pUL99 (also termed pp28) and pUL32 (pp150), which are essential for HCMV virion assembly. We utilized HCMV strains expressing pUL99 or pUL32 carboxyl-terminal green fluorescent protein fusion proteins from their native location in the HCMV genome. Based on the presence of ubiquitin in the pUL99 immunoisolation, we discovered that this viral protein colocalizes with components of the cellular endosomal sorting complex required for transport (ESCRT) pathway during the initial stages of virion assembly. We identified the nucleocapsid and a large number of tegument proteins as pUL32 binding partners, suggesting that events controlling trafficking of this viral protein in the cytoplasm regulate nucleocapsid/tegument maturation. The finding that pUL32, but not pUL99, associates with clathrin led to the discovery that the two viral proteins traffic via distinct pathways during the early stages of virion assembly. Additional investigation revealed that the majority of the major viral glycoprotein gB initially resides in a third compartment. Analysis of the trafficking of these three viral proteins throughout a time course of virion assembly allowed us to visualize their merger into a single large cytoplasmic structure during the late stages of viral assembly. We propose a model of HCMV virion maturation in which multiple components of the virion traffic independently of one another before merging.
Highlights
The assembly of infectious virus particles is a complex event
Targeted Proteomics Approach for Studying Virus-Host Associations—We have previously described a strategy that utilizes a GFP tag to both visualize the dynamic localization of viral proteins and isolate virus-host protein complexes at different times of infection
To gain insights into processes occurring during human cytomegalovirus (HCMV) virion assembly, we generated two viruses, BADinUL99GFP and BADinUL32GFP, containing the GFP coding region fused to the carboxyl terminus of the pUL99 and pUL32, respectively (Fig. 1A)
Summary
The assembly of HCMV particles is a process that has limited homology to cellular events. It comes as no surprise that HCMV encodes proteins that are essential for the orchestration of virion assembly One such protein, pUL99 ( termed pp28), is encoded by the UL99 ORF and resides in the cytoplasm of infected cells [9]. In its absence, tegumented capsids accumulate in the cytoplasm of infected cells [11] This function may require its interaction with the cellular chaperone BiP as pUL99 binds to BiP during infection and disruption of BiP function was shown to inhibit HCMV assembly [12]. Despite its critical role in envelopment, pUL99 is dispensable for cell-to-cell spread of HCMV, a process that does not require enveloped nucleocapsids [13] Another HCMV protein integral to the assembly process is pUL32 ( termed pp150), encoded by the UL32 ORF. We demonstrate that as infection progresses in time these three distinct compartments merge into a single vesicle, which may represent the final site of virion assembly
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