A Targeted Spatial-Temporal Proteomics Approach Implicates Multiple Cellular Trafficking Pathways in Human Cytomegalovirus Virion Maturation

Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Molecular & Cellular Proteomics (Impact Factor: 6.56). 12/2009; 9(5):851-60. DOI: 10.1074/mcp.M900485-MCP200
Source: PubMed


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.

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    • "This can be done either by co-immunoprecipitation of tagged or antibody-reactive viral components[20], far Western analy- sis[102], or using mass spectrometry-based technologies[11,76]. In the field of human and animal virus-host interactions, significant progress has been made through proteomic studies utilizing co-immunoprecipitation coupled to mass spectrometry, demonstrating the value of these approaches for studying these unique and highly recalcitrant systems148149150151. Analysis of large data sets, like those often generated in proteomic experiments, remains a challenge in the " -omic " era. "
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    • "Human cytomegalovirus AD169, Towne, TB40/e and FIX strains have been reported earlier192526. A version of AD169 virus where pp150 tegument protein has been fused with eGFP (BAD32 virus) was obtained from Moorman laboratory at University of North Carolina18. CMV virions were purified using established protocols for herpesviruses272829 with some modifications. "
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    • "The behavior of RL13 observed in this report is more similar to Stanton et al.'s data for infected cells, showing a MW above 100 kDa and a sub-cellular co-localization with early endosomes and the TGN. This last observation would be consistent with a default mechanism of internalization similar to what was described for other HCMV envelope proteins [36]. We currently have no explanation for the slight discrepancies between Stanton et al. and our report that may be due to the different expression system used or, at least for the confocal analysis, to the protein derived from a different strain. "
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