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. 2010 May;9(5):851-60.
doi: 10.1074/mcp.M900485-MCP200. Epub 2009 Dec 20.

A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation

Nathaniel J Moorman  1 , Ronit Sharon-Friling, Thomas Shenk, Ileana M Cristea
Affiliations

A targeted spatial-temporal proteomics approach implicates multiple cellular trafficking pathways in human cytomegalovirus virion maturation

Nathaniel J Moorman et al. Mol Cell Proteomics. 2010 May.

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.

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Figures

Fig. 1.
Fig. 1.
Construction and characterization of GFP-tagged HCMV recombinant viruses. A, schematic depiction of the UL99 and UL32 genomic loci of HCMV. GFP was fused to the carboxyl termini of the UL99 and UL32 ORFs to generate the BADinUL99GFP (left panel) and BADinUL32GFP (right panel) strains. B, growth kinetics and viral yield were measured over multiple rounds of viral replication for BADinUL99GFP, BADinUL32GFP, and parental BADwt. Fibroblasts were infected at a multiplicity of 0.1 pfu/cell. Supernatants were harvested at the indicated times, and virus in the supernatants was quantified by the median tissue culture infective dose (TCID50) method. Error bars indicate standard deviations from two experiments titered in duplicate. C, fibroblasts were infected with either the parental strain, BADinUL99GFP, or BADinUL32GFP. At 72 hpi, the localization of pUL99 and pUL32 was determined by immunofluorescence using either antibodies specific for the viral proteins or, in the case of the GFP fusion viruses, the fluorescent signal of the tagged viral protein. Nuclear DNA is stained blue with DAPI. WT, wild type.
Fig. 2.
Fig. 2.
pUL99-interacting partners at 72 hpi. A, UL99GFP-interacting proteins were isolated by affinity purification, resolved by gel electrophoresis, and identified by sequential MS and MS/MS analyses. *, peptides corresponding to ubiquitin (UBQ) were detected between 50- and 75-kDa molecular mass markers, and therefore its exact location could not be determined. B, representative MALDI LTQ Orbitrap CID spectra of [M + H]+ ions of peptides corresponding to ubiquitin. Following CID, the generated fragment ions were detected at the linear trap level.
Fig. 3.
Fig. 3.
pUL32 associates with clathrin and components of viral tegument at 72 hpi. A, UL32-GFP-interacting proteins were isolated via affinity purifications, separated by gel electrophoresis, and identified by mass spectrometry. B, representative MALDI LTQ Orbitrap CID spectra of [M + H]+ ions of peptides corresponding to clathrin (CHC), obtained from the region indicated in A. Following CID, the generated fragment ions were detected at the linear trap level.
Fig. 4.
Fig. 4.
HCMV pUL99 colocalizes with ubiquitin-binding proteins of ESCRT pathway. A, at 72 hpi, UL99-GFP colocalized with ubiquitin (UBQ), Tsg101, Hrs1, and TGN46. Nuclear DNA is stained blue with DAPI. TGN46 is a cellular marker of the trans-Golgi complex, previously shown to colocalize with pUL99 during viral assembly. B, Western blot analysis of virion preparations indicating the presence of ubiquitinated proteins at ∼28, 50, and 160 kDa. M, medium alone; 5K, 25K, and 50K, the number (×1000) of infectious virions loaded in each lane.
Fig. 5.
Fig. 5.
Brefeldin A sensitivity defines pUL99 and pUL32 as residing in separate vesicles at 72 hpi. Fibroblasts were infected with either BADinUL99GFP or BADinUL32GFP. 3 h prior to fixation, cells were treated with brefeldin A (BFA; 3 μg/ml) and fixed, and localization of the pUL99 and pUL32 proteins was determined. Nuclear DNA is stained blue with DAPI.
Fig. 6.
Fig. 6.
HCMV pUL99, pUL32, and gB reside in separate compartments at early stages of viral assembly. Primary human fibroblasts were infected with the indicated virus. 72 h later, cells were fixed and stained with the indicated antibodies. Nuclear DNA is stained blue with DAPI.
Fig. 7.
Fig. 7.
pUL32-, pUL99-, and gB-containing compartments merge as infection progresses. Infected fibroblasts were assayed for the indicated proteins at 96 (top panels) or 120 hpi (bottom panels). Nuclear DNA is stained blue with DAPI. The white arrow points to an example of pUL99 and pUL32 vesicles in the process of merging.
Fig. 8.
Fig. 8.
Proposed model of trafficking events involved in HCMV virion assembly.
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References

    1. Britt W. J., Boppana S. (2004) Human cytomegalovirus virion proteins. Hum. Immunol 65, 395–402 - PubMed
    1. Gibson W. (2008) Structure and formation of the cytomegalovirus virion. Curr. Top. Microbiol. Immunol 325, 187–204 - PubMed
    1. Kalejta R. F. (2008) Tegument proteins of human cytomegalovirus. Microbiol. Mol. Biol. Rev 72, 249–265, table of contents - PMC - PubMed
    1. Das S., Vasanji A., Pellett P. E. (2007) Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus. J. Virol 81, 11861–11869 - PMC - PubMed
    1. Homman-Loudiyi M., Hultenby K., Britt W., Söderberg-Nauclér C. (2003) Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-Golgi network 46, and mannosidase II. J. Virol 77, 3191–3203 - PMC - PubMed

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