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 &amp 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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    • "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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    ABSTRACT: Human cytomegalovirus (CMV) is a herpesvirus that causes major health problems in neonates as well as in immunocompromised individuals. At present, a vaccine is not available for CMV infection and the available antiviral drugs suffer from toxicity, poor efficacy and resistance. Here, we chemically conjugated a monoclonal antibody raised against CMV surface glycoprotein (gB) with gold nanoparticles (GNP) and characterized the potential of this gB-GNP conjugate for antiviral activity against CMV. The gB-GNP blocks viral replication, virus-induced cytopathogenic effects and virus spread in cell culture without inducing cytotoxicity. High concentrations of gB-GNP that coat the surface of virus particles block virus entry, whereas lower concentrations block a later stage of virus life cycle. Also, cells treated with gB-GNP gain resistance to CMV infection. In addition, infected cells when bound to gB-GNP can be selectively lysed after exposing them to specific wavelength of laser (nanophotothermolysis). Thus, we have not only designed a potential antiviral strategy that specifically blocks CMV infection at multiple stages of virus life cycle, but we have also characterized a technique that can potentially be useful in eliminating CMV infected cells from donor tissue during transplant or transfusion.
    Scientific Reports 07/2014; 4:5550. DOI:10.1038/srep05550 · 5.58 Impact Factor
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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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    ABSTRACT: The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.
    PLoS ONE 11/2012; 7(11):e50166. DOI:10.1371/journal.pone.0050166 · 3.23 Impact Factor
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    • "However, the molecular mechanisms of HCMV morphogenesis remain undefined. Importantly, it has been reported that parallel distinct trafficking pathways merge viral proteins into a single structure at the perinuclear assembly compartment (Moorman et al., 2010). IF analysis of HCMV-infected cells revealed STX3 located at the cell membrane and intracellularly at the assembly site (Fig. 1), a distribution consistent with previous data describing a plasma membrane localization of STX3 in epithelial cells (Low et al., 1996; Li et al., 2002) as well as an intracellular distribution in gastric parietal cells, normal rat kidney fibroblasts and photoreceptor cells (Peng et al., 1997; Band and Kuismanen , 2005; Kwok et al., 2008). "
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    ABSTRACT: As an enveloped virus, replication of human cytomegalovirus (HCMV) is dependent on interaction with cellular membrane systems. Its final envelopment occurs into intracellular membranes prior to its secretion. However the mechanisms underlying these processes are poorly understood. Here, we show that HCMV infection induces expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3), a component of the cellular machinery for membrane fusion. STX3 was located at the plasma membrane and at the assembly site where it was found associated with virus wrapping membranes by immunogold labelling. Depletion of STX3 using RNA interference reduced HCMV production, while expression of a STX3 construct resistant to RNAi inhibition enhanced virus production. Ultrastructural examination of the assembly site in HCMV-infected STX3-depleted cells showed fewer mature virions and more viruses undergoing final envelopment. In contrast, silencing of STX3 did not affect herpes simplex virus type 1 production. The mechanism through which STX3 affected HCMV morphogenesis likely involved late endosomes/lysosomes since STX3 depletion reduced the expression of lysosomal membrane glycoproteins. Our results demonstrate a function for STX3 in HCMV morphogenesis, and unravel a new role for this SNARE protein in late endosomes/lysosomes compartments.
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