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

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. 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. Viruses are obligate intracellular parasites that rely on the host cell for many of the processes essential for their replication. Viruses subvert cellular pathways detrimental to virus growth and hijack complex cellular machinery necessary for efficient viral replication. This complex relationship with the host cell is born of necessity; viruses contain relatively little genetic material, which frequently encodes for a limited number of proteins. Understanding the molecular events at the interface of the virus-host relationship is crucial for the identification of targets for future therapeutics.One of the most complex processes in the viral life cycle is the assembly of infectious viral particles. Multiple events must be coordinated in time and space for the correct incorporation of the viral genetic material into an infectious unit, or virion. For example, the virions of herpesviruses are composed of three essential elements: (i) the viral DNA and its surrounding protein shell, which together form the nucleocapsid, (ii) a layer of proteins surrounding the nucleocapsid termed the tegument, and (iii) a lipid bilayer, or envelope, surrounding the tegumented nucleocapsid in which viral glycoproteins are embedded (1Britt W.J. Boppana S. Human cytomegalovirus virion proteins.Hum. Immunol. 2004; 65: 395-402Crossref PubMed Scopus (50) Google Scholar, 2Gibson W. Structure and formation of the cytomegalovirus virion.Curr. Top. Microbiol. Immunol. 2008; 325: 187-204PubMed Google Scholar, 3Kalejta R.F. Tegument proteins of human cytomegalovirus.Microbiol. Mol. Biol. Rev. 2008; 72 (table of contents): 249-265Crossref PubMed Scopus (124) Google Scholar). Although the formation of the nucleocapsid occurs in the nucleus, the herpesvirus tegument is acquired as the nucleocapsid moves into the cytoplasm through an undefined mechanism (2Gibson W. Structure and formation of the cytomegalovirus virion.Curr. Top. Microbiol. Immunol. 2008; 325: 187-204PubMed Google Scholar, 3Kalejta R.F. Tegument proteins of human cytomegalovirus.Microbiol. Mol. Biol. Rev. 2008; 72 (table of contents): 249-265Crossref PubMed Scopus (124) Google Scholar). Once in the cytoplasm, the tegumented nucleocapsid acquires its envelope, again through a poorly defined event.Although it has been known for some time that multiple HCMV 1The abbreviations used are:HCMVhuman cytomegalovirusESCRTendosomal sorting complex required for transportGFPgreen fluorescence proteinBACbacterial artificial chromosomepfuplaque-forming unit(s)hpihours postinfectionBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLTQlinear trap quadrupoleDAPI4′,6′-diamidino-2-phenylindole dihydrochlorideBiPimmunoglobulin heavy chain binding proteingBglycoprotein B.1The abbreviations used are:HCMVhuman cytomegalovirusESCRTendosomal sorting complex required for transportGFPgreen fluorescence proteinBACbacterial artificial chromosomepfuplaque-forming unit(s)hpihours postinfectionBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLTQlinear trap quadrupoleDAPI4′,6′-diamidino-2-phenylindole dihydrochlorideBiPimmunoglobulin heavy chain binding proteingBglycoprotein B. proteins important for viral assembly localize to a juxtanuclear site termed the assembly zone, only recently have cellular markers for the HCMV assembly zone been described (4Das S. Vasanji A. Pellett P.E. Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus.J. Virol. 2007; 81: 11861-11869Crossref PubMed Scopus (147) Google Scholar, 5Homman-Loudiyi M. Hultenby K. Britt W. Söderberg-Nauclér C. 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. 2003; 77: 3191-3203Crossref PubMed Scopus (110) Google Scholar, 6Sanchez V. Greis K.D. Sztul E. Britt W.J. Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly.J. Virol. 2000; 74: 975-986Crossref PubMed Scopus (272) Google Scholar, 7Seo J.Y. Britt W.J. Cytoplasmic envelopment of human cytomegalovirus requires the postlocalization function of tegument protein pp28 within the assembly compartment.J. Virol. 2007; 81: 6536-6547Crossref PubMed Scopus (44) Google Scholar). This work has led to the view that HCMV virion maturation occurs by progressing through the endoplasmic reticulum to the Golgi apparatus (4Das S. Vasanji A. Pellett P.E. Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus.J. Virol. 2007; 81: 11861-11869Crossref PubMed Scopus (147) Google Scholar, 5Homman-Loudiyi M. Hultenby K. Britt W. Söderberg-Nauclér C. 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. 2003; 77: 3191-3203Crossref PubMed Scopus (110) Google Scholar) and then via the ESCRT pathway to its site of release into the extracellular space (8Tandon R. AuCoin D.P. Mocarski E.S. Human cytomegalovirus exploits ESCRT machinery in the process of virion maturation.J. Virol. 2009; 83: 10797-10807Crossref PubMed Scopus (73) Google Scholar). This work represents a major step forward in our understanding of the spatial requirements for virion maturation. However, the molecular events underlying this process remain poorly defined.The assembly of HCMV particles is a process that has limited homology to cellular events. Therefore, it comes as no surprise that HCMV encodes proteins that are essential for the orchestration of virion assembly. One such protein, pUL99 (also termed pp28), is encoded by the UL99 ORF and resides in the cytoplasm of infected cells (9Murphy E. Yu D. Grimwood J. Schmutz J. Dickson M. Jarvis M.A. Hahn G. Nelson J.A. Myers R.M. Shenk T.E. Coding potential of laboratory and clinical strains of human cytomegalovirus.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14976-14981Crossref PubMed Scopus (410) Google Scholar). pUL99 is required for the envelopment of capsids to generate virions (10Jones T.R. Lee S.W. An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.J. Virol. 2004; 78: 1488-1502Crossref PubMed Scopus (40) Google Scholar, 11Silva M.C. Yu Q.C. Enquist L. Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids.J. Virol. 2003; 77: 10594-10605Crossref PubMed Scopus (151) Google Scholar). In its absence, tegumented capsids accumulate in the cytoplasm of infected cells (11Silva M.C. Yu Q.C. Enquist L. Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids.J. Virol. 2003; 77: 10594-10605Crossref PubMed Scopus (151) Google Scholar). 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 (12Buchkovich N.J. Maguire T.G. Paton A.W. Paton J.C. Alwine J.C. The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the HCMV assembly compartment.J. Virol. 2009; 83: 11421-11428Crossref PubMed Scopus (66) Google Scholar). Despite its critical role in envelopment, pUL99 is dispensable for cell-to-cell spread of HCMV, a process that does not require enveloped nucleocapsids (13Silva M.C. Schröer J. Shenk T. Human cytomegalovirus cell-to-cell spread in the absence of an essential assembly protein.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2081-2086Crossref PubMed Scopus (55) Google Scholar).Another HCMV protein integral to the assembly process is pUL32 (also termed pp150), encoded by the UL32 ORF. Like pUL99, this protein is required for the production of HCMV virions (14Yu D. Silva M.C. Shenk T. Functional map of human cytomegalovirus AD169 defined by global mutational analysis.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12396-12401Crossref PubMed Scopus (332) Google Scholar, 15Dunn W. Chou C. Li H. Hai R. Patterson D. Stolc V. Zhu H. Liu F. Functional profiling of a human cytomegalovirus genome.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14223-14228Crossref PubMed Scopus (518) Google Scholar, 16Tandon R. Mocarski E.S. Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150.J. Virol. 2008; 82: 9433-9444Crossref PubMed Scopus (40) Google Scholar, 17Meyer H.H. Ripalti A. Landini M.P. Radsak K. Kern H.F. Hensel G.M. Human cytomegalovirus late-phase maturation is blocked by stably expressed UL32 antisense mRNA in astrocytoma cells.J. Gen. Virol. 1997; 78: 2621-2631Crossref PubMed Scopus (26) Google Scholar). pUL32 is capable of directly binding to the nucleocapsid, and this event occurs shortly after nucleocapsid assembly (18Baxter M.K. Gibson W. Cytomegalovirus basic phosphoprotein (pUL32) binds to capsids in vitro through its amino one-third.J. Virol. 2001; 75: 6865-6873Crossref PubMed Scopus (50) Google Scholar). Despite its localization to both the nucleus and the cytoplasm during infection (19Hensel G. Meyer H. Gärtner S. Brand G. Kern H.F. Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32).J. Gen. Virol. 1995; 76: 1591-1601Crossref PubMed Scopus (52) Google Scholar, 20Sampaio K.L. Cavignac Y. Stierhof Y.D. Sinzger C. Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements.J. Virol. 2005; 79: 2754-2767Crossref PubMed Scopus (109) Google Scholar), pUL32 acts primarily in the cytoplasm where it may affect the localization and stability of the nucleocapsid (16Tandon R. Mocarski E.S. Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150.J. Virol. 2008; 82: 9433-9444Crossref PubMed Scopus (40) Google Scholar, 21AuCoin D.P. Smith G.B. Meiering C.D. Mocarski E.S. Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.J. Virol. 2006; 80: 8199-8210Crossref PubMed Scopus (63) Google Scholar). In the absence of this viral protein, nucleocapsids are found primarily in the nucleus with few viral particles in the cytoplasm (21AuCoin D.P. Smith G.B. Meiering C.D. Mocarski E.S. Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.J. Virol. 2006; 80: 8199-8210Crossref PubMed Scopus (63) Google Scholar).To generate additional insights into the molecular events controlling HCMV virion assembly, we used a targeted genetic-proteomic approach to identify protein binding partners for pUL99 and pUL32 in the context of viral infection. This work led to the identification of multiple distinct steps in the virion maturation process. The two viral proteins traffic through two separate compartments at the early stages of viral assembly. Although the tegument and nucleocapsid traffic with pUL32 in a clathrin-associated, brefeldin-insensitive vesicle, pUL99 localizes to a distinct brefeldin-sensitive structure that colocalizes with markers of the ESCRT trafficking pathway. The viral envelope glycoprotein B (gB), which is encoded by the UL55 ORF, did not substantially colocalize with pUL99 or pUL32 at early stages of the assembly process. However, 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. Viruses are obligate intracellular parasites that rely on the host cell for many of the processes essential for their replication. Viruses subvert cellular pathways detrimental to virus growth and hijack complex cellular machinery necessary for efficient viral replication. This complex relationship with the host cell is born of necessity; viruses contain relatively little genetic material, which frequently encodes for a limited number of proteins. Understanding the molecular events at the interface of the virus-host relationship is crucial for the identification of targets for future therapeutics. One of the most complex processes in the viral life cycle is the assembly of infectious viral particles. Multiple events must be coordinated in time and space for the correct incorporation of the viral genetic material into an infectious unit, or virion. For example, the virions of herpesviruses are composed of three essential elements: (i) the viral DNA and its surrounding protein shell, which together form the nucleocapsid, (ii) a layer of proteins surrounding the nucleocapsid termed the tegument, and (iii) a lipid bilayer, or envelope, surrounding the tegumented nucleocapsid in which viral glycoproteins are embedded (1Britt W.J. Boppana S. Human cytomegalovirus virion proteins.Hum. Immunol. 2004; 65: 395-402Crossref PubMed Scopus (50) Google Scholar, 2Gibson W. Structure and formation of the cytomegalovirus virion.Curr. Top. Microbiol. Immunol. 2008; 325: 187-204PubMed Google Scholar, 3Kalejta R.F. Tegument proteins of human cytomegalovirus.Microbiol. Mol. Biol. Rev. 2008; 72 (table of contents): 249-265Crossref PubMed Scopus (124) Google Scholar). Although the formation of the nucleocapsid occurs in the nucleus, the herpesvirus tegument is acquired as the nucleocapsid moves into the cytoplasm through an undefined mechanism (2Gibson W. Structure and formation of the cytomegalovirus virion.Curr. Top. Microbiol. Immunol. 2008; 325: 187-204PubMed Google Scholar, 3Kalejta R.F. Tegument proteins of human cytomegalovirus.Microbiol. Mol. Biol. Rev. 2008; 72 (table of contents): 249-265Crossref PubMed Scopus (124) Google Scholar). Once in the cytoplasm, the tegumented nucleocapsid acquires its envelope, again through a poorly defined event. Although it has been known for some time that multiple HCMV 1The abbreviations used are:HCMVhuman cytomegalovirusESCRTendosomal sorting complex required for transportGFPgreen fluorescence proteinBACbacterial artificial chromosomepfuplaque-forming unit(s)hpihours postinfectionBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLTQlinear trap quadrupoleDAPI4′,6′-diamidino-2-phenylindole dihydrochlorideBiPimmunoglobulin heavy chain binding proteingBglycoprotein B.1The abbreviations used are:HCMVhuman cytomegalovirusESCRTendosomal sorting complex required for transportGFPgreen fluorescence proteinBACbacterial artificial chromosomepfuplaque-forming unit(s)hpihours postinfectionBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLTQlinear trap quadrupoleDAPI4′,6′-diamidino-2-phenylindole dihydrochlorideBiPimmunoglobulin heavy chain binding proteingBglycoprotein B. proteins important for viral assembly localize to a juxtanuclear site termed the assembly zone, only recently have cellular markers for the HCMV assembly zone been described (4Das S. Vasanji A. Pellett P.E. Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus.J. Virol. 2007; 81: 11861-11869Crossref PubMed Scopus (147) Google Scholar, 5Homman-Loudiyi M. Hultenby K. Britt W. Söderberg-Nauclér C. 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. 2003; 77: 3191-3203Crossref PubMed Scopus (110) Google Scholar, 6Sanchez V. Greis K.D. Sztul E. Britt W.J. Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly.J. Virol. 2000; 74: 975-986Crossref PubMed Scopus (272) Google Scholar, 7Seo J.Y. Britt W.J. Cytoplasmic envelopment of human cytomegalovirus requires the postlocalization function of tegument protein pp28 within the assembly compartment.J. Virol. 2007; 81: 6536-6547Crossref PubMed Scopus (44) Google Scholar). This work has led to the view that HCMV virion maturation occurs by progressing through the endoplasmic reticulum to the Golgi apparatus (4Das S. Vasanji A. Pellett P.E. Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus.J. Virol. 2007; 81: 11861-11869Crossref PubMed Scopus (147) Google Scholar, 5Homman-Loudiyi M. Hultenby K. Britt W. Söderberg-Nauclér C. 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. 2003; 77: 3191-3203Crossref PubMed Scopus (110) Google Scholar) and then via the ESCRT pathway to its site of release into the extracellular space (8Tandon R. AuCoin D.P. Mocarski E.S. Human cytomegalovirus exploits ESCRT machinery in the process of virion maturation.J. Virol. 2009; 83: 10797-10807Crossref PubMed Scopus (73) Google Scholar). This work represents a major step forward in our understanding of the spatial requirements for virion maturation. However, the molecular events underlying this process remain poorly defined. human cytomegalovirus endosomal sorting complex required for transport green fluorescence protein bacterial artificial chromosome plaque-forming unit(s) hours postinfection 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol linear trap quadrupole 4′,6′-diamidino-2-phenylindole dihydrochloride immunoglobulin heavy chain binding protein glycoprotein B. human cytomegalovirus endosomal sorting complex required for transport green fluorescence protein bacterial artificial chromosome plaque-forming unit(s) hours postinfection 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol linear trap quadrupole 4′,6′-diamidino-2-phenylindole dihydrochloride immunoglobulin heavy chain binding protein glycoprotein B. The assembly of HCMV particles is a process that has limited homology to cellular events. Therefore, it comes as no surprise that HCMV encodes proteins that are essential for the orchestration of virion assembly. One such protein, pUL99 (also termed pp28), is encoded by the UL99 ORF and resides in the cytoplasm of infected cells (9Murphy E. Yu D. Grimwood J. Schmutz J. Dickson M. Jarvis M.A. Hahn G. Nelson J.A. Myers R.M. Shenk T.E. Coding potential of laboratory and clinical strains of human cytomegalovirus.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14976-14981Crossref PubMed Scopus (410) Google Scholar). pUL99 is required for the envelopment of capsids to generate virions (10Jones T.R. Lee S.W. An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.J. Virol. 2004; 78: 1488-1502Crossref PubMed Scopus (40) Google Scholar, 11Silva M.C. Yu Q.C. Enquist L. Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids.J. Virol. 2003; 77: 10594-10605Crossref PubMed Scopus (151) Google Scholar). In its absence, tegumented capsids accumulate in the cytoplasm of infected cells (11Silva M.C. Yu Q.C. Enquist L. Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids.J. Virol. 2003; 77: 10594-10605Crossref PubMed Scopus (151) Google Scholar). 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 (12Buchkovich N.J. Maguire T.G. Paton A.W. Paton J.C. Alwine J.C. The endoplasmic reticulum chaperone BiP/GRP78 is important in the structure and function of the HCMV assembly compartment.J. Virol. 2009; 83: 11421-11428Crossref PubMed Scopus (66) Google Scholar). Despite its critical role in envelopment, pUL99 is dispensable for cell-to-cell spread of HCMV, a process that does not require enveloped nucleocapsids (13Silva M.C. Schröer J. Shenk T. Human cytomegalovirus cell-to-cell spread in the absence of an essential assembly protein.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2081-2086Crossref PubMed Scopus (55) Google Scholar). Another HCMV protein integral to the assembly process is pUL32 (also termed pp150), encoded by the UL32 ORF. Like pUL99, this protein is required for the production of HCMV virions (14Yu D. Silva M.C. Shenk T. Functional map of human cytomegalovirus AD169 defined by global mutational analysis.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12396-12401Crossref PubMed Scopus (332) Google Scholar, 15Dunn W. Chou C. Li H. Hai R. Patterson D. Stolc V. Zhu H. Liu F. Functional profiling of a human cytomegalovirus genome.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14223-14228Crossref PubMed Scopus (518) Google Scholar, 16Tandon R. Mocarski E.S. Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150.J. Virol. 2008; 82: 9433-9444Crossref PubMed Scopus (40) Google Scholar, 17Meyer H.H. Ripalti A. Landini M.P. Radsak K. Kern H.F. Hensel G.M. Human cytomegalovirus late-phase maturation is blocked by stably expressed UL32 antisense mRNA in astrocytoma cells.J. Gen. Virol. 1997; 78: 2621-2631Crossref PubMed Scopus (26) Google Scholar). pUL32 is capable of directly binding to the nucleocapsid, and this event occurs shortly after nucleocapsid assembly (18Baxter M.K. Gibson W. Cytomegalovirus basic phosphoprotein (pUL32) binds to capsids in vitro through its amino one-third.J. Virol. 2001; 75: 6865-6873Crossref PubMed Scopus (50) Google Scholar). Despite its localization to both the nucleus and the cytoplasm during infection (19Hensel G. Meyer H. Gärtner S. Brand G. Kern H.F. Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32).J. Gen. Virol. 1995; 76: 1591-1601Crossref PubMed Scopus (52) Google Scholar, 20Sampaio K.L. Cavignac Y. Stierhof Y.D. Sinzger C. Human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements.J. Virol. 2005; 79: 2754-2767Crossref PubMed Scopus (109) Google Scholar), pUL32 acts primarily in the cytoplasm where it may affect the localization and stability of the nucleocapsid (16Tandon R. Mocarski E.S. Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150.J. Virol. 2008; 82: 9433-9444Crossref PubMed Scopus (40) Google Scholar, 21AuCoin D.P. Smith G.B. Meiering C.D. Mocarski E.S. Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.J. Virol. 2006; 80: 8199-8210Crossref PubMed Scopus (63) Google Scholar). In the absence of this viral protein, nucleocapsids are found primarily in the nucleus with few viral particles in the cytoplasm (21AuCoin D.P. Smith G.B. Meiering C.D. Mocarski E.S. Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation.J. Virol. 2006; 80: 8199-8210Crossref PubMed Scopus (63) Google Scholar). To generate additional insights into the molecular events controlling HCMV virion assembly, we used a targeted genetic-proteomic approach to identify protein binding partners for pUL99 and pUL32 in the context of viral infection. This work led to the identification of multiple distinct steps in the virion maturation process. The two viral proteins traffic through two separate compartments at the early stages of viral assembly. Although the tegument and nucleocapsid traffic with pUL32 in a clathrin-associated, brefeldin-insensitive vesicle, pUL99 localizes to a distinct brefeldin-sensitive structure that colocalizes with markers of the ESCRT trafficking pathway. The viral envelope glycoprotein B (gB), which is encoded by the UL55 ORF, did not substantially colocalize with pUL99 or pUL32 at early stages of the assembly process. However, 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. We thank members of the Cristea and Shenk laboratories for helpful advice, comments, and criticisms. We also thank Joe Goodhouse for assistance with microscopy (Microscopy Core Facility at Princeton University). We also thank the many scientists in both the HCMV and proteomics field upon whose work this study builds. Supplementary Material Download .zip (.2 MB) Help with zip files Download .zip (.2 MB) Help with zip files