Biochemical and structural characterization of the capsid-bound tegument proteins of human cytomegalovirus

Yu, Xuekui; Shah, Sanket; Lee, Manfred; Dai, Wei; Lo, Pierrette; Britt, William J.; Zhu, Hua; Liu, Fenyong; Zhou, Z. Hong

doi:10.1016/j.jsb.2011.03.006

Cited by 49 publications

(67 citation statements)

References 47 publications

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“…Other known capsid proteins (MCP, mCP, mCP-BP, and SCP) were readily identified as expected, and also the betaherpesvirus-specific tegument protein pp150 was found to be associated with capsids. This is in line with data reporting that attachment of pp150 to capsids starts already in the cell nucleus, presumably stabilizing C capsids during nuclear egress (12,13,47,48). However, we cannot exclude that a portion of pp150 detected here originates from some cytoplasmic capsids copurifying with the nuclear capsids and not solely from C capsids.…”

Section: Generation Of Hcmv Mutants Expressing a Ul77-mgfp Fusion Prosupporting

confidence: 88%

“…For HCMV, addition of the EGFP to pp150 (as well as tagging of the envelope protein gM with mCherry) provided a valuable tool to study virus entry and intracellular particle movement (47,56). pp150 was recently defined as the innermost tegument protein of HCMV (12), and although its addition to capsids commences in the nucleus, the majority of pp150 is acquired in the cytoplasm. Thus, the UL77-mGFP virus described here represents the first HCMV mutant with a fluorescently labeled capsid.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, HCMV encodes betaherpesvirus-specific tegument proteins: e.g., pp150 (UL32) and pUL96. Recently it was shown that pp150 is the most inner tegument protein forming a netlike layer around the capsids (12). A follow-up study disclosed the SCP as being necessary for recruitment of pp150 to capsids (13), thus providing an explanation of why in betaherpesviruses SCP is essential (14), while in HSV-1 it is dispensable for viral growth.…”

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confidence: 99%

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The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Borst

Bauerfeind

Binz

et al. 2016

J Virol

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Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.T he life cycle of human cytomegalovirus (HCMV), the prototype member of the betaherpesviruses, comprises a nuclear phase that includes transcription of viral genes, replication of the double-stranded DNA genome, assembly of procapsids, packaging of the viral DNA into the preformed capsids, and maturation of the DNA-filled c...

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Section: Generation Of Hcmv Mutants Expressing a Ul77-mgfp Fusion Prosupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Borst

Bauerfeind

Binz

et al. 2016

J Virol

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“…To gain insight into the working principle of pp150-mediated control in the pre-IE phase of infection, we addressed the question as to whether pp150 acts in trans like other CMV tegument proteins with known regulatory function or in cis, as suggested by its tight interaction with capsids in vitro (16) and its localization within the inner, detergent-resistant tegument layer of virus particles (20). We designed a coinfection experiment where pp150-WT and pp150-RXL mutant viruses were tested for their ability to influence each other in the presence or absence of cyclin A2-CDK activity.…”

Section: Resultsmentioning

confidence: 99%

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2–CDK-dependent sensor of the host cell cycle and differentiation state

Bogdanow

Weisbach

Einem

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDKpp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of selflimitation and adaptation to its human host.H erpesviruses are highly adapted to their hosts. This is due to a long coevolutionary process and based on an intricate molecular virus-host interaction network. Interestingly, a significant fraction of herpesviral gene products is already present in the very first phase of infection, after virus entry but before the onset of de novo viral gene expression. These factors originate from the tegument, a characteristic, protein-rich layer within herpesvirus particles, occupying the space between nucleocapsid and virus envelope. Tegument proteins serve a number of important functions. They are involved in the cytoplasmic transport of nucleocapsids, nuclear delivery of viral genomes, immune evasion, and transactivation of immediate early (IE) gene transcription (1). Importantly, they also provide a unique opportunity for the virus to flexibly respond to different cell types and states immediately after entry and set the course for a productive or nonproductive infection at the earliest possible stage.A compelling example for a tegument-derived sensor of the host cell milieu is the 71-kDa phosphoprotein (pp71, also referred to as the UL82 gene product "pUL82") of human cytomegalovirus (HCMV, human herpesvirus 5), a widespread human pathogen. Permissiveness for HCMV depends on the cellular differentiation state (2) and is intimately l...

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“…Indeed, HCMV SCP is required for the association of pp150 to the capsid (4). In this scenario, the extensive cementing network formed by SCP and pp150 in cytomegalovirus may provide additional strength that is needed for the encapsidation of its genome, which is the largest among all herpesviruses (49).…”

Section: Discussionmentioning

confidence: 99%

CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid

Dai

Gong

Xiao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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With just one eighth the size of the major capsid protein (MCP), the smallest capsid protein (SCP) of human tumor herpesviruses-Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV)-is vital to capsid assembly, yet its mechanism of action is unknown. Here, by cryoEM of KSHV at 6-Å resolution, we show that SCP forms a crown on each hexon and uses a kinked helix to cross-link neighboring MCP subunits. SCP-null mutation decreased viral titer by 1,000 times and impaired but did not fully abolish capsid assembly, indicating an important but nonessential role of SCP. By truncating the C-terminal half of SCP and performing cryoEM reconstruction, we demonstrate that SCP's N-terminal half is responsible for the observed structure and function whereas the C-terminal half is flexible and dispensable. Serial truncations further highlight the critical importance of the N-terminal 10 aa, and cryoEM reconstruction of the one with six residues truncated localizes the N terminus of SCP in the cryoEM density map and enables us to construct a pseudoatomic model of SCP. Fitting of this SCP model and a homology model for the MCP upper domain into the cryoEM map reveals that SCP binds MCP largely via hydrophobic interactions and the kinked helix of SCP bridges over neighboring MCPs to form noncovalent cross-links. These data support a mechanistic model that tumor herpesvirus SCP reinforces the capsid for genome packaging, thus acting as a cementing protein similar to those found in many bacteriophages.Kaposi's sarcoma-associated herpesvirus | smallest capsid protein | capsid assembly | capsid stabilization | cementing protein H erpesviridae is a large family of dsDNA viruses containing several widespread human pathogens. It is classified into three subfamilies, namely alpha-, beta-, and gammaherpesviruses (1). Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is a member of the oncogenic gammaherpesvirus subfamily (2). All herpesviruses share the same architecture with a genome-containing capsid surrounded by a poorly defined tegument layer, which in turn is enclosed in a lipid envelope. The capsids of different herpesviruses are similar in composition and structure (3-5), each composed of at least five capsid proteins (encoding genes of KSHV given in parentheses): the major capsid protein (MCP, ORF25) in hexameric and pentameric capsomers, triplex proteins 1 and 2 (ORF62 and ORF26, respectively) forming heterotrimers in a 1:2 ratio to connect the capsomers, the smallest capsid protein (SCP, ORF65) previously shown to decorate tips of hexons (6-9), and the portal protein (ORF43) forming a dodecameric structure for DNA genome packaging at one of the 12 icosahedral vertices (10).Assembly pathway of herpesvirus capsid resembles what has been established for dsDNA bacteriophages such as T4, λ, and P22 (11, 12): a spherical, porous intermediate named "procapsid" is first assembled around a core of scaffold proteins; the scaffold proteins are then cut by a maturational protease and ext...

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Biochemical and structural characterization of the capsid-bound tegument proteins of human cytomegalovirus

Cited by 49 publications

References 47 publications

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2–CDK-dependent sensor of the host cell cycle and differentiation state

CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid

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