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

Abstract

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...

Human Cytomegalovirus pUL93 Links Nucleocapsid Maturation and Nuclear Egress

“…We recently discovered that pUL93 is required for viral genome cleavage and packaging, similar to its homolog pUL17 in HSV-1 (17). These findings are supported by another recent study which found that both pUL93 and pUL77 are required for genome encapsidation (18). pUL77 has been found to bind double-stranded DNA and interact with DNA-packaging motor components as well as with major capsid protein, suggesting that pUL77 helps retain viral DNA during capsid assembly (19).…”

“…Mouse monoclonal antibody to ␣-tubulin (MA1 19401; Fisher Scientific, Waltham, MA) was used to confirm equal loading of SDS gels in IB experiments. For co-IP and IB in the infection setting, mouse monoclonal anti-UL93 antibody, kindly provided by Eva Maria Borst (18), mouse monoclonal anti-lamin A/C antibody (MA3 1000; Fisher Scientific, Waltham, MA), and rabbit anti-UL50, anti-UL53, and anti-UL97 antibodies, kindly provided by Don Coen (9), were used as the primary antibodies. Peroxidase-labeled goat anti-mouse IgG (PI31444; Fisher Scientific, Waltham, MA) or peroxidase-labeled goat anti-rabbit IgG (PI31460; Fisher Scientific, Waltham, MA) was used as the secondary antibody in IB.…”

Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

“…Since no complementing cell lines are available to reconstitute the corresponding virus mutants, we applied the recently described adenovirus-mediated transfection protocol (adenofection) which allows direct investigation of BAC-transfected cells. During previous analyses, we have shown that upon adenofection, viral gene expression occurs with kinetics similar to that of infected cells, that the majority of adenofected cells successfully enters the late phase of the HCMV infection cycle, and that adenofection is well suited to study the consequences of deletion of various essential genes from the viral genome (18,46). Human telomerase reverse transcriptase-immortalized-RPE-1 cells (RPE-1 cells) were adenofected with the recombinant HCMV BAC genomes lacking either UL51, UL56, or UL89 or with the parental BAC pHG-UL51-SF/HA.…”

“…Overall, our data argue in favor of a model of the HCMV terminase as a multiprotein complex in which the individual subunits stabilize each other upon assembly. Such a setting was also proposed for other CMV protein complexes, namely, for the murine CMV (MCMV) US22 protein family (60) and for the HCMV major and small capsid proteins (18), as well as for the cellular anaphase-promoting complex, a multiprotein assembly targeted by HCMV (61).…”

“…These essential proteins are pUL52, whose specific role is not defined yet (17), as well as pUL77 and pUL93, which are capsid constituents presumably building the capsid vertex-specific complex (18)(19)(20)(21). Moreover, we recently identified pUL51 as a third component of the HCMV terminase, as it forms a complex with pUL56 and pUL89 in infected cells and was found to be crucial for viral genome cleavage-packaging (22).…”