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.

Importance: The 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.

FIG 1

Characteristics of HCMV mutants expressing a UL77-mGFP fusion protein. (A) Construction of the recombinant HCMV BACs pHB5-UL77-mGFP-2 and pHB5-UL77-mGFP-3, which differ in the position of the mGFP sequence inserted into the UL77 open reading frame (ORF). The schematic drawing of the respective BACs is given to the right (pHB5, parental genome), with the expected BglII restriction fragments indicated as black lines. Gray boxes represent repeat regions of the HCMV genome. The left part shows the BglII restriction pattern of the parental BAC (lane 1) compared to that of the mutant genomes (lanes 2 and 3). (Please note the shift of a 7.5-kbp fragment in pHB5 to 8.3 kbp in the UL77-mGFP mutants.) (B) Growth kinetics of the parental virus HB5 (black diamonds) and the HCMV mutants expressing the UL77-mGFP fusion proteins (open triangles and squares, respectively). Fibroblasts were infected at a multiplicity of infection (MOI) of 0.1, supernatants were taken at the time points indicated postinfection, and titers were determined by plaque assay. The values for time point 0 indicate the inoculation doses. (C) Expression of the UL77-mGFP fusion protein and the UL93 protein after infection of fibroblasts with HB5-UL77-mGFP-3 (MOI of 1). Cell lysates were prepared on days 1 to 5 p.i. and analyzed for the indicated proteins by immunoblotting. pUL77-mGFP was detected with a GFP-specific antibody. The nonspecific reactivity with a cellular protein is marked with an open circle. The HCMV early protein pUL44 served as control for viral gene expression and GAPDH as a loading control. Molecular mass markers (in kildodaltons) are given to the left. n.i., noninfected fibroblasts. The asterisk denotes the putative 48-kDa version of pUL93. (D) Expression kinetics of the UL77-mGFP and UL93 proteins. IE, immediate early (i.e., treatment with cycloheximide/actinomycin D); E, early (i.e., treatment with phosphonoacetic acid); L, late (i.e., protein expression in the absence of inhibitors). Fibroblasts were infected with HB5-UL77-mGFP-3 at an MOI of 1, kept with or without the respective inhibitors, followed by preparation of cell lysates on day 3 p.i. and analysis by immunoblotting. IE1, UL44, and UL52 are representative IE, E, and L viral proteins. The open arrow depicts the full-length UL93 ORFs (codons 1 to 594). A putative internal initiation codon at position 173 would give rise to the synthesis of a 48-kDa protein. The part of the UL93 ORF marked in gray was used to express a recombinant protein employed for generation of the specific antibody.

FIG 2

Subcellular localization of pUL77-mGFP in infected cells. (A) Fibroblasts were infected with HB5-UL77-mGFP-3 (MOI, 0.1) and fixed on days 2 to 6 p.i. The UL77-mGFP fusion protein was detected by fluorescence microscopy, and labeling of the viral IE1 protein with a specific Ab was used to delineate the nuclei of infected cells. (B) Fibroblasts infected with HB5-UL77-mGFP-3 at an MOI of 0.1 were fixed on day 5 p.i. The UL77-mGFP fusion protein was detected via the GFP fluorescence, To-Pro-3 was used to stain nucleic acid, and capsid proteins were visualized with Abs directed against the major capsid protein (MCP; upper panel) or the small capsid protein (SCP; lower panel). Scale bars, 5 μm.

FIG 3

Detection of pUL77-mGFP and pUL93 in nuclear capsids. Capsids isolated from the nuclei of fibroblasts infected with HB5-UL77-mGFP-3 or the parental virus HB5 (MOI of 1) were visualized by negative staining and electron microscopy (scale bars, 50 nm) (A) and by immunoblotting (B) for the presence of the indicated proteins. Lysates of HB5-UL77-mGFP-3-infected fibroblasts and of noninfected cells served as controls. The 48-kDa UL93 product is marked with an asterisk. Two percent of the cell lysates and 8% of the capsid preparations were loaded. (C) Immunogold labeling of isolated nuclear capsids using a GFP antibody followed by EM (top). The gold particles attached to the different capsid types were quantified as shown in the lower part (“total,” label on all capsid types). GuHCl, treatment with guanidinium hydrochloride. The numbers of capsids analyzed are as follows: HB5 total, 41; 77GFP total, 56; 77GFP A, 26; 77GFP B, 7; 77GFP C, 23; and 77GFP total plus GuHCl (right), 15. For statistical analysis, the unpaired t test was applied. ***, P < 0.0001. Scale bars, 50 nm. (D) Comparison of pUL77-mGFP and pUL93 amounts of gradient-purified A and B capsids by immunoblotting. Following adjustment to equal MCP levels, the samples were loaded in duplicates onto the gel.

FIG 4

Detection of pUL77-mGFP and pUL93 in virus particles and identification of viral proteins associated with pUL77-mGFP. (A) Virus particles purified from the supernatant of fibroblasts infected with the parental virus HB5 or the HB5-UL77-mGFP mutants (MOI of 1) were analyzed by immunoblotting (right). The lysates of cells the virions were derived from were used for comparison (left). Two percent of the cell lysates and 10% of the virus preparations were analyzed. (B) Investigation into pUL77-mGFP and pUL93 levels in gradient-purified A and B capsids and virions by immunoblotting. Each sample was loaded twice onto the gel, and signals were quantified with the ImageJ 1.46r software, using membranes exposed for a few seconds only. The lower part depicts the relative abundance of pUL77-mGFP and pUL93 as obtained after dividing the respective signals by the MCP signal. (C) Live-cell image of HFFs infected with HB5-UL77-mGFP-3 on day 5 p.i. The dotted line marks the cell nucleus, and the arrow points to the viral assembly compartment. (D) Viral proteins associated with the UL77-mGFP fusion protein in infected cells. Fibroblasts were infected with HB5 or HB5-UL77-mGFP-3 at an MOI of 1 and lysed on day 5 p.i. pUL77-mGFP was pulled down using GFP-trap beads and copurifying proteins were analyzed by immunoblotting. One percent of the cell lysates before pulldown (input) and 8% of the material eluted from the beads (bound) were loaded onto the gels. An asterisk indicates the 48-kDa version of pUL93.

FIG 5

Localization and interaction of pUL77-mGFP in the absence of individual proteins of the encapsidation network. (A) Subnuclear distribution of the UL77-mGFP fusion protein in RPE-1 cells adenofected with the parental HB5 BAC, the HB5-UL77-mGFP-3 BAC, or mutant BAC genomes derived thereof. pUL77-mGFP was detected by GFP fluorescence, and viral replication compartments were visualized with an antibody specific for the terminase subunit pUL89. For all images, the same microscope settings were used. Scale bars, 10 μm. (B) Immunoblot analysis of total lysates of RPE-1 cells 5 days after adenofection with the indicated BACs. Please note that the pUL52 and pp150 MAbs exhibit some cross-reactivity with cellular proteins of RPE-1 cells migrating with similar mobility to the respective viral protein. The presence of capsids in the respective cell lysates as assessed by negative staining and EM is depicted at the bottom. The scheme at the bottom shows the UL93 ORF of the ΔUL93 mutant carrying an inserted stop codon (asterisk), which results in the synthesis of a truncated 42-kDa protein. (The gray box depicts the part used to generate the pUL93-specific MAb.) (C) Pulldown of pUL77-mGFP from the given cell lysates by GFP-trap followed by investigation of viral proteins associated with pUL77-mGFP by immunoblotting.

FIG 6

Analysis of genome cleavage and capsid formation in the absence of pUL77 or pUL93. (A) Total DNA of RPE-1 cells adenofected with the parental HG BAC or the ORF UL77- or UL93-deleted genomes was analyzed on day 5 posttransfection by pulsed-field gel electrophoresis followed by Southern blotting using an HCMV-specific probe. (B) The RPE-1 cells were adenofected as in panel A and examined by EM for the presence of different capsid species in the nucleus. Open arrowhead, A capsid; gray arrowhead, B capsid; black arrowhead, DNA-filled C capsid. Scale bar, 100 nm. (C) Quantification of capsid types shown in panel B. The number of capsids per cell nucleus (n = 12 cells, with each dot representing a section through one nucleus) was determined, and the percentage of the capsid types (A, B, and C) is depicted (percentage of total capsids).