The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

Abstract

dCleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

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

“…To this end, PCR products were generated with the primers UL93-N.for (5=-CGCGGATCCTTCTATGCCGTCTTCACTACG-3=) and UL93-N.rev (5=-CGCAAGCTTGCGACTGCGCCAAAAGGAATT-3=) or UL93-C.for (5=-CGCGGATCCGAGCTGAGCTACGATGACCAC-3=) and UL93-C.rev (5=-CGCAAGCTTAAGATCGTCGAACGGCAAGC G-3=) and pHB5 as the template and were cloned into plasmid pQE-30 (Qiagen, Hilden, Germany), giving rise to pQE-UL93-N and pQE-UL93-C. Expression of the recombinant proteins pUL93-N and pUL93-C and purification, immunization of mice, and generation of hybridoma cultures was done as reported previously (18). Antibodies reactive with the recombinant proteins were finally evaluated with HCMV-infected cells by immunoblotting (IB) and immunofluorescence (IF) microscopy done as described previously (18,21,29).…”

“…Expression of the recombinant proteins pUL93-N and pUL93-C and purification, immunization of mice, and generation of hybridoma cultures was done as reported previously (18). Antibodies reactive with the recombinant proteins were finally evaluated with HCMV-infected cells by immunoblotting (IB) and immunofluorescence (IF) microscopy done as described previously (18,21,29). Hybridoma cultures secreting pUL93-specific antibodies were only obtained after immunization with pUL93-N, but not with pUL93-C. Other antibodies used were GFP mouse MAb (Santa Cruz; catalog no.…”

“…Polyclonal rabbit sera raised against the HCMV mCP or the mCP-BP were generously provided by Wade Gibson (Johns Hopkins University, Baltimore, MD). Generation of MAbs specific for UL52, UL56, and UL89 was described previously (18). The kinetic class of HCMV protein expression was determined by applying inhibitors of early or late gene expression (cycloheximide/ actinomycin D or phosphonoacetic acid) as given elsewhere (21).…”

“…These are as follows: (i) the terminase subunits pUL56 and pUL89, which bind to the concatemeric viral DNA and cleave it into unitlength genomes (15)(16)(17); (ii) pUL51, which interacts with pUL56 and pUL89 and may represent a third subunit of the terminase complex (18); (iii) the portal protein pUL104, present at one capsid vertex (19,20); and (iv) pUL52, whose function is elusive, yet essential for genome cleavage-packaging (21). For only two of those proteins, pUL51 and pUL52, are HCMV mutants available that allow analysis of the phenotypic consequences caused by the lack of either of these proteins (18,21). This is in part due to the difficulties in generating complementing systems for essential HCMV proteins (22).…”

Treatment of CMV infection after allogeneic hematopoietic stem cell transplantation

“…Member of the new antiviral class of quinazolines, it acts after viral DNA synthesis by inhibiting the subunit protein pUL56 that, together with pUL89, is a key element of the enzyme complex named terminase, [72,73] directly involved in the cleavage and package of viral DNA chains in the virionic capside [74,75]. Because of its distinct mechanism of action, it does not show cross-resistance with other antiviral drugs and there are reports of clinically relevant activity against GCV-, FOS-and CDF-resistant CMV strains [76,77].…”