Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.

Surface biotinylation of human cytomegalovirus (HCMV)-infected fibroblasts under pulse-chase conditions was used to define the cellular route of the dominant viral envelope glycoprotein gB into the cytoplasmic compartment of viral maturational envelopment. The results showed that a major fraction of gB was re-internalized from the infected cell surface prior to incorporation into the viral envelope. Viral particles carrying biotinylated gB were subsequently released into the culture medium. Viral release appeared to be inhibited in the presence of gB-specific antibody or when infected cultures were incubated at room temperature, but was not reduced by inhibitors of cellular glycoprotein transport. To our knowledge this is the first report describing that HCMV gB is retrieved from the infected cell surface prior to viral envelopment.

Herpesviral DNA packaging is a complex process involving binding and cleavage of DNA containing the specific DNA-packaging motifs, pac1 and pac2, and packaging of the resulting unit-length genomes into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of human cytomegalovirus the terminase consists of the proteins pUL56 and pUL89. While pUL56 (i) mediates the specific binding to pac sequences on the concatamers, (ii) provides energy for the translocation of the DNA to the procapsids and (iii) associates itself with the capsid for enabling the entry of the DNA into the procapsid, pUL89 is mainly required to effect DNA cleavage. Based on the limited efficacy of the current drugs ganciclovir, cidofovir and foscarnet, new antiviral therapeutics appear to be in demand. Inhibitors targeting pUL56 and/or pUL89 may offer an attractive alternative since mammalian cell DNA replication does not involve cleavage of concatameric DNA. Drugs targeted to terminase-like proteins should therefore be safe and highly selective.

Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity. Human cytomegalovirus (HCMV)1 DNA replication results in the formation of large head-to-tail DNA concatemers. The subsequent maturation into unit-length molecules involves site-specific cleavage at sequence motifs (pac motifs) located within the a sequence of the terminal and internal repeat segments (1-4). Unit-length genomes are then encapsidated into preassembled capsids. DNA-packaging is a complex biological process. While it is commonly accepted that ATP hydrolysis is the driving force behind it, the molecular mechanisms of DNA translocation and genome packaging remain unclear. In general, the following five steps are involved. (i) The recognition of concatemeric DNA by a specific protein (complex) able to (ii) bind and cut the DNA at specific sequence motifs (packaging signals, e.g. pac1 and pac2), (iii) translocation of the DNAprotein complex into the procapsid, (iv) packaging of one unitlength genome, and (v) completion of the packaging process by cutting off excess DNA at the portal region (5-7).Recently, we have demonstrated that the HCMV pUL56 gene product (pUL56), the homolog of the HSV-1 open reading frame UL28, is associated with sequence-specific binding of DNA containing packaging motifs leading to the suggestion that pUL56 plays a key role in DNA packaging (7-9). Comparable results were reported of the HSV-1 pUL28, demonstrating a direct interaction of pUL28 with DNA containing the pac1 motif (10). Furthermore, by the use of viral mutants it was demonstrated that the deletion of pUL28 leads to nuclear accumulation of naked nucleocapsids and uncleaved concatemeric DNA (11, 12). In addition, it has been noted that the HCMV UL...

Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.

Human cytomegalovirus (HCMV) terminase is composed of subunits pUL56 (130 kDa) and pUL89 ( approximately 75 kDa), encoded by the UL56 and UL89 genes. In a recent investigation, we demonstrated that the main ATPase activity is associated with the large terminase subunit pUL56. The protein has two putative ATP-binding sites, which were suggested to be composed of the sequence (amino acids 463-470) for ATP-binding site 1 and YNETFGKQ (amino acids 709-716) for the second site. We now demonstrate using a 1.5 kb fragment encoding the C-terminal half of pUL56 that ATP-binding site 1 is not critical for the function, whereas ATP-binding site 2 is required for the enzymatic activity. Mutation G714A in this protein reduced the ATPase activity to approximately 65% and the double mutation G714A/K715N showed a reduction up to 75%. However, the substitution of E711A revoked the effect of the substitutions. The functional character of the ATP-binding site was demonstrated by transfer of YNETFGKQLSIACLR (709-723) to glutathione-S-transferase (GST). Interestingly, vanadate, an ATPase inhibitor, has the ability to block the ATPase activity of pUL56 as well as of Apyrase, while the antitumor ATP-mimetic agent geldanamycin, did not affect the ATP-binding of pUL56. Furthermore, in contrast to an inactive control compound, the specific HCMV terminase inhibitor BDCRB showed a partial inhibition of the pUL56-specific ATPase activity. Our results clearly demonstrated that (i) the enzymatic activity of the terminase subunit pUL56 could be inhibited by vanadate, (ii) only the ATP-binding site 2 is critical for the pUL56 function and (iii) glycine G714 is an invariant amino acid.

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in thesecis-acting motifs, 5′-TAAAAA-3′ (pac 1) and 5′-TTTTAT-3′ (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.