Resistance of human cytomegalovirus to the benzimidazole L-ribonucleoside maribavir maps to UL27

1-(beta-D-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(beta-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.     

Resistance of Human Cytomegalovirus to Benzimidazole Ribonucleosides Maps to Two Open Reading Frames: UL89 and UL56

2,5,6-Trichloro-1-β-d-ribofuranosyl benzimidazole (TCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA processing and packaging. Resistance to the 2-bromo analog (BDCRB) has been mapped to the UL89 open reading frame (ORF), and this gene product was proposed as the viral target of the benzimidazole nucleosides. In this study, we report the independent isolation of virus that is 20- to 30-fold resistant to TCRB (isolate C4) and the characterization of the virus. The six ORFs known to be essential for viral DNA cleavage and packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced from wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 was identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstrated that each of these mutations individually caused ∼10-fold resistance to the benzimidazoles and that the combination of both mutations caused ∼30-fold resistance. The rate and extent of replication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Mapping of resistance to UL56 supports and extends recent work showing that UL56 codes for a packaging motif binding protein which also has specific nuclease activity (E. Bogner et al., J. Virol. 72:2259–2264, 1998). Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimidazole ribonucleoside binding site. The results also suggest that the gene products of UL89 and UL56 may be antiviral drug targets.Human cytomegalovirus (HCMV) can cause significant morbidity and mortality in immunocompromised populations (3). It is a common opportunistic disease in patients with AIDS and is often a factor in their death (38). HCMV infection has been implicated in increased risk of organ rejection following heart (28) and kidney transplants (8) and in restenosis of diseased arteries following angioplasty (41, 63). It is also a leading cause of birth defects (16).Current therapies for HCMV infection include ganciclovir (GCV) (22), cidofovir (30), and foscarnet (20). Each of these drugs has several limitations to its use: none are orally bioavailable, all have dose-limiting toxicity, and resistance has developed to each (26). Because all three of these drugs inhibit viral replication through an interaction with the virally encoded DNA polymerase (25, 31, 37), the possibility of cross-resistance exists. Thus, additional drugs with unique mechanisms of action are needed for the treatment of HCMV infections.In 1995, we reported that 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB; Fig. ​Fig.1)1) and the 2-chloro analog [2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole TCRB] are potent and selective inhibitors of HCMV replication (55). These compounds have a novel mechanism of action, which unlike the current therapies for HCMV infection, does not involve inhibition of DNA synthesis. The benzimidazole ribonucleosides prevent the cleavage of high-molecular-weight viral DNA concatemers to monomeric genomic lengths (57). Resistance to BDCRB has been mapped to the HCMV UL89 open reading frame (ORF), which, by analogy to gene gp17 from bacteriophage T4, may be a terminase (23, 57). Consequently, we have proposed that the benzimidazole ribonucleosides inhibit the product of this gene and that the UL89 gene product is involved in the viral DNA concatemer cleavage process (57). Open in a separate windowFIG. 1Structure of benzimidazole ribonucleosides. TCRB, R = Cl; BDCRB, R = Br.HCMV replication proceeds in a manner which is conserved among herpesviruses. The virally encoded DNA polymerase produces large, complex head-to-tail concatemers (10, 29, 33) which must be cleaved into genomic-length pieces before insertion into preformed capsids (59). With herpes simplex virus type 1 (HSV-1), temperature-sensitive mutants which are unable to cleave and package the concatemeric DNA have been derived (1, 2, 4, 45, 49, 50, 61). By this process, six HSV-1 genes have been found to be involved in concatemer cleavage and packaging. They are UL6, UL15, UL25, UL28, UL32, and UL33. In addition, recent studies in Homa’s laboratory have established that the product of UL25 is required for viral DNA encapsidation but not cleavage (39). Homologs of these genes exist in HCMV and are UL104, UL89, UL77, UL56, UL52, and UL51, respectively (18).In our continuing investigation of the mode of action of benzimidazole nucleosides, we report herein the independent isolation of HCMV strains resistant to TCRB, characterization of these strains, and identification of the mutations responsible for the development of resistance. The results demonstrate that the mechanism of action of the benzimidazole ribonucleosides is more complex than previously proposed and that a second gene product implicated in DNA cleavage and packaging is involved.     

Identification of acetylated, tetrahalogenated benzimidazole D-ribonucleosides with enhanced activity against human cytomegalovirus

DNA packaging is the key step in viral maturation and involves binding and cleavage of viral DNA containing specific DNA-packaging motifs. This process is mediated by a group of specific enzymes called terminases. We previously demonstrated that the human cytomegalovirus (HCMV) terminase is composed of the large subunit pUL56 and the small subunit pUL89. While the large subunit mediates sequence-specific DNA binding and ATP hydrolysis, pUL89 is required only for duplex nicking. An excellent inhibitor targeting HCMV terminase is 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB), but it was not developed as an antiviral drug due to its metabolic cleavage in experimental animals. We now have tested several new benzimidazole d-ribonucleosides in order to determine whether these compounds represent new, potent inhibitors. Analysis by bioluminometric ATPase activity assays identified two of the new compounds with a high inhibitory effect, 2-bromo-4,5,6-trichloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) benzimidazole (BTCRB) and 2,4,5,6-tetrachloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl benzimidazole (Cl(4)RB). By using viral plaque formation, viral yield, and viral growth kinetics, we demonstrated that the two compounds BTCRB and Cl(4)RB had antiviral activities similar to that of BDCRB. Interestingly, BTCRB retained its inhibitory activity after preincubation with HFF cells. By use of electron microscopy, we observed an increase of B capsids and a lack of cytoplasmic capsids in the presence of the compounds that correlated with the virus yield. Furthermore, cleavage of concatenated DNA was inhibited by both compounds, and inhibition by BTCRB was shown to be dose dependent. These results demonstrate that the new compounds are highly active against HCMV and act by mechanisms similar but not identical to those of BDCRB.     

Dramatic effects of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole riboside on the genome structure, packaging, and egress of guinea pig cytomegalovirus

The halogenated benzimidazoles BDCRB (2-bromo-5,6-dichloro-1-beta-D-riborfuranosyl benzimidazole riboside) and TCRB (2,5,6-trichloro-1-beta-D-riborfuranosyl benzimidazole riboside) were the first compounds shown to inhibit cleavage and packaging of herpesvirus genomes. Both inhibit the formation of unit length human cytomegalovirus (HCMV) genomes by a poorly understood mechanism (M. R. Underwood et al., J. Virol. 72:717-715, 1998; P. M. Krosky et al., J. Virol. 72:4721-4728, 1998). Because the simple genome structure of guinea pig cytomegalovirus (GPCMV) provides a useful model for the study of herpesvirus DNA packaging, we investigated the effects of BDCRB on GPCMV. GPCMV proved to be sensitive to BDCRB (50% inhibitory concentration = 4.7 microM), although somewhat less so than HCMV. In striking contrast to HCMV, however, a dose of BDCRB sufficient to reduce GPCMV titers by 3 logs (50 microM) had no effect on the quantity of GPCMV genomic DNA that was formed in infected cells. Electron microscopy revealed that this DNA was in fact packaged within intranuclear capsids, but these capsids failed to egress from the nucleus and failed to protect the DNA from nuclease digestion. The terminal structure of genomes formed in the presence of BDCRB was also altered. Genomes with ends lacking a terminal repeat at the right end, which normally exist in an equimolar ratio with those having one copy of the repeat at the right end, were selectively eliminated by BDCRB treatment. At the left end, BDCRB treatment appeared to induce heterogeneous truncations such that 2.7 to 4.9 kb of left-end-terminal sequences were missing. These findings suggest that BDCRB induces premature cleavage events that result in truncated genomes packaged within capsids that are permeable to nuclease. Based on these and other observations, we propose a model for duplication of herpesvirus terminal repeats during the cleavage and packaging process that is similar to one proposed for bacteriophage T7 (Y. B. Chung, C. Nardone, and D. C. Hinkle, J. Mol. Biol. 216:939-948, 1990).     

Identification of the interaction domain of the small terminase subunit pUL89 with the large subunit pUL56 of human cytomegalovirus

The small terminase subunit pUL89 of human cytomegalovirus (HCMV) is thought to be required for cleavage of viral DNA into unit-length genomes in the cleavage/packaging process. Immunoprecipitations with a UL89-specific antibody demonstrated that pUL89 occurs predominantly as a monomer of approximate M(r) 75.000 together with a dimer of approximate 150.000. This was confirmed by gel permeation chromatography. In view of its putative function, pUL89 needs to be transported into the nucleus. By use of laser scanning confocal microscopy, pUL89 was found to be predominantly localized throughout the nucleus and in particular in viral replication centers of infected cells. By immunofluorescence, we demonstrated that both terminase subunits co-localized in viral replication centers. Furthermore, analysis with pUL89 GST-fusion protein mutants showed that amino acids 580-600 may represent the interaction domain with pUL56. To verify this result, a recombinant HCMV genome was constructed in which the UL89 open reading frame was disrupted. By transfection of the deletion BACmid alone, we showed that it has a lethal phenotype. Cotransfection assays demonstrated that, in contrast to pUL89 wild-type, a plasmid construct encoding a pUL89 variant without aa 580-590 as well as one encoding a variant without aa 590-600 could not complement the HCMV-pUL89 null genome, thus, suggesting that the 20 aa sequence GRDKALAVEQFISRFNSGYIK is sufficient for the interaction with pUL56 and in conclusion required for DNA packaging.     

Interaction of the putative human cytomegalovirus portal protein pUL104 with the large terminase subunit pUL56 and its inhibition by benzimidazole-D-ribonucleosides

Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-D-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.     

Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus

Human cytomegalovirus (HCMV) terminase is composed of subunits pUL56 (130 kDa) and pUL89 (~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 ~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.     

The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase

Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.     

The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure

Herpesvirus DNA packaging involves binding and cleavage of DNA containing the specific DNA-packaging motifs. Here we report a first characterization of the terminase subunits pUL56 and pUL89 of human cytomegalovirus (HCMV). Both gene products were shown to have comparable nuclease activities in vitro. Under limiting protein concentrations the nuclease activity is enhanced by interaction of pUL56 and pUL89. High amounts of 2-bromo-5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole partially inhibited the pUL89-associated nuclease activity. It was demonstrated that pUL56 is able to bind to nucleocapsids in vivo. Electron microscopy (EM) and image analysis of purified pUL56 revealed that the molecules occurred as a distinct ring-shaped structure with a pronounced cleft. EM analysis of purified pUL89 demonstrated that this protein is also a toroidal DNA-metabolizing protein. Upon interaction of pUL56 with linearized DNA, the DNA remains uncut while the cutting event itself is mediated by pUL89. Using biochemical assays in conjunction with EM pUL56 was shown to (i) bind to DNA and (ii) associate with the capsid. In contrast to this, EM analysis implied that pUL89 is required to effect DNA cleavage. The data provide the first insights into the terminase-dependent viral DNA-packaging mechanism of HCMV.     

Human cytomegalovirus UL97 D605E polymorphism has a high prevalence in immunocompetent Japanese infants and children

There is no existing data on UL97 mutation in human cytomegalovirus (HCMV) isolates obtained from individuals who have never been exposed to ganciclovir (GCV). UL97 codons 439 to 645 from 61 CMV isolates from 61 immunocompetent Japanese infants and children were sequenced directly. No known GCV resistance mutations were found, meaning that the UL97 mutation had resulted from the use of GCV. On the other hand, a mutation at codon 605 (D to E) was frequently identified (56/61: 91.8%). This could be a genetic marker for HCMV in East Asian counties, because of its low prevalence in the strains of HCMV circulating in Western countries.     

Human Cytomegalovirus pUL93 Is Required for Viral Genome Cleavage and Packaging

Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that the absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit-length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging.     

Design, synthesis, and antiviral evaluation of some polyhalogenated indole C-nucleosides

2,5, 6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5, 6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5, 6-dichloro-3-(beta-D-ribofuranosyl)indole (12). 3-(beta-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.     

ATPase activity of the terminase subunit pUL56 of human cytomegalovirus.

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.     

The Gene Product of Human Cytomegalovirus Open Reading Frame UL56 Binds the pac Motif and Has Specific Nuclease Activity

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 these cis-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.Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (2). For example, transplant and AIDS patients may develop life-threatening diseases as a consequence of primary infection or reactivation of latent infection. Present therapeutic approaches are limited, and new strategies that may result from a better understanding of the molecular events involved in viral maturation are needed.The HCMV virion consists of an envelope, an amorphous tegument, and an icosahedral nucleocapsid, which is assembled in the nuclei of infected cells. The precise molecular events of HCMV capsid assembly and subsequent DNA packaging are not well understood. It is generally accepted that viral DNA is packaged into a procapsid consisting of major capsid protein (UL86), minor capsid protein (UL85), minor capsid protein-binding protein (UL46), smallest capsid protein (UL47/48), assembly protein (UL80.5), and proteinase precursor protein (UL80a) (8). The assembly protein is removed during DNA insertion. It is unclear how the concatenated viral DNA contacts empty capsids and is cleaved and packaged into the capsid.Recent studies with herpes simplex virus type 1 (HSV-1) mutants that were temperature sensitive suggest that cleavage of the concatenated DNA does not occur in the absence of packaging (1). One possible model would be the involvement of cleavage packaging protein(s) which could facilitate incorporation of DNA into the procapsid by attaching to a specific motif within the viral genome. With HSV-1, the UL36 gene product (ICP1) and a smaller protein (possibly encoded by UL37) are part of a complex that recognizes the HSV-specific a sequence and are required for cleavage and packaging of viral DNA from concatemers (6, 7). In addition, the HSV-1 ICP 18.5 (UL28) gene product and the pseudorabies virus (PrV) homolog (16) were also reported to play an important role in DNA packaging (1, 14). Addison et al. (1) demonstrated that empty capsids were observed under conditions nonpermissive for the expression of the HSV-1 ICP 18.5 gene product. The HSV-1 ICP 18.5 mutants failed to cleave concatenated viral DNA in noncomplementing cells, suggesting that cleavage and packaging require ICP 18.5. Similar results were reported by Mettenleiter et al. (14) for PrV mutant protein. These observations suggest that the HSV-1 UL36, UL37, and UL28 gene products are involved in cleavage and packaging of concatenated viral DNA.In a recent study, we identified and partially characterized the gene product of HCMV UL56 (4). The HCMV UL56 gene product of 130 kDa is the homolog of the HSV-1 UL28 gene product. It is therefore postulated that UL56 possesses properties comparable to those of HSV-1 UL28, implying an involvement in cleavage and packaging of DNA. The HCMV genomic a sequence is a short sequence located at both termini of the genome and repeated in an inverted orientation at the L-S junction. The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging of progeny viral DNA and circularization of the viral genome. The HCMV a sequence contains two conserved motifs, pac 1 and pac 2, which are required for cleavage and packaging of the viral DNA (18). Both sequence motifs are located on one side of the cleavage site. The pac 1 and pac 2 motifs have an AT-rich core flanked by a GC-rich sequence. During the initial step of viral DNA packaging, a capsid-associated protein may bind to the pac sequences and may be involved in cleavage of the viral DNA concatemer.In this study, electrophoretic mobility shift assays (EMSAs) were performed with DNA probes spanning the region of these cis-acting elements. These studies demonstrate that specific proteins from HCMV-infected nuclear extracts or baculovirus-UL56-infected cell extracts bind to the pac motifs. Using affinity-purified monospecific antibodies, we show that p130 is present in specific DNA-protein complexes containing the pac motifs of the viral genome. Furthermore, evidence is presented for a sequence-specific endonuclease activity of recombinant HCMV p130, using circular plasmid DNA bearing the a sequence as a substrate.     

Linker insertion mutations in the herpes simplex virus type 1 UL28 gene: effects on UL28 interaction with UL15 and UL33 and identification of a second-site mutation in the UL15 gene that suppresses a lethal UL28 mutation

The UL28 protein of herpes simplex virus type 1 (HSV-1) is one of seven viral proteins required for the cleavage and packaging of viral DNA. Previous results indicated that UL28 interacts with UL15 and UL33 to form a protein complex (terminase) that is presumed to cleave concatemeric DNA into genome lengths. In order to define the functional domains of UL28 that are important for DNA cleavage/packaging, we constructed a series of HSV-1 mutants with linker insertion and nonsense mutations in UL28. Insertions that blocked DNA cleavage and packaging were found to be located in two regions of UL28: the first between amino acids 200 to 400 and the second between amino acids 600 to 740. Insertions located in the N terminus or in a region located between amino acids 400 and 600 did not affect virus replication. Insertions in the carboxyl terminus of the UL28 protein were found to interfere with the interaction of UL28 with UL33. In contrast, all of the UL28 insertion mutants were found to interact with UL15 but the interaction was reduced with mutants that failed to react with UL33. Together, these observations were consistent with previous conclusions that UL15 and UL33 interact directly with UL28 but interact only indirectly with each other. Revertant viruses that formed plaques on Vero cells were detected for one of the lethal UL28 insertion mutants. DNA sequence analysis, in combination with genetic complementation assays, demonstrated that a second-site mutation in the UL15 gene restored the ability of the revertant to cleave and package viral DNA. The isolation of an intergenic suppressor mutant provides direct genetic evidence of an association between the UL28 and UL15 proteins and demonstrates that this association is essential for DNA cleavage and packaging.     

Design,Synthesis, and Antiviral Evaluation of Some Polyhalogenated Indole C-nucleosides

2,5,6-Trichloro-1-(β-D-ribofuranosyl)benzimidazole (TCRB), 2-bromo-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BDCRB) and 2-benzylthio-5,6-dichloro-1-(β-D-ribofuranosyl)benzimidazole (BTDCRB) are benzimidazole nucleosides that exhibit strong and selective anti-HCMV activity. Polyhalogenated indole C-nucleosides were prepared as 1-deaza analogs of the benzimidazole nucleosides TCRB and BDCRB. A mild Knoevenagel coupling reaction between an indol-2-thione and a ribofuranose derivative was developed for the synthesis of 2-benzylthio-5,6-dichloro-3-(β-D-ribofuranosyl)indole (12). 3-(β-D-ribofuranosyl)-2,5,6-trichloroindole (16) was prepared from 12 in 4 steps. A Lewis acid-mediated glycosylation method was then developed to prepare the targeted 2-haloindole C-nucleoside 16 stereoselectively in four steps from the corresponding 2-haloindole aglycons. Only 12 was active against HCMV but it also was somewhat cytotoxic.     

Analysis of the quaternary structure of the putative HCMV portal protein PUL104

In this report we analyze the UL104 open reading frame of human cytomegalovirus (HCMV) genome that encodes the putative portal protein. An affinity-purified monospecific antiserum directed against a GST-UL104 fusion protein identified proteins of approximate M(r) 73000 and 145000 in HCMV-infected cells and purified virions. Furthermore, using an in vitro assay the ability of pUL104 to bind double-stranded DNA was shown. Analysis under native conditions of pUL104 revealed that the monomeric and dimeric forms of the protein also form high molecular weight complexes upon sucrose gradient centrifugation. The protein has been purified from recombinant baculovirus UL104 infected cells. The quaternary structure of rpUL104 was investigated by gel permeation chromatography and electron microscopy. The purified rpUL104 was found to assemble into high molecular weight complexes, a prerequisite of portal proteins which form channels for DNA import into capsids.