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.     

Role of a mutation in human cytomegalovirus gene UL104 in resistance to benzimidazole ribonucleosides

The benzimidazole D-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV "terminase" and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA.     

Human cytomegalovirus DNA replicates after early circularization by concatemer formation, and inversion occurs within the concatemer.

To determine the replicative mechanism for human cytomegalovirus (HCMV) DNA, field inversion gel electrophoresis was used to separate HCMV replicative DNAs during lytic infection. Unit-length circular HCMV genomes lacking terminal restriction fragments were detected starting 4 h after infection even when cells were treated with aphidicolin, phosphonoacetic acid, or cycloheximide. Viral DNA synthesis began 24 h after infection and produced large amounts of high-molecular-weight replicative DNA that was a precursor of progeny genomes. Replicative DNA contained rare terminal restriction fragments, and long-arm termini were much less frequent than short-arm termini. Replicative DNA was not composed of unit-length circles because low-dose gamma irradiation of replicative DNA generated numerous random high-molecular-weight fragments rather than unit-length molecules. PacI digestion of replicative DNA from a recombinant HCMV with two closely spaced PacI sites revealed that replicative DNA is concatemeric and genome segment inversion occurs after concatemer synthesis. These results show that after circularization of the parental genome, DNA synthesis produces concatemers and genomic inversion occurs within concatemeric DNA. The results further suggest that concatemers acquire genomic termini during the cleavage/packaging process which preferentially inserts short-arm termini into empty capsids, causing a predominance of short-arm termini on the concatemer.     

A 128-base-pair sequence containing the pac1 and a presumed cryptic pac2 sequence includes cis elements sufficient to mediate efficient genome maturation of human cytomegalovirus

Herpesvirus DNA replication proceeds via concatemeric replicative intermediates that are comprised of head-to-tail linked genomes. Genome maturation is carried out by the terminase, an enzyme complex that mediates both the insertion of concatemer DNA into capsids and its subsequent cleavage to release genomes within these capsids. This cleavage is sequence specific, but the governing cis-acting DNA sequences are only partially characterized. Two highly conserved motifs, the pac1 and pac2 motifs, lie near the ends of herpesvirus genomes and are known to be critical for genome maturation. In murine cytomegalovirus, poorly conserved sequences distal to the pac2 motif up to 150 bp from the point of cleavage are also important for cleavage. Here, we sought to identify the cleavage/packaging signals of human cytomegalovirus. Our results show that a previously proposed pac2-like poly(A) tract is dispensable for cleavage/packaging function and suggest that human cytomegalovirus may utilize a cryptic pac2 motif that lacks a poly(A) tract characteristic of pac2 motifs in other herpesviruses. Additional distal sequences 47 to 100 bp from the point of cleavage were found to enhance cleavage efficiency. These results should facilitate the identification of trans-acting factors that bind to these cis elements and elucidation of their functions. Such information will be critical for understanding the molecular basis of this complex process.     

The ends on herpesvirus DNA replicative concatemers contain pac2 cis cleavage/packaging elements and their formation is controlled by terminal cis sequences

Herpesviruses have large double-stranded linear DNA genomes that are formed by site-specific cleavage from complex concatemeric intermediates. In this process, only one of the two genomic ends are formed on the concatemer. Although the mechanism underlying this asymmetry is not known, one explanation is that single genomes are cleaved off of concatemer ends in a preferred direction. This implies that cis elements control the direction of packaging. Two highly conserved cis elements named pac1 and pac2 lie near opposite ends of herpesvirus genomes and are important for cleavage and packaging. By comparison of published reports and by analysis of two additional herpesviruses, we found that pac2 elements lie near the ends formed on replicative concatemers of four herpesviruses: herpes simplex virus type 1, equine herpesvirus 1, guinea pig cytomegalovirus, and murine cytomegalovirus. Formation of pac2 ends on concatemers depended on terminal cis sequences, since ectopic cleavage sites engineered into the murine cytomegalovirus genome mediated formation of pac2 ends on concatemers regardless of the orientation of their insertion. These findings are consistent with a model in which pac2 elements at concatemer ends impart a directionality to concatemer packaging by binding proteins that initiate insertion of concatemer ends into empty capsids.     

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.     

Characterization of guinea pig cytomegalovirus DNA.

The genome of guinea pig cytomegalovirus (GPCMV) was analyzed and compared with that of human cytomegalovirus (HCMV). GPCMV and HCMV DNAs were isolated from virions and further purified by CsCl centrifugation. Purified GPCMV DNA sedimented as a single peak in a neutral sucrose gradient and was infectious when transfected into guinea pig embryo fibroblast cells. The cytopathology was characteristic of that seen after infection with GPCMV. Virus DNA purified from virions isolated from infected GPEF or 104C1 cells had a CsCl buoyant density of 1.713 g/cm3, which corresponds to a guanine plus cytosine content of 54.1%. The CsCl buoyant density of GPCMV DNA was slightly less than that of HCMV DNA (1.716 g/cm3), but sufficiently different so that the two virus DNA peaks did not coincide. GPCMV DNA cosedimented with T4 DNA in a neutral sucrose gradient. Restriction endonuclease cleavage of GPCMV or HCMV DNAs with HindIII, XbaI, or EcoRI yielded fragments easily separable by agarose gel electrophoresis and ranging from 1.0 X 10(6) to 25.8 X 10(6) daltons. The number, size, and molarity of GPCMV DNA fragments generated by restriction enzymes were determined. Hybridization of restriction endonuclease-cleaved GPCMV DNA with radioactively labeled HCMV DNA and, conversely, hybridization of restriction endonuclease-cleaved HCMV DNA with radioactively labeled GPCMV DNA indicated sequence homology between the two virus DNAs.     

Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.     

Packaging of genomic and amplicon DNA by the herpes simplex virus type 1 UL25-null mutant KUL25NS

The herpes simplex virus type 1 (HSV-1) mutant KUL25NS, containing a null mutation within the UL25 gene, was isolated and characterized by McNab and coworkers (A. R. McNab, P. Desai, S. Person, L. L. Roof, D. R. Thomsen, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998). This mutant was able to cleave the concatemeric products of viral DNA replication into monomeric units, but in contrast to wild-type (wt) HSV-1, they were degraded by DNase treatment, indicating that they were not stably packaged into virus capsids. I have examined the packaging of the KUL25NS genome and an HSV-1 amplicon in cells infected with the mutant virus. In contrast to the previous results, a low level of KUL25NS DNA was resistant to DNase digestion, indicating that it was retained in capsids. The proportion of this packaged DNA present as full-length genomes was much lower than in cells infected by wt HSV-1, and there was a significant overrepresentation of the long terminus and underrepresentation of the short terminus. KUL25NS was less impaired in stably packaging amplicon DNA than in packaging its own genome, and the packaged molecules contained approximately equimolar amounts of the two terminal fragments. Below about 100 kbp, the packaged amplicon molecules exhibited an abundance and size distribution similar to those generated using wt HSV-1 as a helper, but the mutant was relatively impaired in packaging longer amplicon molecules. Both packaged genomic and amplicon DNAs were retained in the nuclei of KUL25NS-infected cells. These results suggest that the UL25 protein may play an important role during the later stages of the head-filling process, prior to release of capsids into the cytoplasm.     

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.     

New endogenous herpesvirus of guinea pigs: biological and molecular characterization.

Two known guinea pig herpesviruses, guinea pig cytomegalovirus (GPCMV) and guinea pig herpes-like virus (GPHLV), and well characterized. A third herpesvirus (GPXV) was originally isolated from leukocytes of healthy strain 2 guinea pigs. Growth of GPXV in guinea pig embryo fibroblastic cells produced a characteristic cytopathic effect. Electron microscopy of guinea pig cells infected with GPXV revealed the morphological development of a herpesvirus. Cross-neutralization tests and immunoferritin electron microscopy demonstrated that GPXV, GPCMV, and GPHLV were serologically distinct herpeviruses of guinea pigs. To confirm the distinction between these three herpesviruses, DNA genomes were compared by CsCl equilibrium buoyant density measurements and restriction endonuclease cleavage analysis. 32P-labeled viral DNA ws obtained from nucleocapsids isolated from virus-infected cells, and the buoyant density of GPXV DNA differed from that of GPCMV and GPHLV. Cleavage of viral DNAs with restriction endonucleases followed by gel electrophoresis revealed distinct patterns for each virus.     

Assembly of herpes simplex virus capsids using the human cytomegalovirus scaffold protein: critical role of the C terminus.

An essential step in assembly of herpes simplex virus (HSV) type 1 capsids involves interaction of the major capsid protein (VP5) with the C terminus of the scaffolding protein (encoded by the UL26.5 gene). The final 12 residues of the HSV scaffolding protein contains an A-X-X-F-V/A-X-Q-M-M-X-X-R motif which is conserved between scaffolding proteins found in other alphaherpesviruses but not in members of the beta- or gamma-herpesviruses. Previous studies have shown that the bovine herpesvirus 1 (alphaherpesvirus) UL26.5 homolog will functionally substitute for the HSV UL26.5 gene (E. J. Haanes et al., J. Virol. 69:7375-7379, 1995). The homolog of the UL26.5 gene in the human cytomegalovirus (HCMV) genome is the UL80.5 gene. In these studies, we tested whether the HCMV UL80.5 gene would substitute for the HSV UL26.5 gene in a baculovirus capsid assembly system that we have previously described (D. R. Thomsen et al., J. Virol. 68:2442-2457, 1994). The results demonstrate that (i) no intact capsids were assembled when the full-length or a truncated (missing the C-terminal 65 amino acids) UL80.5 protein was tested; (ii) when the C-terminal 65 amino acids of the UL80.5 protein were replaced with the C-terminal 25 amino acids of the UL26.5 protein, intact capsids were made and direct interaction of the UL80.5 protein with VP5 was detected; (iii) assembly of intact capsids was demonstrated when the sequence of the last 12 amino acids of the UL80.5 protein was changed from RRIFVA ALNKLE to RRIFVAAMMKLE; (iv) self-interaction of the scaffold proteins is mediated by sequences N terminal to the maturation cleavage site; and (v) the UL26.5 and UL80.5 proteins will not coassemble into scaffold structures. The results suggest that the UL26.5 and UL80.5 proteins form a scaffold by self-interaction via sequences in the N termini of the proteins and emphasize the importance of the C terminus for interaction of scaffold with the proteins that form the capsid shell.     

Characterization of the guinea pig cytomegalovirus genome by molecular cloning and physical mapping.

Fragments of guinea pig cytomegalovirus (GPCMV) DNA produced by HindIII or EcoRI restriction endonuclease digestion were cloned into vectors pBR322 and pACYC184, and recombinant fragments representing ca. 97% of the genome were constructed. Hybridization of 32P-labeled cloned and gel-purified HindIII, EcoRI, and XbaI fragments to Southern blots of HindIII-, EcoRI-, and XbaI-cleaved GPCMV DNA verified the viral origin of cloned fragments and allowed construction of HindIII, EcoRI, and XbaI restriction maps. On the basis of the cloning and mapping experiments, the size of GPCMV DNA was calculated to include 239 kilobase pairs, corresponding to a molecular weight of 158 X 10(6). No cross-hybridization between any internal fragments was seen. We conclude that the GPCMV genome consists of a long unique sequence with terminal repeat sequences but without internal repeat regions. In addition, GPCMV DNA molecules exist in two forms. In the predominant form, the molecules demonstrate sequence homology between the terminal fragments; in the minor population, one terminal fragment is smaller by 0.7 X 10(6) daltons and is not homologous with the fragment at the other end of the physical map. The structural organization of GPCMV DNA is unique for a herpesvirus DNA, similar in its simplicity to the structure reported for murine cytomegalovirus DNA and quite dissimilar from that of human cytomegalovirus DNA.     

Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging

Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.     

Characterization of ICP6::lacZ insertion mutants of the UL15 gene of herpes simplex virus type 1 reveals the translation of two proteins.

The herpes simplex virus type 1 (HSV-1) UL15 gene is a spliced gene composed of two exons and is predicted to encode an 81-kDa protein of 735 amino acids (aa). Two UL15 gene products with molecular masses of 75 and 35 kDa have been observed (J. Baines, A. Poon, J. Rovnak, and B. Roizman, J. Virol. 68:8118-8124, 1994); however, it is not clear whether the smaller form represents a proteolytic cleavage product of the larger form or whether it is separately translated. In addition, an HSV-1 temperature-sensitive mutant in the UL15 gene (ts66.4) is defective in both cleavage of viral DNA concatemers into unit-length monomers and packaging of viral DNA into capsids (A. Poon and B. Roizman, J. Virol. 67:4497-4503, 1993; J. Baines et al., J. Virol. 68:8118-8124, 1994). In this study, we detected two UL15 gene products of 81 and 30 kDa in HSV-1-infected cells, using a polyclonal antibody raised against a maltose binding protein fusion construct containing UL15 exon 2. In addition, we report the isolation of two HSV-1 insertion mutants, hr81-1 and hr81-2, which contain an ICP6::lacZ insertion in UL15 exon 1 and exon 2 and thus would be predicted to encode C-terminally truncated peptides of 153 and 509 aa long, respectively. hr81-1 and hr81-2 are defective in DNA cleavage and packaging and accumulate only B capsids. However, both mutants are able to undergo wild-type levels of DNA replication and genomic inversion, suggesting that genomic inversion is a result of DNA replication rather than of DNA cleavage and packaging. We also provide evidence that the 81- and 30-kDa proteins are the products of separate in-frame translation events from the UL15 gene and that the 81-kDa full-length UL15 protein is required for DNA cleavage and packaging.     

The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome

Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.     

Cryomicroscopy of human cytomegalovirus virions reveals more densely packed genomic DNA than in herpes simplex virus type 1

All members of the herpesvirus family have a characteristic virion structure, comprising a DNA containing, icosahedral capsid, embedded in a proteinaceous layer (tegument) and surrounded by a lipid envelope. Human cytomegalovirus (HCMV, the prototypic beta-herpesvirus) has a genome that is significantly larger (>50 %) than that of the alpha-herpesvirus HSV-1. Although the internal volume of the HCMV capsid is approximately 17 % larger than that of HSV-1, this slight increase in volume does not provide adequate space to encapsidate the full length HCMV genome at the same packing density as HSV-1.We have investigated the nature of DNA packing in HCMV and HSV-1 virions by electron-cryomicroscopy and image processing. Radial density profiles calculated from projection images of HCMV and HSV-1 capsids suggest that there is no increase in the volume of the HCMV capsid upon DNA packaging. Packing density of the viral DNA was assessed for both HCMV and HSV-1 by image analysis of both full and empty particles. Our results for packing density in HSV-1 are in good agreement with previously published measurements, showing an average inter-layer spacing of approximately 26 A. Measurements taken from our HCMV images, however, suggest that the viral genomic DNA is more densely packed, with an average inter-layer spacing of approximately 23 A. We propose therefore, that the combination of greater volume in HCMV capsids and increased packing density of viral DNA accounts for its ability to encapsidate a large genome.     

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.