Nuts and bolts of human cytomegalovirus lytic DNA replication.

HCMV lytic DNA replication is complex and highly regulated. The cis-acting lytic origin of DNA replication (oriLyt) contains multiple repeat motifs that comprise two main functional domains. The first is a bidirectional promoter element that is responsive to UL84 and IE2. The second appears to be an RNA/DNA hybrid region that is a substrate for UL84. UL84 is required for oriLyt-dependent DNA replication along with the six core proteins, UL44 (DNA processivity factor), UL54 (DNA polymerase), UL70 (primase), UL105 (helicase), UL102 (primase-associated factor) and UL57 (single-stranded DNA-binding protein). UL84 is an early protein that shuttles from the nucleus to the cytoplasm, binds RNA, suppresses the transcriptional activation function of IE2, has UTPase activity and is proposed to be a member of the DExH/D box family of proteins. UL84 is a key factor that may act in concert with the other core replication proteins to initiate lytic replication by altering the conformation of an RNA stem loop structure within oriLyt. In addition, new data suggests that UL84 interacts with at least one member of the viral replication proteins and several cellular encoded proteins.

Generation of a life-expanded rhesus monkey fibroblast cell line for the growth of rhesus rhadinovirus (RRV).

RRV, the rhesus macaque equivalent to HHV-8 or kaposi's sarcoma-associated herpesvirus (KSHV) was recently isolated from a simian immunodeficiency virus (SIV) infected macaque with a lymphoproliferative disorder. The growth of RRV in tissue culture requires propagation of primary rhesus monkey fibroblasts (RFs). In an effort to extend the life of these primary cells in tissue culture, the catalytic subunit of telomerase (hTERT) was introduced into RF cells using a recombinant retrovirus. This new cell line, Telo-RFs, have currently been passed in tissue culture over 80 times compared to a maximum passage number of 38 for wild type RFs, remain fully permissive for RRV DNA replication and production of infectious virus. Viral gene expression of immediate-early and early RNA transcripts was virtually identical to that observed in wild-type (wt) RFs. In addition, transfection experiments show that telo-RFs are easily and more efficiently transfected than wtRFs.

Identification of the rhesus macaque rhadinovirus lytic origin of DNA replication.

We have identified a lytic origin of DNA replication (oriLyt) for rhesus macaque rhadinovirus (RRV), the rhesus macaque homolog of human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. RRV oriLyt maps to the region of the genome between open reading frame 69 (ORF69) and ORF71 (vFLIP) and is composed of an upstream A+T-rich region followed by a short (300-bp) downstream G+C-rich DNA sequence. A set of overlapping cosmids corresponding to the entire genome of RRV was capable of complementing oriLyt-dependent DNA replication only when additional ORF50 was supplied as an expression plasmid in the transfection mixture, suggesting that the level of ORF50 protein originating from input cosmid DNA was insufficient. The requirement of RRV ORF50 in the cotransfection replication assay may also suggest a direct role for this protein in DNA replication. RRV oriLyt shares a high degree of nucleotide sequence and G+C base distribution with the corresponding loci in HHV-8.

A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency.

Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCDelta97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in trans in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, the UL97 gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.

Identification of persistent RNA-DNA hybrid structures within the origin of replication of human cytomegalovirus.

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the cis-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites XhoI at nucleotide (nt) 93799 and SacI at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the SphI (nt 92636) and BamHI (nt 93513) and is approximately 300 bases long. This region was previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.

Generation of a nude mouse tumor model for in vivo replication of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a common opportunistic infection resulting in retinitis in 15%-40% of AIDS patients. Several anti-HCMV therapies are currently available, and new treatments are in various stages of development. An HCMV animal model involving in vivo infection of human cells without the dependence on human fetuses or multiple surgical procedures has been developed. A human glioblastoma cell line that is permissive for HCMV replication (U373MG) was adapted to grow as a subcutaneous tumor in nude mice. These tumors arise in approximately 7 days and grow progressively. An evaluation of HCMV DNA replication demonstrated an increase in the accumulation of HCMV DNA within infected tumors from 48 to 168 h after infection. Immunohistochemical analysis showed focal areas of HCMV infection in which expression of immediate-early and late antigens was detected. In addition, it was demonstrated that ganciclovir inhibited HCMV DNA replication in vivo in a dose-dependent manner.

Antisense approach for the treatment of cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common viral opportunistic infection in patients suffering with acquired immunodeficiency virus (AIDS). HCMV is a systemic infection that may infect several sites in the body, including the retina, gastrointestinal tract, lungs, liver, and central nervous system. Retinitis is the most frequent manifestation of HCMV infection, occurring in 15-40% of all patients. HCMV is progressive and destroys the retina, eventually leading to blindness. Although, there are several drugs available to treat this disease, they are often of limited efficacy and have significant side-effects. Antisense oligonucleotides represent a novel alternative to the currently available drugs. Due to their high affinity and specificity to target the HCMV RNAs, interest in antisense technology to treat HCMV infections has been intense during the past few years. Two antisense drugs are currently in clinical trials, ISIS 2922 (Formivirsen) and GEM 132.

Characterization of the human cytomegalovirus UL105 gene and identification of the putative helicase protein.

We have characterized the transcription unit for the human cytomegalovirus UL105 gene and identified its putative protein product. The UL105 gene product is proposed to mediate helicase activity in the assembled helicase-primase complex. The two other putative proteins in this complex are the gene products of UL102 (primase-associated factor) and UL70 (primase). Using Northern blot analysis we have determined that the UL105 transcript is a 3.4-kb message that can be detected as early as 24 hr postinfection in the presence of phosphonoformic acid but not in the presence of cycloheximide. Subsequent primer-extension analysis showed a transcriptional start site upstream of a consensus TATA sequence and just downstream of a CCAAT box sequence motif. In addition, we have identified an infected cell protein with an approximate molecular weight (M(r)) of 110 kDa using anti-peptide antiserum. This same antiserum detected proteins of the same M(r) as those produced from two expression systems where the protein from the 981-amino-acid UL105 open reading frame was overexpressed, suggesting that the ATG located at nt 151,850 of the genomic sequence is utilized in the context of the virus genome.

Potent antiviral activity of an antisense oligonucleotide complementary to the intron-exon boundary of human cytomegalovirus genes UL36 and UL37.

An antisense phosphorothioate oligonucleotide complementary to the intron-exon boundary of human cytomegalovirus genes UL36 and UL37 (UL36ANTI) reduced the yield of infectious virus by 99% and inhibited human cytomegalovirus DNA replication at a concentration of 0.08 microM. In addition, oligonucleotides with base substitutions which resulted in base pair mismatches showed lesser degrees of activity, indicating a sequence-specific antisense mechanism. UL36ANTI was also shown to inhibit DNA replication of ganciclovir-resistant strains and human cytomegalovirus clinical isolates.

Human cytomegalovirus UL102 gene.

We have identified and characterized the transcript for the human cytomegalovirus (HCMV) UL102 gene. The UL102 gene product is proposed to encode the primase-associated factor. The primase-associated factor is one of the three components of the helicase-primase complex, along with UL105 (helicase) and UL70 (primase). In order to characterize the UL102 transcription unit we used single-stranded antisense RNA probes to identify an abundant 2.7-kb transcript originating from the UL102 region. This transcript can be initially detected at 24 h postinfection and in the presence of phosphonoformic acid but not in the presence of cycloheximide. A 2.7-kb cDNA clone containing this transcript was isolated from a 72-h HCMV (strain Towne) cDNA library. Sequence analysis of this clone revealed a continuous unspliced transcript between the region of UL101X and UL102; the only in-frame translational stop codon is 2,619 bp downstream from the first ATG in the message. Genome sequencing of the UL102 region from strains AD169 and Towne revealed that the UL101X stop codon TAA was actually TAC and that the cDNA and genomic sequences were in agreement. The cDNA clone starts 5 nucleotides (nt) upstream of the UL101X ATG, continues through the putative ATG of UL102, and ends 97 nt downstream of the putative termination codon of the UL102 open reading frame. Primer extension analysis indicated a transcriptional start site 23 nt upstream of the UL101X open reading frame.

Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication.

It was previously reported that the region encoding human cytomegalovirus (HCMV) genes UL36 to UL38 was required for origin-dependent DNA replication. These genes encode transactivators that upregulate viral and cellular transcription. However, their requirement for viral DNA replication has not been demonstrated. We have now used an antisense phosphorothioate oligonucleotide complementary to the intron-exon boundary of the UL36 and UL37 unspliced RNA to show that these gene products are required for HCMV DNA replication. Southern analysis showed that this oligonucleotide almost completely inhibits HCMV DNA replication when used at concentrations as low as 0.08 microM. The ability of this oligonucleotide to inhibit DNA replication was not the result of an inhibition of virus adsorption. Southern blots showed no impairment of viral adsorption or internalization in the presence of either specific or nonspecific phosphorothioate oligonucleotides. In addition, Northern (RNA) blots confirm that this antisense compound specifically reduced UL36 mRNA in treated cells to undetectable levels while the steady-state levels of immediate-early transcripts IE1 and IE2 were unaffected. These results demonstrate that the UL36 and UL37 gene products provide an essential function in initiation of HCMV DNA replication.

Eleven loci encoding trans-acting factors are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA replication.

Recently we described the use of human cytomegalovirus (HCMV) cosmid clones in a cotransfection assay of HCMV oriLyt replication (G. S. Pari, M. A. Kacica, and D. G. Anders, J. Virol. 67:2575-2582, 1993). We have now used this assay to identify 11 distinct required loci encoding trans-acting factors sufficient for transient complementation of oriLyt-dependent DNA replication. This set includes all of the virus genes essential to initiate and perform DNA synthesis together with the virus genes required to express these replication functions from their native promoters. Six of the identified loci span open reading frames (ORFs) that encode homologs or probable homologs of herpes simplex virus type 1 replication genes, consistent with predictions based on sequence similarities and biochemical properties. These include the DNA polymerase UL54 and polymerase-associated protein UL44, the single-stranded-DNA-binding protein UL57, and proposed subunits of a helicase-primase complex, UL70, UL105, and UL101-102. Frameshift mutations in any one of these essential ORFs abrogated complementation of DNA replication. Three required loci, UL36-38, IRS1 (or TRS1), and IE1/IE2, encode known regulatory proteins. The remaining two loci span ORFs UL84 and UL112-113 and encode early temporal class nucleus-associated proteins of unknown function. Neither of these genes have been implicated previously in DNA replication or in regulating gene expression, nor have counterparts in herpes simplex virus type 1 or Epstein-Barr virus been described. The results presented here will facilitate investigation of the mechanisms and regulation of HCMV lytic-phase DNA replication.

Open reading frames UL44, IRS1/TRS1, and UL36-38 are required for transient complementation of human cytomegalovirus oriLyt-dependent DNA synthesis.

Previous results showed that plasmids containing human cytomegalovirus (HCMV) oriLyt are replicated after transfection into permissive cells if essential trans-acting factors are supplied by HCMV infection (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373-3384, 1992). We have now used oriLyt as a reporter of HCMV DNA replication in a transient complementation assay in which cotransfected cosmid clones, instead of HCMV infection, provided essential trans-acting factors. Complemented replication was oriLyt dependent and phosphonoformic acid sensitive and produced tandem arrays typical of HCMV lytic-phase DNA synthesis. Thus, this assay provides a valid genetic test to find previously unidentified genes that are essential for DNA synthesis and to corroborate functional predictions made by nucleotide sequence comparisons and biochemical analyses. Five cosmids were necessary and sufficient to produce origin-dependent DNA synthesis; all but one of these required cosmids contain at least one candidate homolog of herpes simplex virus type 1 replication genes. We further used the assay to define essential regions in two of the required cosmids, pCM1017 and pCM1052. Results presented show that UL44, proposed on the basis of biochemical evidence to be the HCMV DNA polymerase accessory protein, was required for complementation. In addition, three genomic regions encoding regulatory proteins also were needed to produce origin-dependent DNA synthesis in this assay: (i) IRS1/TRS1, which cooperates with the major immediate-early proteins to activate UL44 expression; (ii) UL36-38; and (iii) the major immediate-early region comprising IE1 and IE2. Combined, these results unequivocally establish the utility of this approach for mapping HCMV replication genes. Thus, it will now be possible to define the set of HCMV genes necessary and sufficient for initiating and performing lytic-phase DNA synthesis as well as to identify those virus genes needed for their expression in human fibroblasts.

Human cytomegalovirus major immediate early gene product can induce SV40 DNA replication in human embryonic lung cells.

Previously we had reported that human cytomegalovirus (HCMV) induced replication of plasmids containing the SV40 origin of replication in human fibroblasts that were nonpermissive for SV40 and permissive for HCMV DNA replication. The amplification of SV40 origin-containing plasmids was dependent upon the HCMV-induced expression of T-antigen RNA. From previous studies it was determined that cotransfection of cosmids, containing HCMV genomic DNA, could stimulate SV40 DNA replication and T-antigen production. This indicated that the gene products of HCMV responsible for inducing SV40 DNA replication could be determined. In this study we report that the cotransfection of the major IE gene of HCMV alone was sufficient to facilitate the replication of the SV40 origin-containing plasmid. The HCMV IE1 gene product (i) increased expression of T-antigen RNA and protein and (ii) induced SV40 plasmid DNA replication in a T-antigen-dependent manner. The SV40 replication event was not due only to the expression of T-antigen. When the gene coding for T-antigen was placed under control of the Rous sarcoma viral promoter so that T-antigen expression in HEL cells was constitutive, it was not sufficient to replicate the SV40 plasmid in the absence of the HCMV IE1 protein. Therefore, the major IE gene of HCMV was capable of increasing the expression of T-antigen RNA and facilitating the replication of the SV40 origin. We are currently investigating the mechanism responsible for these observations.

Effect of human cytomegalovirus on replication of SV40 origin and the expression of T antigen.

Human cytomegalovirus (HCMV) induces replication of the cloned SV40 origin of DNA replication in both productive and nonproductive infections. HCMV-induced replication of the SV40 DNA origin required the presence of T antigen. Human embryonic lung (HEL) cells were found to be fully permissive for SV40 origin replication only in the presence of HCMV gene expression. In addition, expression of plasmid encoded SV40 T antigen in HEL cells was only induced in the presence of HCMV. Cotransfection of the SV40-cloned origin of replication and seven overlapping cosmids containing the entire HCMV genome is capable of stimulating replication of the SV40 origin. We conclude that induction of the SV40 origin of replication by HCMV is (i) not due to any component of the HCMV virion, (ii) has a requirement for T antigen expression, (iii) occurs in an SV40 nonproductive cell, when T antigen expression is induced by HCMV gene expression.