Human Cytomegalovirus Replication Cycle Research Articles

Inhibitory effect of hexamethylene bisacetamide on replication of human cytomegalovirus

The effect of hexamethylane bisacetamide (HMBA), a hybrid polar compound, on gene expression and replication of human cytomegalovirus (HCMV) was studied. When HCMV-infected human thyroid papillary carcinoma (TPC-1) and human embryonic lung (HEL) fibroblast cells were maintained with medium containing 2.5 and 5 mM HMBA for 10 days, there was a greater than 2- to 3-log reduction in virus yield compared to that in untreated cells. Infection of TPC-1 cells with HCMV resulted in an establishment of persistent infection and the cells continuously produced virus with titer of over 10(5) PFU/ml, whereas HMBA prevented the infected cells from entering into the persistent infection. Moreover, treatment of the persistently infected cultures with HMBA reduced production of infectious HCMV more efficiently than did ganciclovir, and eventually ceased HCMV production. Western blotting analysis revealed that HMBA blocks accumulation of the immediate early 2 (IE2) protein in TPC-1 cells and delays synthesis of this protein in HEL cells, but has little effect on the level of the IE1 protein during the early times after infection. Synthesis of the viral early and late proteins in both cells was also substantially blocked by HMBA. The results indicate that the inhibition or the delay of the critical IE2 protein synthesis in the presence of HMBA would actually be a process that fails to proceed beyond the IE stages in HCMV replication cycle.

NF-κB activation can mediate inhibition of human cytomegalovirus replication

The activation of NF-kappaB has long been considered a positive factor for human cytomegalovirus (HCMV) replication. The HCMV immediate-early promoter, the initial transcriptional element in the HCMV replication cycle, is activated by the transcription factor NF-kappaB, and several HCMV gene products have been demonstrated to activate this transcription factor. However, the role of NF-kappaB in the full replication cycle of the virus has not been carefully examined. A series of experiments that demonstrate an important inhibitory role of NF-kappaB for HCMV replication in fibroblasts is presented here. Using both genetic and pharmaceutical methods, it was shown that blocking NF-kappaB activation in cell culture does not inhibit HCMV replication, but rather leads to a modest increase in replication. Two cytokines inhibitory for HCMV, tumour necrosis factor-alpha and interferon-gamma, no longer inhibit HCMV when NF-kappaB activation is blocked. Furthermore, forced expression of the NF-kappaB activating IkappaB kinase beta (IKKbeta), but not a kinase inactive mutant, also inhibits HCMV replication. In addition, it was shown that NF-kappaB signalling is essential for the production of an anti-viral factor in the supernatant of HCMV-infected fibroblasts, and identified interferon-beta as this factor. Thus, the role of NF-kappaB in fibroblasts is to activate a host defence against HCMV.

Permissive human cytomegalovirus infection of a first trimester extravillous cytotrophoblast cell line

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection in the United States and Europe. Despite the significant morbidity associated with prenatal HCMV infection, little is known about how the virus infects the fetus during pregnancy. To date, primary human cytotrophoblasts (CTBs) have been utilized to study placental HCMV infection and replication; however, the minimal mitotic potential of these cells restricts experimentation to a few days, which may be problematic for mechanistic studies of the slow-replicating virus. The aim of this study was to determine whether the human first trimester CTB cell line SGHPL-4 was permissive for HCMV infection and therefore could overcome such limitations. HCMV immediate early (IE) protein expression was detected as early as 3 hours post-infection in SGHPL-4 cells and progressively increased as a function of time. HCMV growth assays revealed the presence of infectious virus in both cell lysates and culture supernatants, indicating that viral replication and the release of progeny virus occurred. Compared to human fibroblasts, viral replication was delayed in CTBs, consistent with previous studies reporting delayed viral kinetics in HCMV-infected primary CTBs. These results indicate that SGHPL-4 cells are fully permissive for the complete HCMV replicative cycle. Our findings suggest that these cells may serve as useful tools for future mechanistic studies of HCMV pathogenesis during early pregnancy.

An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function.

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.

Human cytomegalovirus proteins PP65 and IEP72 are targeted to distinct compartments in nuclei and nuclear matrices of infected human embryo fibroblasts.

The cellular distribution of the human cytomegalovirus (HCMV)-specific UL83 phosphoprotein (pp65) and UL123 immediate-early protein (IEp72) in lytically infected human embryo fibroblasts was studied by means of indirect immunofluorescence and confocal microscopy. Both proteins were found to have a nuclear localization, but they were concentrated in different compartments within the nuclei. The pp65 was located predominantly in the nucleoli; this was already evident with the parental viral protein, which was targeted to the above nuclear compartment very soon after infection. The nucleolar localization of pp65 was also observed at later stages of the HCMV infectious cycle. After chromatin extraction (in the so-called in situ nuclear matrices), a significant portion of the pp65 remained associated with nucleoli within the first hour after infection, then gradually redistributed in a perinucleolar area, as well as throughout the nucleus, with a granular pattern. A quite different distribution was observed for IEp72 at very early stages after infection of human embryo fibroblasts with HCMV; indeed, this viral protein was found in bright foci, clearly observable in both non-extracted nuclei and in nuclear matrices. At later stages of infection, IEp72 became almost homogeneously distributed within the whole nucleus, while the foci increased in size and were more evenly spread; in several infected cells some of them lay within nucleoli. This peculiar nuclear distribution of IEp72 was preserved in nuclear matrices as well. The entire set of data is discussed in terms of the necessity of integration for HCMV-specific products into the pre-existing nuclear architecture, with the possibility of subsequent adaptation of nuclear compartments to fit the needs of the HCMV replicative cycle.

Interactions among antiviral drugs acting late in the replication cycle of human cytomegalovirus

This study describes the extent of cross-resistance and interactions for selected inhibitors of human cytomegalovirus (HCMV) DNA synthesis and DNA processing. HCMV isolates resistant to the benzimidazole d-ribonucleoside viral DNA processing inhibitors TCRB and BDCRB were sensitive to BAY 38-4766, a non-nucleoside inhibitor of viral DNA processing. This indicates that these two drug types have distinct interactions with the products of HCMV genes UL56 and UL89 required for viral DNA cleavage and packaging. These virus isolates also were sensitive to ganciclovir (GCV) but slightly resistant to the l-benzimidazole ribonucleoside viral DNA synthesis inhibitor 1263W94. Virus resistant to 1263W94 remained sensitive to BDCRB, GCV, and BAY 38-4766. Examination of drug–drug interactions in cell culture assays measuring inhibition of HCMV replication revealed strong synergism for the combination of BDCRB with 1263W94, and for combinations of 1263W94 with cidofovir (CDV) and foscarnet (PFA), but not with GCV. Combinations of GCV with CDV and PFA were synergistic as well. The combination of GCV with 1263W94 showed additive antiviral interactions, whereas, a combination of BAY 38-4766 with GCV showed antagonism. Interaction of BDCRB with BAY 38-4766 showed a mixed pattern of synergy and antagonism. The antiviral synergy observed between GCV and PFA or CDV serves to validate clinical combination therapies for these drugs. Antagonism seen for BAY 38-4766 with GCV indicates that these two drugs are unlikely to be useful for combination therapies. Notably, 1263W94 demonstrated greater synergy in combination with PFA or CDV than did GCV, suggesting some promise for this benzimidazole l-riboside in such combination therapies.

Efficient downregulation of major histocompatibility complex class I molecules in human epithelial cells infected with cytomegalovirus.

Liver and intestinal epithelial cells are a major target of infection by cytomegaloviruses (CMV), causing severe disease in affected organs of immunocompromised patients. CMV downregulates major histocompatibility complex class I (MHC-I) molecule expression in fibroblasts in order to avoid lysis by CD8(+) cytotoxic T lymphocytes. However, MHC-I expression in human cytomegalovirus (HCMV)-infected hepatic tissue was reported to be increased. As it is unclear at present whether HCMV affects MHC-I expression in epithelial cells, new cell culture models for HCMV infection of differentiated hepatobiliary cell lines were established. HCMV immediate early gene expression was achieved in 60 to 95% of cells. Progression of the HCMV replication cycle differed from prototypic infection of fibroblasts, since structural early and late proteins were produced at low levels and HCMV progeny yielded much lower titres in hepatobiliary cells. In contrast, HCMV glycoproteins, gpUS2, gpUS3, gpUS6 and gpUS11, that downregulate MHC-I expression were synthesized with temporal kinetics and in a similar quantity to that seen in fibroblasts. As a result, HCMV infection led to a drastic and selective downregulation of MHC-I expression in epithelial cells and was uniformly observed irrespective of the hepatic or biliary origin of the cells. The new models document for the first time a stealth function of HCMV in epithelial cells and indicate that the downregulation of MHC-I expression by HCMV can occur in the virtual absence of virus replication.

UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells.

The human cytomegalovirus UL82 gene encodes a protein (pp71) that is localized in the tegument domain of the virus particle. The UL82 gene product is delivered to the nucleus at the time of infection, and it is believed to function in gene activation. We have constructed a human cytomegalovirus mutant, ADsubUL82, that lacks a substantial portion of the UL82 coding region. It was propagated on human diploid fibroblasts expressing the UL82 gene product, and it was possible to produce a mutant virus lacking the UL82 protein by passaging virus stocks for one cycle of growth on normal, noncomplementing fibroblasts. The UL82-deficient mutant displays a multiplicity-dependent growth defect in normal human fibroblasts. The growth of ADsubUL82 is severely restricted at low input multiplicities (0.01-0.1 plaque-forming units per cell), producing a yield that is reduced by a factor of about 10(5) in comparison to wild-type virus. At higher input multiplicities (10 plaque-forming units per cell), ADsubUL82 grew nearly as well as the wild-type virus. By using a human cytomegalovirus gene array, we demonstrated that UL82 functions to facilitate virus mRNA accumulation very early during the human cytomegalovirus replication cycle. The growth phenotype associated with the UL82 mutant seems to result from its inability to efficiently activate human cytomegalovirus immediate early genes.

Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly.

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

Nonnucleoside pyrrolopyrimidines with a unique mechanism of action against human cytomegalovirus.

Based upon a prior study which evaluated a series of nonnucleoside pyrrolo[2,3-d]pyrimidines as inhibitors of human cytomegalovirus (HCMV), we have selected three active analogs for detailed study. In an HCMV plaque-reduction assay, compounds 828, 951, and 1028 had 50% inhibitory concentrations (IC(50)s) of 0.4 to 1.0 microM. Similar results were obtained when 828 and 951 were examined by HCMV enzyme-linked immunosorbent assay (IC(50)s = 1.9 and 0.4 microM, respectively) and when 828 was tested in a viral DNA-DNA hybridization assay (IC(50) = 1.3 microM). In yield-reduction assays with a low multiplicity of infection (MOI), all three compounds caused multiple log(10) reductions in virus titer, and the activities of these compounds were comparable to the activity of ganciclovir (GCV; IC(90) = 0.2 microM). In contrast to the reduction of viral titers by GCV, the reduction of viral titers by 828, 951, and 1028 decreased with increasing MOI. Cytotoxicity in human foreskin fibroblasts and KB cells ranged from 32 to >100 microM. In addition, 828 (the only compound tested) was less toxic against human bone marrow progenitor cells than GCV. Time-of-addition and time-of-removal studies established that the three pyrrolopyrimidines inhibited HCMV replication before GCV had an effect on viral DNA synthesis but after viral adsorption. Compound 828 was equally effective against GCV-sensitive and GCV-resistant HCMV clinical isolates. Combination studies with 828 and GCV showed that the effects of the two compounds on HCMV were additive but not synergistic. Taken together, the data indicate that these pyrrolopyrimidines target a viral protein that is required in an MOI-dependent manner and that is expressed early in the HCMV replication cycle.

Synthesis and antiviral activity of 6-benzoyl-benzoxazolin-2-one and 6-benzoyl-benzothiazolin-2-one derivatives.

The synthesis and antiviral activity of an original series of 6-benzoyl-benzoxazolin-2-one and 6-benzoyl-benzothiazolin-2-one derivatives are described. Several compounds were found to have a selective inhibitory activity against human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) in vitro, being inactive against a variety of other DNA and RNA viruses. 6-(3-fluorobenzoyl)benzoxazolin-2-one, 6-(3-fluorobenzoyl)benzothiazolin-2-one, 6-(3-bromobenzoyl)benzothiazolin-2-one, 6-(3-iodobenzoyl)benzothiazolin-2-one, 3-methyl-6-(3-fluorobenzoyl)benzothiazolin-2-one, 3-benzyl-6-benzoyl-benzothiazolin-2-one, 3-benzyl-6-(3-fluorobenzoyl)benzothiazolin-2-one and 3-benzoyl-6-(3-fluorobenzoyl)benzothiazolin-2-one were the most active of the series against HCMV and VZV with a selectivity index (CC50/IC50) ranging from 10 to 20. They displayed similar activity against thymidine kinase competent (TK+) and deficient (TK-) VZV strains, and also proved to be active against clinical HCMV isolates that were resistant to ganciclovir (GCV). Time-of-addition experiments revealed a site of interaction with the HCMV replicative cycle that may be close or similar to that of GCV and cidofovir (HPMPC). The compounds showed poor, if any, activity against herpes simplex virus type 1 (HSV-1) and HSV-2, and were not inhibitory against human immunodeficiency virus and other DNA and RNA viruses. Therefore, these compounds may represent a novel lead for the development of specific HCMV and VZV drugs.

Strong conservation of the constitutive activity of the IE1/2 transcriptional control region in wild-type strains of human cytomegalovirus.

The IE1/2 transcriptional control region of human cytomegalovirus (HCMV) drives the expression of the HCMV major immediate-early genes (UL123-122), which encode proteins crucial for initiation of the virus replicative cycle. Nucleotide sequence polymorphism in this region of the viral genome could account for variations in the replication of HCMV wild-type strains. In order to test this hypothesis, the constitutive transcription-enhancing activity of the IE1/2 transcriptional control region derived from 12 clinical isolates of HCMV was compared. This was done by PCR amplification of the respective elements followed by cloning up-stream of a beta-globin reporter gene. After transient expression in various cell types, including human teratocarcinoma cell lines, and quantification of RNA levels, the activating function of this complex cis-element was shown to be strongly conserved. This was mirrored by high nucleotide sequence conservation, even within the so-called modulator region. This strong evolutionary conservation of sequence and of transcription-enhancing function strengthens the assumption that the IE1/2 transcriptional control region plays an essential role in initiation of the HCMV replicative cycle.

The Human Cytomegalovirus UL97 Protein Is Phosphorylated and a Component of Virions

The expression of the human cytomegalovirus (HCMV) UL97 open reading frame in infected or transfected cells in the presence of the antiherpes compound ganciclovir (GCV) results in the intracellular phosphorylation of GCV. There are conventional kinase domains within the UL97-encoded protein (pUL97). However, the role of pUL97 in the HCMV replication cycle, and the mechanism by which it causes phosphorylation of GCV, are currently unknown. Herein, the biosynthesis and biogenesis of pUL97 was studied in HCMV-infected cells. pUL97 is expressed with early-late kinetics and is posttranslationally modified by phosphorylation. This phosphorylation occurs within 1 hr after synthesis, affects the electrophoretic mobility of pUL97, and is independent of the presence of other HCMV proteins. pUL97 was localized to the nucleus of infected cells and found in the HCMV virions. Thus, pUL97 is a virion phosphoprotein, and a likely tegument component.

Human cytomegalovirus IE1 and IE2 proteins block apoptosis

Human cytomegalovirus-infected fibroblasts are resistant to the induction of apoptosis by superinfection with a mutant adenovirus unable to produce the viral E1B 19-kDa protein that normally causes an E1A protein-mediated apoptotic response. Two cytomegalovirus gene products that block apoptosis were identified. The IE1 and IE2 proteins each inhibit the induction of apoptosis by tumor necrosis factor alpha or by the E1B 19-kDa-protein-deficient adenovirus but not by irradiation with UV light. Our results suggest a new physiological role for the IE1 and IE2 proteins in the human cytomegalovirus replication cycle.

Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains

Reduction of major histocompatibility complex class I cell surface expression occurs in adenovirus-, herpes simplex virus-, human cytomegalovirus (HCMV)-, and murine cytomegalovirus-infected cell systems. Recently, it was demonstrated that the down-regulation mediated by HCMV infection is posttranslational, as a result of increased turnover of class I heavy chains in the endoplasmic reticulum (M. F. C. Beersma, M. J. E. Bijlmakers, and H. L. Ploegh, J. Immunol. 151:4455-4464, 1993; Y. Yamashita, K. Shimokata, S. Saga, S. Mizuno, T. Tsurumi, and Y. Nishiyama, J. Virol. 68:7933-7943, 1994. To identify HCMV genes involved in class I regulation, we screened our bank of HCMV deletion mutants for this phenotype. A mutant with a 9-kb deletion in the S component of the HCMV genome (including open reading frames IRS1 to US9 and US11) failed to down-regulate class I heavy chains. By examining the effects of smaller deletions within this portion of the HCMV genome, a 7-kb region containing at least nine open reading frames was shown to contain the genes required for reduction in heavy-chain expression. Furthermore, it was determined that at least two independent loci within the 7-kb region were able to cause class I heavy-chain down-regulation. One of these, US11, encodes a 32-kDa glycoprotein which causes down-regulation of class I heavy chains in the absence of other viral gene products. Hence, a specific function associated with a phenotype of the HCMV replicative cycle has been mapped to a dispensable gene region. These loci may be important for evasion of the host's immune response and viral persistence.

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.

Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.

UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression

The UL69 open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of herpes simplex virus, an essential viral regulatory protein involved in the transition from early to late gene expression. Genes with homology to ICP27 have been detected in all subclasses of herpesviruses so far. While the respective proteins in alpha- and gammaherpesviruses have been defined as trans-regulatory molecules, nothing is known about these genes in betaherpesviruses. This study was therefore undertaken in order to investigate expression from the UL69 gene locus of HCMV. Northern (RNA) blot experiments revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early as 7 h after infection. However, these transcripts could not be detected in the presence of cycloheximide. Additional, larger transcripts were present exclusively at late times after infection. To analyze protein expression from the UL69 gene region, the UL69 open reading frame was expressed as a histidine-tagged protein in Escherichia coli. A specific antiserum was generated and used to detect the UL69 protein in HCMV-infected cells which revealed its localization within the intranuclear inclusions that are characteristic for HCMV infection. In cotransfection experiments, an HCMV true late promoter could not be activated by UL69, whereas an early promoter and several heterologous promoters were stimulated about 10-fold. Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the context of the HSV infection, suggesting functional differences between these two proteins. In summary, these experiments define a novel regulatory protein encoded by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression.

Nuclear expression of the lower matrix protein of human cytomegalovirus in peripheral blood leukocytes of immunocompromised viraemic patients.

Peripheral blood leukocytes (PBL), namely polymorphonuclear leukocytes (PMNL), are the major carrier of human cytomegalovirus (HCMV) in the blood of immunocompromised patients with HCMV viraemia. By using monoclonal antibodies (MAbs) directed against different early and late viral proteins, we showed that the protein accumulating in PBL, originally reported to be an immediate early (IE) gene product, is the 65K lower matrix early structural protein (ep65). This protein is detectable by immunofluorescence before IE proteins during early stages of the replication cycle of HCMV in permissive human embryonic lung fibroblast cells. However, the appearance of ep65 in the nucleus within 1 h post-infection in the presence of cycloheximide indicates that it represents uptake from the virus inoculum rather than newly synthetized protein. The ep65 MAbs staining PBL did not react with Vero cells infected with a recombinant vaccinia virus encoding the major IE gene (IE1) product, whereas MAbs reactive with the 72K major IE protein stained only faintly a small number of infected PBL. A group of four ep65 MAbs was tested in competitive binding assays to show that ep65 possesses at least three distinct epitopes. These were recognized by all four MAbs in AD169-infected Vero cell cultures when fixed with formaldehyde, whereas only one MAb recognizing a distinct epitope was reactive with methanol-acetone (MA)-fixed AD169-infected Vero cells. In formalin-fixed PBL the number of infected cells stained by the four ep65 MAbs was about twofold that found using MA-fixed cells. Using fluorescence-activated cell sorter-purified leukocyte subpopulations from viraemic patients with different levels of viraemia, the ratio of ep65-positive to ep65-negative cells was found to be 1:100 to 1:100,000 for PMNL, and only 1:10,000 to 1:100,000 for mononuclear cells.

Establishment and biological characterization of an in vitro human cytomegalovirus latency model

In an attempt to develop an in vitro human cytomegalovirus (HCMV) latency model system, the growth characteristics of HCMV in a human thyroid papillary carcinoma cell line (TPC-1) were examined. When TPC-1 cultures preheated at 40.5° for 48 hr were infected with HCMV and incubated at a supraoptimal temperature (40.5°), the cultures could be maintained for at least 65 days without detection of infectious virus. In contrast, when the infected cultures were incubated at 37°, HCMV persistently infected cultures were established. HCMV was reactivated from the latently infected cultures by decreasing the incubation temperature from 40.5 to 37°, and the cultures subsequently entered into virus persistent infection. Although HCMV-specific polypeptides which comigrate with the immediate early virus polypeptides and nuclear antigens were continuously detectable in the majority (more than 95%) of the cells during the latent period, a detectable level of virus-specified DNA polymerase (one of the early virus proteins) was not induced, suggesting that the blockage of HCMV replication in the latently infected cultures occurs at the early stages of the HCMV replication cycle. Infectious center assay revealed that 0.002 to 0.2% of the cells contain an HCMV genome that can be activated during the latent period. The latently infected cells were susceptible to superinfection with homologous and heterologous strains of HCMV. In persistently infected cultures approximately 38% of the cells were lysed by reaction with HCMV immune serum and complement, whereas complement-mediated immune cytolysis could not be detected in the latently infected cultures. The data presented suggest that a temperature-sensitive cellular function(s) that controls the expression of the HCMV early functions plays an important role in maintenance of the HCMV genome in the latent state and reactivation of HCMV by decreasing the incubation temperature.