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Viral subversion of apoptosis regulation plays an important role in the outcome of host/virus interactions. Although human cytomegalovirus (HCMV) encodes several immediate early (IE) antiapoptotic proteins (IE1, IE2, vMIA and vICA), no proapoptotic HCMV protein has yet been identified. Here we show that US28, a functional IE HCMV-encoded chemokine receptor, which may be involved in both viral dissemination and immune evasion, constitutively induces apoptosis in several cell types. In contrast, none of nine human cellular chemokine receptors, belonging to three different subfamilies, induced any significant level of apoptosis. US28-induced cell death involves caspase 10 and caspase 8 activation, but does not depend on the engagement of cell-surface death receptors of the tumour necrosis factor receptor/CD95 family. US28 cell-death induction is prevented by coexpression of C-FLIP, a protein that inhibits Fas-associated death domain protein (FADD)-mediated activation of caspase 10 and caspase 8, and by coexpression of the HCMV antiapoptotic protein IE1. The use of US28 mutants indicated that the DRY sequence of its third transmenbrane domain, required for constitutive G-protein signalling, and the US28 intracellular terminal domain required for constitutive US28 endocytosis, are each partially required for cell-death induction. Thus, in HCMV-infected cells, US28 may function either as a chemokine receptor, a phospholipase C activator, or a proapoptotic factor, depending on expression levels of HCMV and/or cellular antiapoptotic proteins.

Human cytomegalovirus (HCMV) remains one of the major pathogens in immunocompromised patients (AIDS and transplants) and the main cause for congenital infections leading from slight cognitive defects up to severe mental retardation. The drugs that are currently available for the treatment of HCMV infections, i.e. ganciclovir, foscarnet and cidofovir, are all acting at the level of the viral DNA polymerase. Here we describe an entirely new molecule, the 2-chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxamide (CMV423), that shows very potent in vitro activity against HCMV. CMV423 is highly active against HCMV reference strains and clinical isolates, but also against those strains, isolated from patients or emerging after in vitro selection, that are resistant to either ganciclovir, foscarnet or cidofovir. CMV423 also showed activity in two ex vivo models, that are both highly relevant for the pathophysiology of HCMV, the retinal pigment epithelial and the bone marrow stromal cell assays. Viral antigen expression analysis by flow cytometry, as well as time of addition experiments, confirmed that CMV423 acts on a step of the viral replicative cycle that precedes the DNA polymerase step and, most likely, coincides with the immediate early (IE) antigen synthesis. Finally, CMV423 combined with either ganciclovir, foscarnet or cidofovir in checkerboard experiments demonstrated a highly synergistic activity.

The human cytomegalovirus (HCMV) US28 gene product, pUS28, is a G protein-coupled receptor that interacts with both CC and CX3C chemokines. To date, the role of pUS28 in immune evasion and cell migration has been studied only in cell types that can establish productive HCMV infection. We show that HCMV can latently infect THP-1 monocytes and that during latency US28 is transcribed. We also show that the transcription is sustained during differentiation of the THP-1 monocytes. Since cells expressing pUS28 were previously shown to adhere to immobilized CX3C chemokines (C. A. Haskell, M. D. Cleary, and I. F. Charo, J. Biol. Chem. 275:34183–34189, 2000), we hypothesize that latently infected circulating monocytes express pUS28, thereby enabling adhesion of these cells to CX3C-exposing endothelium. Consequently, the US28-encoded chemokine receptor may play an important role in dissemination of latent HCMV.

The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55 gag -based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1A isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120UG sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLPAs show the expected density (1.14‐1.18 g:ml) on a 10‐60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55 gag autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules. © 2001 Elsevier Science B.V. All rights reserved.

Purpose: To evaluate the contribution of molecular methods for the diagnosis of an acyclovir-resistant HSV-1 bilateral keratitis in an AIDS patient and to report a new point mutation in the nucleotide sequence of the thymidine kinase (tk) gene involved. Methods: A 31 year old HIV-positive female presented with severe, active, bilateral and sight-threatening keratitis of 6 months duration, which was treated unsuccessfully with acyclovir. After corneal biopsy, samples were analysed by standard virological procedures, in situ hybridization, and PCR. The tk gene was cloned and subsequently sequenced. Results: Conventional virological methods remained inconclusive. However, in situ hybridization and PCR rapidly confirmed the diagnosis of HSV-1 keratitis. The tk gene sequence revealed the presence of five variations previously described in two reference strains, but also a new point mutation at nucleotide position 431 which leads to an amino-acid change at position 144 that supported the hypothesis of a putatively altered functional form of the enzyme. Intravenous foscarnet treatment in an induction regimen was effective and cicatrization occurred within 3 weeks. Conclusions: PCR and in situ hybridization are effective and powerful techniques when other virological procedures are non-contributive, particularly in immunocompromised patients previously treated with antiviral drugs. The new point mutation identified in the tk gene may be associated with resistance to acyclovir.

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.

Human cytomegalovirus (HCMV), a betaherpesvirus, has developed several ways to evade the immune system, notably downregulation of cell surface expression of major histocompatibility complex class I heavy chains. Here we report that HCMV has devised another means to compromise immune surveillance mechanisms. Extracellular accumulation of both constitutively produced monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor–superinduced RANTES (regulated on activation, normal T cell expressed and secreted) was downregulated in HCMV-infected fibroblasts in the absence of transcriptional repression or the expression of polyadenylated RNA for the cellular chemokine receptors CCR-1, CCR-3, and CCR-5. Competitive binding experiments demonstrated that HCMV-infected cells bind RANTES, MCP-1, macrophage inflammatory protein (MIP)-1β, and MCP-3, but not MCP-2, to the same receptor as does MIP-1α, which is not expressed in uninfected cells. HCMV encodes three proteins with homology to CC chemokine receptors: US27, US28, and UL33. Cells infected with HCMV mutants deleted of US28, or both US27 and US28 genes, failed to downregulate extracellular accumulation of either RANTES or MCP-1. In contrast, cells infected with a mutant deleted of US27 continues to bind and downregulate those chemokines. Depletion of chemokines from the culture medium was at least partially due to continuous internalization of extracellular chemokine, since exogenously added, biotinylated RANTES accumulated in HCMV-infected cells. Thus, HCMV can modify the chemokine environment of infected cells through intense sequestering of CC chemokines, mediated principally by expression of the US28-encoded chemokine receptor.

OBJECTIVE: The HIV Tat protein is a transcriptional transactivator of the HIV-1 long terminal repeat (LTR) promoter element. Its activity depends on its direct interaction with the trans-activation response (TAR) element, although TAR-independent activation by Tat has been demonstrated in different cells. Herpesviruses in general and human cytomegalovirus (HCMV) in particular are often isolated from HIV-1-infected patients and could play a role in the activation of latent HIV and in a subsequent increase in HIV replication. HCMV immediate early gene products (IE1 and IE2) are nuclear phosphoproteins that play a pivotal role in HCMV replication and have been shown to transregulate both viral and cellular gene expression. It has repeatedly been shown that HCMV IE1/IE2 can independently transactivate HIV-1 LTR. The aim of this study was to investigate IE1/IE2 transactivation of HIV-1 LTR in a CD4+ T-cell line in the absence and presence of HIV-1 Tat to establish whether IE1/IE2 can synergize with Tat. METHODS: HIV-1 LTR transactivation by HCMV IE1/IE2 in the presence and absence of HIV-1 Tat was determined by transient transfection experiments of J-Jhan lymphoblastoid cells with a series of different expression vectors. RESULTS: We found a strong synergistic transactivation between HIV Tat and the IE1-IE2 complex on HIV LTR activity using vectors driven either by wild-type LTR or by the nuclear factor NF-kappa(B) response element-mutated HIV LTR. IE1/IE2 synergism with HIV Tat was also observed in Sp1 binding site-mutated for TAR-deleted LTR, which cannot be activated by Tat alone. This cooperation is abolished when the region in IE2 that binds the TATA box binding protein is deleted. CONCLUSIONS: The results obtained indicate that Sp1-binding and TAR sequences are not strictly required for Tat responsiveness when Tat is directed to the HIV promoter by HCMV IE1-IE2. This synergistic effect is mediated by the IE2 and TATA-binding region, and could play a major role in HIV activation when cells are infected by both viruses, a feature often observed in AIDS patients.

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another.

Cellular immune responses are important in the recovery from human cytomegalovirus (HCMV) infection. However, little is known about the CD4+ T cell response and the target antigens (Ag) recognized. In this paper, we have analysed the proliferative T cell response of healthy HCMV seropositive (HCMV+) blood donors to recombinant immediate-early proteins expressed in transfected astrocytoma cells and to total HCMV Ags expressed in infected astrocytoma cells. We found that CD4+ T cells were the major cell population that proliferated in the presence of IE or total HCMV Ags. Among healthy HCMV seropositive blood donors with anti-HCMV specific proliferative response, 33-44% also responded to IE Ags. Moreover, in high responders, the precursor frequencies of cells which proliferated in the presence of total HCMV, IE, or IE1 Ags were high (1/103 to 1/255, 1/2785 to 1/7744 and 1/5190 to 1/13531, respectively). In some donors, the anti-IE response was variable over time, whereas the anti-total HCMV Ags response remained constant, which suggests regulation of the anti-IE response in immunocompetent subjects. Our results suggest that the CD4+ anti-IE1 response represents a significant part of the anti-HCMV proliferative response, both at the population level, and within individual immune systems.

Human cytomegalovirus (CMV) infection can elicit a transitory, but profound, immunodepression in immunocompetent individuals. Cytopathogenic destruction of CMV-infected leukocytes alone does not seem sufficient to explain this phenomenon, which suggests that immune system mediators (cytokines) may play a role in amplifying local modifications wrought by CMV infection. We reported previously that transforming growth factor beta 1 (TGF-beta 1) stimulates CMV replication (J. Alcami, C. V. Paya, J. L. Virelizier, and S. Michelson, J. Gen. Virol. 74:269-274, 1993). Since TGF-beta 1 can have profound negative effects on cell growth and immune responses, we investigated the induction of TGF-beta 1 following CMV infection of permissive fibroblasts. TGF-beta 1 promoter was activated by immediate-early (IE) proteins in the absence of infection and transactivated at 5 and 9 h after infection. TGF-beta 1 mRNA increased during the early phase of infection, suggesting that this phenomenon is due to enhanced transcription of the TGF-beta 1 gene. A comparative study of the influence of CMV infection and IE protein expression on TGF-beta 1 promoter function in permissive cells pointed to a possible cooperative role between IE proteins and protein(s) expressed during the early phase of viral infection. Induction of TGF-beta 1 by CMV infection could modify infected cells individually, surrounding tissues, and systemic immune reactions to the advantage of virus replication by both upregulating CMV replication and downregulating host immune responses.

The appearance of cytomegalovirus (CMV) proteins in infected fibroblasts was determined by flow cytometry. The sequential production of immediate early (IE), early (E), and late (L) proteins reacting with respective monoclonal antibodies (mAbs) E13, 58/5, and 24/4 was determined in fibroblasts infected with the AD-169 strain of CMV. The percentage of cells expressing CMV proteins and the intensity of fluorescence within cells were determined from day 1 to day 7 post-infection. The effect of interferons (IFNs) alpha, beta, gamma on expression of CMV proteins was analyzed using flow cytometry. IFNs inhibited E and L protein production at days 3 and 6 post-infection in a dose-dependent manner. This inhibitory effect on protein expression was associated with a reduction in release of infectious CMV into culture media. The method described here for detection of CMV proteins using flow cytometry may be useful for basic studies of gene expression and for diagnostic purposes.

Recombinant baculoviruses containing the unspliced gene (Bac-IE1) and a truncated cDNA (Bac-EX4) of the immediate early protein 1 (IE1) of human cytomegalovirus (HCMV) were constructed. The recombinant proteins IE1 and EX4 were expressed in Sf 9 insect cells. Immunoblot analyses using a specific monoclonal antibody or human sera from HCMV seropositive subjects revealed that the IE1 protein had an apparent molecular mass of 71 kDa which was similar to that observed in both HCMV infected human fibroblasts and infected or transfected human astrocytoma cells. Furthermore, HCMV-specific CD4+ T cell clones proliferated in the presence of IE1 or of EX4 used as a control, and appropriate antigen presenting cells. Our data on the IE1 gene provide evidence that two introns can be properly spliced out in baculovirus infected insect cells. The expressed proteins should be useful in further studies on the immune response to the virus.

By using human CD4+ lymphoblastoid T cells transiently cotransfected with human immunodeficiency virus (HIV) and cytomegalovirus (CMV), we tested whether modulation of T-cell activation through the protein kinase C (PKC) or the protein kinase A (PKA) pathway synergized with CMV immediate-early (IE) proteins in HIV long terminal repeat (LTR) transactivation. Stimulation with phorbol myristate acetate, tumor necrosis factor, or cross-linked antibodies to CD3 and CD28 resulted in modest enhancement (two- to fourfold) of the activity of a luciferase expression vector under control of the HIV LTR. Cotransfection of a vector expressing the CMV IE1 and IE2 proteins under the control of their own promoter enhanced HIV LTR activity 16- to 49-fold. Combination of any one of the above stimuli and CMV IE expression amplified HIV LTR activity 99- to 624-fold. Stimulation of PKA-dependent pathways with forskolin, 8-bromo cyclic AMP, or prostaglandin E2 had a minimal effect on HIV LTR activity, whereas such stimuli resulted in synergistic amplification in cells cotransfected with CMV IE (three- to fivefold increases over the effects of CMV IE alone). This synergism was independent of the NF-kappa B binding motifs within the HIV LTR. CMV IE2, but not IE1, protein induced HIV transactivation and synergized with signals modulating T-cell activation. The intense synergism observed was superior to the increase in IE protein expression following PKC activation by phorbol myristate acetate. Treatment of cells with PKC inhibitor GF109203X blocked most of the observed synergism.(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient loti' E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s"1 at 30 °C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and A-chlorosuccinimide. We show that the calmo- dulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase.

A major protein of M r 68,000 with a previously described protein kinase activity (PK68), which is induced in HCMV-infected cells, is shown to be virus encoded by means of hybrid selection. The gene coding for the corresponding 4-kb mRNA was located within map units 0.510 and 0.525. This location is the same as that of pp65, a structural protein with no attributed function. The early nature of the protein was confirmed with its mRNA first appearing 3 hr after infection under cycloheximide reversal and phosphonoacetic acid blocking conditions. Another mRNA, 97 nucleotides smaller and 5′ coterminal with PK68, which codes for a M r 52,000 protein, was also observed.

Aim: To describe the diagnostic and therapeutic management of cytomegalovirus (CMV) anterior uveitis unassociated with retinal necrosis in immunocompetent patients. Methods: Patients referred between 2001 and 2003 for management of unilateral, chronic, recurrent uveitis associated with secondary glaucoma underwent extensive investigation including laboratory tests for herpes virus infections. Specific antiviral treatment was initiated in all cases and the level of ocular inflammation was evaluated during the follow up. Results: Five patients, three men and two women, were included. Median age was 50 years old (range 30–80 years). Anterior unilateral uveitis without iris atrophy was observed in all cases. Uveitis was chronic in three cases and recurrent in two cases. Glaucoma was observed in all patients with a median intraocular pressure of 30 mm Hg (range 22–43 mm Hg). Five patients responded initially to specific anti-CMV therapy. Even though glaucoma surgery was necessary in two cases, both ocular inflammation and glaucoma were controlled in all cases. Relapses occurred in three cases after cessation of therapy, requiring prolonged maintenance therapy with valganciclovir. Conclusions: CMV infection and specific antiviral therapy should be considered in all cases of relapsing or chronic iridocyclitis and secondary glaucoma. Maintenance regimens of valganciclovir may be necessary to prevent further relapses.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

The HCMV genome has evolved with its host by incorporating a series of genes that are homologous to, or functionally mimic, cellular genes. Some are designed to counteract the stress of infection on the host cell, notably the viral antiapoptotic proteins (vICA, vMIA). Others potentially help the infected cell maintain a low immunologic profile. These include virus-encoded chemokine receptors (UL33, UL78, US27, US28), FcRs (gp TRL11/IRL11, gp UL119-118), and proteins that directly or indirectly thwart natural killer cell activity (UL16, gpUL40). In addition, some viral proteins may play a role in immunopathology because of fortuitous cross-reactivity with host cell proteins. This overview discusses how these proteins affect the life of the host cell and its immediate neighbors.

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34 + cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34 + cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34 + progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34 + cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.

The major matrix phosphoprotein 65 (pp65) of cytomegalovirus (CMV) is an important target of HLA-restricted cytotoxic T cells (CTL) after natural infection. A canarypox-CMV pp65 recombinant was studied for its ability to induce CMV pp65-specific CTL, helper T lymphocytes, and antibodies in a phase I clinical trial. Twenty-one CMV-seronegative adult volunteers were randomized to receive immunizations at months 0, 1, 3, and 6 with either canarypox-CMV pp65 or placebo. In canarypox-CMV pp65-immunized subjects, pp65-specific CTL were elicited after only 2 vaccinations and were present at months 12 and 26 in all subjects tested. Cell-depletion studies indicated that the CTL were phenotype CD8 + . Peripheral blood mononuclear cells proliferated in response to stimulation with purified pp65, and antibodies specific for pp65 also were detected. Canarypox-CMV pp65 is the first recombinant vaccine to elicit CMV-specific CTL responses, which suggests the potential usefulness of this approach in preventing disease caused by CMV.

Human cytomegalovirus (CMV) has devised numerous means of escaping immune surveillance. The CMV genome encodes at least 4 genes involved in downregulating surface expression of HLA class I molecules. In addition, it sequesters CC chemokines, induces Fc receptors, interferes with induction of HLA class II antigens, and can inhibit natural killer cell activity. CMV can efficiently block the presentation of immediate early antigens, the first viral proteins to be produced. Together, these mechanisms probably contribute to the ability of CMV to persist in its host and may play a role in the immunopathology of CMV disease.

Human cytomegalovirus (HCMV) is a complex DNA virus encoding more than 200 viral proteins. This highly adapted opportunist agent has developed several ways to evade the immune system. Among all clinical features due to HCMV, retinitis occurs especially in severely immunosuppressed patients, particularly during the end phase of HIV infection. Highly active antiretroviral therapy (HAART) has significantly reduced the incidence of this complication. However, in this HAART era, we observe the emergence of new clinical patterns in patients presenting with cicatricial HCMV retinitis. These patterns could be potentially related to immune mechanisms directed against viral antigens expressed at the surface of retinal cells that are still latently infected without any viral replication. We used a model of human retinal pigment epithelial (RPE) cells to evaluate virus-host interactions in the presence of different cytokines in the eye which play a major role in immunological or infectious conditions. Two different enzymatic pathways seem to be particularly involved during infection. Lack of tryptophan and production of nitric oxide seem to block HCMV replication in RPE cells. We propose a model to explain some of the mechanisms involved during severe immunosuppression and also after immune recovery.

The human cytomegalovirus (HCMV) open reading frame UL83 encodes a phosphoprotein of 64 to 68kDa (pp65) which is a major constituent of this virion and dense bodies. To determine the importance of the HCMV gene in the virus cycle, we studied HCMV replication in astrocytoma cells stably transfected with a retroviral vector carrying an antisense UL83 cDNA. Reverse transcription-PCR detected antisense RNA in the cytoplasm. The steady-state level of a 4-kb RNA containing coding sequences for pp65 was significantly reduced after infection of antisense cells. Concomitant with this, levels of expression of pp65 and pp71 (UL82) were severely reduced. Extracellular HCMV production was almost completely blocked, irrespective of the multiplicity of infection or the time after infection studied. The block occurred at an early phase, since immediate-early protein synthesis occurred normally, while several late proteins (e.g., pp150 [ppUL32] and assembly protein [UL80]) were absent or strongly inhibited. Normal replication of herpes simplex virus and of a pp65 deletion mutant of HCMV (RVAd65), lacking target sequences of antisense RNA, demonstrated the specificity of the block for wild-type HCMV in the antisense-stabilized cells and indicated that the block was not due to indirect interference with cellular genes. Our results appear to contradict those of Schmolke et al (S. Schmolke, H.F. Kern, P. Drescher, G. Jahn, and B. Plachter, J. Virol. 69:5959-5968, 1995), which show that UL83 is a nonessential gene for HCMV replication in vitro. This contradiction is discussed in light of the fact that the 4-kb mRNA, which codes for pp65 and was targeted in UL83-antisense cell lines, may be a bicistronic mRNA which also codes for pp71 (UL82). Thus, interference of expression from the genes encoding pp65 and pp71 by blocking of this putative bicistronic message leads to severe impairment of viral replication.

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.

We evaluated a semiquantitative PCR assay prospectively in 40 liver transplant recipients as an aid in making a prompt diagnosis of cytomegalovirus (CMV) infection. For 2 months after transplantation, clinical specimens from patients were tested weekly by PCR, virus isolation from peripheral blood and urine, and CMV serology. The incidence of active CMV infection was 70%. The levels of CMV DNA determined by hybridization of PCR samples and densitometric scanning of blots were assigned a score of 1 to 4 by comparison with four external standards amplified in parallel and corresponding to a range of 80 to 80,000 genomes. The first detection of CMV in blood by PCR occurred at a mean of 15 days, and high-level PCR scores of 3 or 4 were obtained 21 days after transplantation, whereas viremia occurred 33 days after transplantation. Significantly higher levels of CMV DNA were seen in patients with CMV disease (P < 0.05) than in asymptomatic patients. The prevalence of symptomatic CMV infection was 30%. The positive predictive value of PCR was 48%, while the negative predictive value was 100%. After treatment, the clearance of CMV DNA was always observed and the disappearance of symptoms occurred concomitantly with undetectable PCR signals.

The polymerase chain reaction (PCR) as applied to human cytomegalovirus (HCMV) detection should provide a valuable tool for rapid, reliable diagnosis of infection, thereby allowing prompt treatment. However, to date the high sensitivity of this technique and the lack of semi-quantitative interpretation have hindered establishing its validity for diagnosing systemic infection. We describe a rapid, simple, semi-quantative PCR technique for HCMV detection. The validity of the technique was tested objectively by analyzing over 2000 leukocytes specimens by PCR and comparing the results with virus isolation from urine and blood in concomitant samples in the absence of any clinical data. It could thus be established that this technique had a sensitivity and specificity of 97%. When the PCR signal corresponded to ⩾ 8000 genome equivalents for 10 4 leukocytes, the predictive value for viremia was 86%. This semi-quantitative PCR technique should allow rapid diagnosis of systemic infection and provide a reliable means of monitoring clearance of CMV from blood during drug therapy.

To describe and evaluate a polymerase chain reaction (PCR) method for early diagnosis and prompt management of cytomegalovirus (CMV) retinitis in HIV-infected patients.A total of 110 HIV-infected patients (Centers for Disease Control and Prevention stages II to IV) were sampled sequentially for isolation of CMV from peripheral blood leukocytes (PBL; n = 560) and for amplification of CMV DNA in PBL. Semiquantitative analysis of the PCR product was performed and each PCR-positive specimen was assigned a score between 1+ and 4+ (corresponding to four points on a standard curve of dilutions: 80, 800, 8000 and 80,000 CMV genome copies).Levels of CMV DNA in blood increased with HIV infection stage. We focused on eight patients who developed one or more episodes of retinitis during longitudinal follow-up, in whom we found a strong correlation between viraemia, high PCR signal (3+ or 4+) (P0.0001) and clinical symptoms. Relapse was preceded by an increase in CMV DNA and resolution correlated with clearance of CMV DNA from blood.Persistent high PCR levels always preceded virus isolation and may be the first indication of organ involvement and thus early treatment. PCR scores were consistently useful as indicators of drug efficacy and for monitoring of treatment.

We studied the effect of two cytokines, basic fibroblastic growth factor (bFGF) and transforming growth factor beta (TGF-beta) on human cytomegalovirus (HCMV) replication in cultured human lung fibroblasts. We show that TGF-beta increases HCMV production, probably by a transcriptional mechanism, and that bFGF represses HCMV replication in a dose-dependent manner. These actions were antagonistic and the mechanisms involved were independent of the effects of these factors on cell DNA synthesis and proliferation.

Cytomegalovirus (CMV) infection remains a major cause of morbidity and mortality in renal transplant recipients, and may be associated with reduced graft survival [1, 2]. Restriction enzyme analysis of CMV isolates [3–5] has clearly established that the donor kidney may be the source of CMV contamination, especially in CMV seronegative recipients who are at high risk for serious CMV disease [6]. This study, based on gene amplification by the polymerase chain reaction (PCR) [7], was undertaken to determine the incidence of CMV genome in kidneys from 30 patients. PCR has been successfully applied to the identification of CMV genome in urine, blood and saliva [8–13]. Since for ethical reasons we were reluctant to perform donor kidney biopsies in this initial study, we selected samples from patients undergoing a kidney biopsy justified by renal disease symptoms or a nephrectomy for cancer. The results show that kidneys from 8 of 20 seropositive patients contained CMV genetic material by Southern blot analysis. Unexpectedly, the latter could also be detected in 3 of 10 seronegative patients.

Human cytomegalovirus (HCMV) induction of growth factors in fibroblasts was investigated after establishing serum-free culture conditions conducive to viral replication. HCMV infection induced a type II heparin-binding growth factor which stimulated human endothelial cell proliferation.

A herpes-type virus that was originally isolated from a cell culture (designated K9V) derived from a tumor biopsy specimen from a patient with Kaposi's sarcoma was partially characterized. The host range of K9V, as determined by the induction of virus-specific cytopathology, synthesis of antigens, and plaque formation, was limited to human cells and particularly to fibroblasts. Immunofluorescence and complement fixation assays confirmed the specificity of the presence of cytomegalovirus (CMV)-type antigens in K9V-infected human fibroblasts. In addition, the density of K9V DNA was consistent with the density of CMV DNA. However, some peculiarities were observed in the K9V strain of CMV. The virus seemed more cell-associated in human fibroblasts than were known laboratory strains: The spread of cytopathology was slow and did not always involve the whole cell sheet, and the total regression of cytopathology with the establishment of a persistent infection was common. Similar characteristics have recently been observed in the Mj strain of CMV, which has been shown to be oncogenic in human fibroblasts.

Summary In this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid-cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available.

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.

A human cytomegalovirus (HCMV)-induced polypeptide of 68,000 Da (p68) with protein kinase activity was identified using a monoclonal antibody (F6b) produced against HCMV-infected cell proteins. p68 was detected by immunoprecipitation from 3 to 120 hr after infection and was induced by several strains of human and not simian CMV. Protein kinase activity was associated almost exclusively with nuclear HCMV-induced p68. Enzyme activity with ATP and casein as phosphate donor and acceptor, respectively, exhibited an optimum pH between 6 and 6.5. It was Mg2+ dependent and cAMP independent. The KATPm of 45 microM at pH 6.5 indicated a relatively high affinity of p68 for the nucleotide. p68 also transferred phosphate to phosvitin and light chains of F6b, as well as autophosphorylating at threonine and serine residues.

Human lung epithelial cells were productively infected with human cytomegalovirus in vitro. Infectious virus was released up to 8 weeks postinfection. The cells retained their morphological characteristics throughout the period of observation, while simultaneously bearing all the features typical of cytomegalovirus infection.

New ultrastructual aspects of cytomegalovirus nucleocapsid assembly are illustrated in cells from a Kaposi sarcoma. The occurrence of viral deoxyribonucleoproteins in the form of strands and penetration of these strands into open capsids is illustrated. The geometrical arrangement of the core material of the nucleocapsid is aslo emphasized.

Summary We have shown that human cytomegalovirus (CMV) infection increases, through transactivation, the functioning of long terminal repeat (LTR) elements of the human immunodeficiency virus (HIV) genome in vitro, a finding implying that CMV infection may be a direct co-factor in the pathogenesis of acquired immunodeficiency syndrome. Human MRC-5 fibroblasts were transiently transfected with a vector containing the LTR of the LAV-1 isolate of HIV and the gene coding for the enzyme chloramphenicol-acetyl-transferase (CAT). The HIV LTR-driven synthesis of CAT observed in control MRC-5 cells was much enhanced when the cells were infected with the AD169 strain of human CMV. In contrast, CAT synthesis driven by regulatory sequences from either mouse mammary tumour virus or hepatitis B was not increased. The effects of CMV were clearly associated with the early phases of infection (before CMV DNA replication occurs), since it was observed as early as 8 h after infection in CMV-permissive MRC-5 cells, and in non-permissive LTK cells which expressed only immediate-early and early CMV antigens. Our results indicate that, even under non-permissive conditions, CMV is able to transactivate HIV LTR, a finding which opens up the possibility that co-infection of human cells in vitro with both viruses results in enhanced replication of HIV, even in cell types with low permissivity to CMV.

Summary A battery of 24 anti-human cytomegalovirus (HCMV) monoclonal antibodies (McAb) has been characterized. By immunofluorescence, 7 categories of McAb were defined according to their ability to detect HCMV antigens at different locations and times after infection. Eight McAb reacted with 4 laboratory strains and 10 fresh isolates of HCMV, illustrating their potential diagnostic value. Five McAb had group-specific virus neutralizing activity, one of which showed some type-specificity. Reactivity of these McAb was also tested in indirect haemagglutination and enzyme immunoassay. None reacted in the former test, and all but 6 reacted in the latter assay. The potential value of the various tests used in screening for HCMV McAb is discussed.

DURING a comparison of cytomegalovirus (CMV)-specific haemagglutinating and immunofluorescent antibody titres in human sera, 3 of 60 sera tested by indirect immunofluorescence gave homogenous nuclear staining in 100% of infected cells within 1 h after infection (p.i.). These antigens differ from EBNA-type antigens described recently by Geder1 in CMV-infected cells 3 h p.i. in that anticomplement immunofluorescence2 is not required for their detection. They differ from ‘early antigens’ (EA)3 in that they appear much sooner and are located uniquely in the nucleus. Finally, they differ from ‘late antigens’ (LA) for they appear in the absence of DNA synthesis. By analogy to antigens described for the pseudo-rabies system by Ben-Porat et al.4 and for herpes simplex virus type 1 by Beth et al.5, we term these antigens ‘immediate early antigens’ (IEA).