PKCε as a novel promoter of skeletal muscle differentiation and regeneration.

INTRODUCTION: Satellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε-HMGA1 signaling axis in myoblast differentiation and regeneration processes. METHODS: PKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKCε and HMGA1 in myogenic differentiation. RESULTS: PKCε expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKCε blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKCε impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration. CONCLUSION: This work identifies the PKCε-HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.

Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

PKCε is a negative regulator of PVAT-derived vessel formation.

RATIONALE: Vessel formation is a crucial event in tissue repair after injury. Thus, one assumption of innovative therapeutic approaches is the understanding of its molecular mechanisms. Notwithstanding our knowledge of the role of Protein Kinase C epsilon (PKCε) in cardio-protection and vascular restenosis, its role in vessel progenitor differentiation remains elusive. OBJECTIVE: Given the availability of PKCε pharmacological modulators already tested in clinical trials, the specific aim of this study is to unravel the role of PKCε in vessel progenitor differentiation, with implications in vascular pathology and vasculogenesis. METHODS AND RESULTS: Mouse Peri-Vascular Adipose Tissue (PVAT) was used as source of mesenchymal vessel progenitors. VEGF-induced differentiation of PVAT cells down-regulates both PKCε and p-PAK1 protein expression levels. PKCε overexpression and activation: i) reduced the expression levels of SMA and PECAM in endothelial differentiation of PVAT cells; ii) completely abrogated tubules formation in collagen gel assays; iii) increased the expression of p-PAK1. CONCLUSION: PKCε negatively interferes with vessel progenitor differentiation via interaction with PAK-1.

The role of PKCε-dependent signaling for cardiac differentiation.

Protein kinase Cepsilon (PKCε) exerts a well-known cardio-protective activity in ischemia-reperfusion injury and plays a pivotal role in stem cell proliferation and differentiation. Although many studies have been performed on physiological and morphological effects of PKCε mis-expression in cardiomyocytes, molecular information on the role of PKCε on early cardiac gene expression are still lacking. We addressed the molecular role of PKCε in cardiac cells using mouse cardiomyocytes and rat bone marrow mesenchymal stem cells. We show that PKCε is modulated in cardiac differentiation producing an opposite regulation of the cardiac genes NK2 transcription factor related, locus 5 (nkx2.5) and GATA binding protein 4 (gata4) both in vivo and in vitro. Phospho-extracellular regulated mitogen-activated protein kinase 1/2 (p-ERK1/2) levels increase in PKCε over-expressing cells, while pkcε siRNAs produce a decrease in p-ERK1/2. Indeed, pharmacological inhibition of ERK1/2 rescues the expression levels of both nkx2.5 and gata4, suggesting that a reinforced (mitogen-activated protein kinase) MAPK signaling is at the basis of the observed inhibition of cardiac gene expression in the PKCε over-expressing hearts. We demonstrate that PKCε is critical for cardiac cell early gene expression evidencing that this protein is a regulator that has to be fine tuned in precursor cardiac cells.

Neuroendocrine regulation and tumor immunity.

The morphogenetic events leading to the transendothelial passage of lymphoid and tumoral cells are analyzed in light of a very recent and global theory of intercellular communication designated as the Triune Information Network (TIN). The TIN system is based on the assumption that cell-cell interactions primarily occur through cell surface informations or topobiological procesess, whose mechanisms rely upon expression of adhesion molecules, and are regulated by an array of locally-borne (autocrine/paracrine signals and autonomic inputs) and distantly-borne (endocrine secretions) messages. The final aim of the TIN is to control homeostatic functions crucial for the organism survival, like morphogenesis. Knowledge of the TIN signals involved in lymphoid and tumoral cell intravasation might offer a new perspetive to study the mechanisms of tumor immunity. Recognition of tumor target cells by immune cytotoxic effectors, in fact, can be considered a notable case of TIN-mediated cell to cell interaction. In particular, Natural Killer (NK) cells play a role in the cell-mediated control of tumor growth and metastatic spreading. Cell targeting and killing are dependent on the different NK cell receptors and on the efficacy of NK cells after cytokine and monoclonal antibody administration in cancer therapy. Since efficacy of NK cell-based immunotheraphy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell based immunotherapy.

TNF-related apoptosis-inducing ligand (TRAIL) and erythropoiesis: a role for PKC epsilon.

The regulation of the hematopoietic stem cell pool size and the processes of cell differentiation along the hematopoietic lineages involve apoptosis. Among the different factors with a recognized activity on blood progenitor cells, TRAIL - a member of the TNF family of cytokines - has an emerging role in the modulation of normal hematopoiesis.PKC(epsilon) levels are regulated by EPO in differentiating erythroid progenitors and control the protection against the apoptogenic effect of TRAIL. EPO-induced erythroid CD34 cells are insensitive to the apoptogenic effect of TRAIL between day 0 and day 3, due to the lack of specific surface receptors expression. Death receptors appear after day 3 of differentiation and consequently erythroid cells become sensitive to TRAIL up to day 9/10, when the EPO-driven up-regulation of PKC epsilon intracellular levels inhibits the TRAIL-mediated apoptosis, via Bcl-2. In the time interval between day 3 and 9, therefore, the number of erythroid progenitors can be limited by the presence of soluble or membrane-bound TRAIL present in the bone marrow microenvironment.

The response of human natural killer cells to interleukin-2.

Natural killer cells play a key role in the defence of organisms against virus infections and in the control of tumor onset. Interleukin-2 is a multifunctional inflammatory cytokine able to activate natural killer cells, essentially inducing cell proliferation, lymphokine-activated-killer cell generation and cytokine production. Here we discuss some signaling events generated by interleukin-2 in the cell nucleus of primary human natural killer cells, specifically focusing on the lipid signal transduction and the induction of the cyclic adenosine-5'-monophosphate response element binding protein transcription factor. The implications of these nuclear events in the response of natural killer cells to interleukin-2 are also discussed.

Epitope mapping of human herpesvirus-7 gp65 using monoclonal antibodies.

Human herpesvirus (HHV)-7 encodes a unique 65-kDa heparin-binding glycoprotein, designated gp65. This molecule is thought to play a role in virus attachment and entry. To obtain reagents to map the structure and function of HHV-7 gp65, we produced monoclonal antibodies to this molecule. Ten monoclonal antibodies reacting with gp65 on ELISA were subdivided in four groups on the basis of their isotype and differential reactivity with (i) native versus denatured forms of gp65, and (ii) mature (virion-associated) versus immature (cell-associated) forms of the molecule. We were able to map the binding epitopes for eight of these ten antibodies, and these were found to cluster to one site on gp65 (amino acids 239-278); within this region, the antibodies reacted with at least three distinct domains (244-251, 255-262, 263-278). The reasons for the apparent immunodominance of this region are uncertain. Taken together, this panel of antibodies constitutes an extensive and well-characterized set of HHV-7 specific antibodies that may have utility for future analyses of the structure/function of gp65, and for studies on the virus life cycle.

Human herpesvirus 7 induces the functional up-regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) coupled to TRAIL-R1 down-modulation in CD4(+) T cells.

Human herpesvirus 7 (HHV-7) is endemic in the adult human population. Although HHV-7 preferentially infects activated CD4(+) T lymphocytes, the consequence of T-cell infection for viral pathogenesis and immunity are still largely unknown. HHV-7 infection induces apoptosis mostly in uninfected bystander cells but not in productively infected CD4(+) T cells. To dissect the underlying molecular events, the role of death-inducing ligands belonging to the tumor necrosis factor (TNF) cytokine superfamily was investigated. HHV-7 selectively up-regulated the expression of TNF-related apoptosis-inducing ligand (TRAIL), but not that of CD95 ligand or TNF-alpha in lymphoblastoid (SupT1) or primary activated CD4(+) T cells. Moreover, in a cell-to-cell-contact assay, HHV-7-infected CD4(+) T lymphocytes were cytotoxic for bystander uninfected CD4(+) T cells through the TRAIL pathway. By contrast, HHV-7 infection caused a marked decrease of surface TRAIL-R1, but not of TRAIL-R2, CD95, TNF-R1, or TNF-R2. Of note, the down-regulation of TRAIL-R1 selectively occurred in cells coexpressing HHV-7 antigens that became resistant to TRAIL-mediated cytotoxicity. These findings suggest that the TRAIL-mediated induction of T-cell death may represent an important immune evasion mechanism of HHV-7, helping the virus to persist in the host organism throughout its lifetime.

Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the human myeloid K562 cell line. TRAIL stimulated caspase 3 and nitric oxide synthase (NOS) activities, and both pathways cooperate in mediating inhibition of K562 survival/growth. This was demonstrated by the ability of z-VAD-fmk, a broad inhibitor of effector caspases, and N-nitro-L-arginine methyl ester (L-NAME), an NOS pharmacologic inhibitor, to completely (z-VAD-fmk) or partially (L-NAME) suppress the TRAIL-mediated inhibitory activity. Moreover, z-VAD-fmk was able to block TRAIL-mediated apoptosis and cell cycle abnormalities and increase of NOS activity. The addition of the NO donor sodium nitroprusside (SNP) to K562 cells reproduced the cytostatic effect of TRAIL without inducing apoptosis. When TRAIL was associated to SNP, a synergistic increase of apoptosis and inhibition of clonogenic activity was observed in K562 cells as well as in other myeloblastic (HEL, HL-60), lymphoblastic (Jurkat, SupT1), and multiple myeloma (RPMI 8226) cell lines. Although SNP greatly augmented TRAIL-mediated antileukemic activity also on primary leukemic blasts, normal erythroid and granulocytic cells were less sensitive to the cytotoxicity mediated by TRAIL with or without SNP. These data indicate that TRAIL promotes cytotoxicity in leukemic cells by activating effector caspases, which directly lead to apoptosis and stimulate NO production, which mediates cell cycle abnormalities. Both mechanisms seem to be essential for TRAIL-mediated cytotoxicity.

Human primary CD4 + T cells activated in the presence of IFN-alpha 2b express functional indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the catabolism of tryptophan. By creating a local microenvironment in which levels of tryptophan are low, IDO-expressing antigen-presenting cells (APC) could regulate T cell activation. This may be relevant to control both viral and bacterial replication as well as neoplastic cell growth. Interferon-alpha (IFN-alpha) is an antiviral cytokine affecting cellular differentiation. In addition, it reduces proliferation of CD4(+) T cells by several molecular mechanisms. To dissect the molecular steps responsible for the INF-mediated antiproliferative activity, we sought to determine whether activated primary CD4(+) T cells in the presence of IFN-alpha would produce IDO. We demonstrate here that IDO mRNA is not present in resting CD4(+) T cells. Stimulation with anti-CD3 plus interleukin-2 (IL-2) induces expression of IDO mRNA (about 2000 copies/150,000 cells), as determined by semiquantitative RT-PCR. When cells were stimulated in the presence of IFN-alpha, expression of IDO mRNA was significantly increased (more than 12,000 copies/150,000 cells). Functional analysis of IDO activity paralleled the results obtained with RT-PCR, demonstrating increased production of active enzyme in CD4(+) T cells stimulated in the presence of IFN-alpha. Our results indicate that IFN-alpha modulates levels of IDO produced by activated CD4(+) T cells. This would likely affect bystander cells by modifying levels of tryptophan in the local microenvironment.

HIV-1 Tat protein down-regulates CREB transcription factor expression in PC12 neuronal cells through a phosphatidylinositol 3-kinase/AKT/cyclic nucleoside phosphodiesterase pathway.

The addition of low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein to PC12 neuronal cells stimulated a rapid (peak at 5 min) elevation of the cAMP intracellular levels, which in turn induced the phosphorylation of CREB transcription factor (peak at 15 min) on serine-133 (Ser-133). On the contrary, at later time points (60-120 min) Tat induced a significant decline of intracellular cAMP with respect to the basal levels observed in control cells treated with bovine serum albumin. In blocking experiments performed with pharmacological inhibitors, Tat decreased the intracellular levels of cAMP and CREB Ser-133 phosphorylation through a signal transduction pathway involving the sequential activation of phosphatidylinositol 3-kinase, AKT, and cyclic nucleoside phosphodiesterases. Moreover, in transient transfection experiments, Tat inhibited transcription of CREB promoter in a manner strictly dependent on the presence of the cAMP-responsive elements (CRE) in the CREB promoter. Consistently, the expression of endogenous CREB protein was significantly reduced in PC12 cells by prolonged (24-48 h) treatment with Tat. This decline in the expression of CREB, which plays an essential role in the survival and function of neuronal cells, anticipated a progressive increase of apoptosis in Tat-treated cells. Although obtained in a neuronal cell line, our findings might help to explain some aspects of the pathogenesis of HIV-1-associated dementia.

Pivotal role of cyclic nucleoside phosphodiesterase 4 in Tat-mediated CD4+ T cell hyperactivation and HIV type 1 replication.

We show here that HIV type 1 (HIV-1) Tat protein, in combination with anti-CD3/CD28 mAbs, promotes IL-2 production and proliferation of primary CD4(+) T lymphocytes, obtained from HIV-1-seronegative donors. This effect was observed when Tat was immobilized on a solid support, but it was not observed with soluble Tat. Such hyperactivation was accomplished by recruiting the rolipram-sensitive cyclic nucleoside phosphodiesterase 4 and resulted in increased susceptibility to HIV-1 infection. Accordingly, rolipram potently inhibited HIV-1 replication in cultures stimulated by anti-CD3/CD28 +/- Tat. These results add to the concept that decreasing Tat activity is an important addition to anti-HIV-1 therapy, and they suggest a target for anti-HIV-1 chemotherapy, phosphodiesterase 4.

Infection of CD34(+) hematopoietic progenitor cells by human herpesvirus 7 (HHV-7).

To investigate the tropism of the T-lymphotropic human herpesvirus 7 (HHV-7) for hematopoietic progenitors, cord blood CD34(+) cells were inoculated in vitro with HHV-7 and then induced to differentiate along the granulocytic and erythroid lineages by the addition of appropriate cytokine cocktails. In semisolid assays, HHV-7 modestly affected the growth of committed (granulocytic/macrophagic and erythroid) progenitors, whereas it significantly decreased the number of pluripotent (granulocytic/erythroid/ monocytic/megakaryocytic) progenitors. Such inhibitory effect was completely abrogated by incubating HHV-7 inoculum with anti-HHV-7 neutralizing serum. In liquid cultures, HHV-7 hastened maturation along the myeloid but not the erythroid lineage, as demonstrated by the up-regulation of CD33 early myeloid antigen at day 7 of culture, and of CD15 and CD14 antigens at day 15. Moreover, HHV-7 messenger RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells maturating along both the myeloid and the erythroid lineages. To evaluate the relevance of these in vitro findings, the presence of HHV-7 was investigated in bone marrow (BM) unfractionated mononuclear cells (MCs) as well as in purified CD34(+) and CD34(-) cell subsets, obtained from 14 normal adult donors. HHV-7 DNA was detected by DNA-PCR in 4 of 7 BMMC samples, and it was found to be associated with both the CD34(-) (2 of 7) and the CD34(+ )(1 of 7) fractions. These data indicate that HHV-7 infects BM cells in vivo and shows the ability to affect the survival/differentiation of CD34(+) hematopoietic progenitors in vitro by inhibiting more ancestral progenitors and perturbing the maturation of myeloid cells.

HIV-1 Tat-mediated inhibition of the tyrosine hydroxylase gene expression in dopaminergic neuronal cells.

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

OBJECTIVE: To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. METHODS: CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. RESULTS: Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. CONCLUSIONS: Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.

Human herpesvirus 6: An emerging pathogen.

Infections with human herpesvirus 6 (HHV-6), a beta-herpesvirus of which two variant groups (A and B) are recognized, is very common, approaching 100% in seroprevalence. Primary infection with HHV-6B causes roseola infantum or exanthem subitum, a common childhood disease that resolves spontaneously. After primary infection, the virus replicates in the salivary glands and is shed in saliva, the recognized route of transmission for variant B strains; it remains latent in lymphocytes and monocytes and persists at low levels in cells and tissues. Not usually associated with disease in the immunocompetent, HHV-6 infection is a major cause of opportunistic viral infections in the immunosuppressed, typically AIDS patients and transplant recipients, in whom HHV-6 infection/reactivation may culminate in rejection of transplanted organs and death. Other opportunistic viruses, human cytomegalovirus and HHV-7, also infect or reactivate in persons at risk. Another disease whose pathogenesis may be correlated with HHV-6 is multiple sclerosis. Data in favor of and against the correlation are discussed.

Trafficking to the plasma membrane of the seven-transmembrane protein encoded by human herpesvirus 6 U51 gene involves a cell-specific function present in T lymphocytes.

The sequence of human herpesvirus 6 (HHV-6) U51 open reading frame predicts a protein of 301 amino acid residues with seven transmembrane domains. To identify and characterize U51, we derived antipeptide polyclonal antibodies and developed a transient expression assay. We ascertained that U51 was synthesized in cord blood mononuclear cells infected with either variant A- or variant B-HHV-6 and was transported to the surface of productively infected cells. When synthesized in transient expression systems, U51 intracellular trafficking was regulated in a cell-type-dependent fashion. In human monolayer HEK-293 and 143tk- cells, U51 accumulated predominantly in the endoplasmic reticulum and failed to be transported to the cell surface. In contrast, in T-lymphocytic cell lines J-Jhan, Molt-3, and Jurkat, U51 was successfully transported to the plasma membrane. We infer that transport of U51 to the cell surface requires a cell-specific function present in activated T lymphocytes and T-cell lines.

The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells.

We report on the functional cloning of a hitherto unknown member of the immunoglobulin (Ig) superfamily selected for its ability to confer susceptibility to herpes simplex virus (HSV) infection on a highly resistant cell line (J1.1-2 cells), derived by exposure of BHKtk- cells to a recombinant HSV-1 expressing tumor necrosis factor alpha (TNF-alpha). The sequence of herpesvirus Ig-like receptor (HIgR) predicts a transmembrane protein with an ectodomain consisting of three cysteine-bracketed domains, one V-like and two C-like. HIgR shares its ectodomain with and appears to be an alternative splice variant of the previously described protein PRR-1 (poliovirus receptor-related protein). Both HIgR and PRR-1 conferred on J1.1-2 cells susceptibility to HSV-1, HSV-2, and bovine herpesvirus 1. The viral ligand of HIgR and PRR-1 is glycoprotein D, a constituent of the virion envelope long known to mediate viral entry into cells through interaction with cellular receptor molecules. Recently, PRR-1, renamed HveC (herpesvirus entry mediator C), and the related PRR-2, renamed HveB, were reported to mediate the entry of HSV-1, HSV-2, and bovine herpesvirus 1, and the homologous poliovirus receptor was reported to mediate the entry of pseudorabies virus (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998; M. S. Warner, R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear, Virology 246:179-189, 1998). Here we further show that HIgR or PRR-1 proteins detected by using a monoclonal antibody to PRR-1 are widely distributed among human cell lines susceptible to HSV infection and commonly used for HSV studies. The monoclonal antibody neutralized virion infectivity in cells transfected with HIgR or PRR-1 cDNA, as well as in the human cell lines, indicating a direct interaction of virions with the receptor molecule, and preliminarily mapping this function to the ectodomain of HIgR and PRR-1. Northern blot analysis showed that HIgR or PRR-1 mRNAs were expressed in human tissues, with the highest expression being detected in nervous system samples. HIgR adds a novel member to the cluster of Ig superfamily members able to mediate the entry of alphaherpesviruses into cells. The wide distribution of HIgR or PRR-1 proteins among human cell lines susceptible to HSV infection, coupled with the neutralizing activity of the antibody in the same cells, provides direct demonstration of the actual use of this cluster of molecules as HSV-1 and HSV-2 entry receptors in human cell lines. The high level of expression in samples from nervous system makes the use of these proteins in human tissues very likely. This cluster of molecules may therefore be considered to constitute bona fide receptors for HSV-1 and HSV-2.

Temporal mapping of transcripts in herpesvirus 6 variants.

To define the molecular features characteristic of the early stages of infection of lymphocytes with human herpesvirus 6 (HHV-6) variant A or B, we studied the temporal regulation of expression of selected sets of viral genes. Thus, U42, U94, U89-U90, U73, and U39 are alpha genes since their transcripts (i) were made in the presence of inhibitors of protein synthesis and (ii) were detected 3 h after infection of untreated cells. U41, U53, U31, and U19 are beta genes since their expression is inhibited by cycloheximide but not by phosphonoacetate, an inhibitor of DNA synthesis. U100 is a gamma gene since its spliced transcript encoding the structural glycoprotein gp82/105 was first detected 16 h after infection of untreated cells but could not be detected in cells treated with phosphonoacetate. HHV-6 variants differ in the transcription patterns of their genes. U16-U17 originates a splice transcript and is regulated as alpha in HHV-6B and as beta in HHV-6A. U91 generates two transcripts, amplified as 476- and 374-bp PCR fragments. The 476-bp fragment is alpha in HHV-6A-infected cells but beta in HHV-6B-infected cells. Conversely, the 374-bp fragment is beta in HHV-6A-infected cells and alpha in HHV-6B-infected cells. Furthermore, the spliced product of U18-U19-U20 (526 bp) is beta in HHV-6A-infected cells, but only a partially spliced form (1.9 kb) was detected at late stages of infection in HHV-6B. HHV-6 transcription was also studied in nonproductive lymphoid cells, and the same transcription pattern detected during lytic infection was observed. Also, HHV-6 variants maintain the differences in U91, U16-17, and U18-U19-U20. We conclude that, as expected from the sequencing data, gene expression is generally similar in HHV-6 variants. However, transcription of selected genes in HHV-6A and HHV-6B differs with respect to temporal regulation and splicing pattern. Furthermore, the identification of viral functions expressed during the different stages of lytic replication suggests that reverse transcription-PCR for HHV-6 genes is a useful diagnostic approach to differentiate between latent and productive HHV-6 infection.