The thymidylate synthase inhibitor ZD1694 potently inhibits murine and human cytomegalovirus replication in quiescent fibroblasts.

Tomudex (ZD1694) is a quinazoline-based folate analog and a powerful inhibitor of cellular thymidylate synthase and is approved in Europe for use in oncology. Here the first evidence of its activity against murine and human cytomegalovirus (MCMV and HCMV) is reported. ZD1694 irreversibly inhibited the replication and DNA synthesis of both viruses in quiescent fibroblasts. The corresponding 50% effective concentrations were 0.006 and 0.002 microM respectively, whereas the 50% cytotoxic concentration was >10 microM for both murine and human quiescent fibroblasts. A similar antiviral effect was observed against two ganciclovir-resistant HCMV strains isolated from AIDS patients. Taken as a whole these results demonstrate that cellular thymidylate synthase plays an essential role in viral replication and that ZD1694 merits further investigation as anticytomegaloviral agent.

Constitutive expression of the interferon-inducible protein p202 in NIH 3T3 cells affects cell cycle progression.

p202 is a protein expressed in murine cells after Interferon treatment. Although the function of p202 is still basically unknown, its ability to bind the hypophosphorylated form of the retinoblastoma protein pRb suggests a possible role in the control of cell proliferation. To investigate the role of p202 we have generated several cell clones of NIH 3T3 fibroblasts that constitutively express p202. Here we show that proliferation of quiescent cells on stimulation by serum addition is strongly inhibited by constitutive p202 expression. Moreover, when growth arrested cells are stimulated to proliferate, expression of p202 inhibits G0/G1 progression into the S phase and the cells accumulate with a DNA content that is equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that p202 may play a negative role in growth regulation.

cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products.

Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with p(delta)ACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of p(delta)ACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, beta-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21ras. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.

Modulation of HIV-LTR activity by ras oncogenes.

Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a v-Ha-ras gene, together with a plasmid carrying the human immunodeficiency virus (HIV) long terminal repeat (LTR) linked to the chloramphenicol acetyl transferase (CAT) reporter gene, significantly stimulated CAT activity. High HIV LTR activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. By contrast an inactivated form of ras (Ha-ras Asn-17) did not stimulate the HIV-LTR but strongly inhibited its basal activity. Activation of the p21ras protein may thus be one of the signals that regulate LTR driven transcription during HIV infection.

Characterization of nuclear factors involved in 202 gene induction by IFN-alpha, viruses or dsRNA in murine leukemia cells.

When treated with IFN-alpha, L1210 leukemia cells express high levels of the mouse 202 gene mRNA after a few hours. Three tandem copies of a 43 bp fragment (GAbox) homologous to the IFN-stimulatable response element (ISRE), located in the 5'-flanking region of the 202 gene, were linked to the reporter CAT gene and transiently transfected into L1210 cells. The data suggest that the GA box is sufficient to confer transcriptional inducibility upon IFN stimulation. Binding assays, using the labeled GA box as a probe, demonstrated the presence of a retarded complex, designated GAbfl, in the nuclear extracts of L1210 cells treated with IFN-alpha. This complex is absent in the extracts of L1210 cells treated with ssRNA viruses or synthetic dsRNA. Moreover, photoaffinity cross-linking experiments revealed that GAbfl contains a protein of about 50 kDa. Altogether these results demonstrate that antiviral state induction by IFN-alpha in L1210 cells is preceded by GAbfl binding to the ISRE of the IFN-inducible genes.

Trans-activation of the mouse cytomegalovirus immediate early gene enhancer by ras oncogenes.

The ras gene family encodes 21K proteins that reside on the inner face of the plasma membrane and bind GTP and GDP with an equally high affinity. Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a viral Harvey-ras (v-Ha-ras) cDNA, together with a plasmid (pCMVCAT) carrying the immediate early (IE) enhancer of the murine cytomegalovirus (MCMV) linked to the chloramphenicol acetyltransferase (CAT) reporter gene strongly stimulated CAT activity. Basal levels of pCMVCAT expression as well as trans-activation by v-ras plasmid were both inhibited by cotransfection of an expression vector containing the dominant inhibitory mutant gene Ha-ras Asn-17. This indicates that the p21ras protein is responsible for these activities. High pCMVCAT activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. To define the cis-acting DNA elements in the MCMV IE enhancer responsible for this trans-activation by p21ras protein, we constructed several plasmids containing the CAT gene under control of MCMV IE enhancers that were deleted in different regions. The CAT assays demonstrated that several sequences were responsive to p21ras protein. These sequences are scattered throughout the IE enhancer, upstream of the transcription start site, and contain responsive elements that are homologous to the binding sites for cellular transcription factors such as NF kappa B, AP1, ATF and SP1. Activation of the p21ras protein may thus be one of the signals that regulate IE genes transcription during MCMV infection.

[Protein p53 inhibits the activity of the enhancer of the immediate-early genes of murine cytomegalovirus]. / La proteina p53 inibisce l'attivita' dell'enhancer dei geni immediati precoci del citomegalovirus murino.

The protein encoded by the tumor suppressor gene p53 can complex and functionally interact with cytomegalovirus proteins produced during the immediate-early phase of infection. The functions of these complex are unclear but there is some evidence to suggest that binding of p53 to these viral proteins may inactivate p53 functions. Recent reports have shown that p53 is involved in regulation of transcription. In this study we have considered the possibility that p53 may regulate transcription of cytomegalovirus immediate early genes which play a crucial role for virus replication. Here we report that experiments in which NIH 3T3 cells were cotransfected with a p53 expression plasmid together with a reporter gene linked to the mouse cytomegalovirus immediate-early enhancer/promoter revealed that wild type p53 could efficiently reduce the transcriptional activity of this viral regulatory sequence. By contrast expression of a mutated p53 correlated with a much smaller reduction of transcription. Deletion mutants analysis of the enhancer revealed that repression of transcription by p53 requires a minimal promoter containing an SP1 consensus sequence and a TATA box.

[Serum stimulates the transcriptional activity of the enhancer of the immediate-early genes of the murine cytomegalovirus through p21 ras]. / Il siero stimola l'attivita' trascrizionale dell'enhancer dei geni immediati precoci del citomegalovirus murino tramite la p21ras.

The analysis of the MCMV IE enhancer revealed the presence of many putative binding sites for the transcription factors AP-1 and NFkB. Previous studies suggested that such factors represent a final target for the metabolic cascade triggered by serum and growth factors. On these basis we wanted to verify if serum stimulates the transcriptional activity of the MCMV IE enhancer through p21ras and AP-1 and NFkB according to the actual model of transduction of the mitogenic signal. Our data demonstrate that serum stimulates the MCMV IE enhancer through a pathway in which the p21ras is involded, as demonstrated by using the dominant inhibitory mutant ras(Asn 17). Moreover deletion mutant analysis of the enhancer showed that the serum responsive region lies between nucleotides -1280 and -285 and contains a high concentration of putative AP-1 and NFkB binding sites.

Effect of interferon-alpha on immediate early gene expression of murine cytomegalovirus.

Interferon-alpha (IFN-alpha) significantly reduced the replication of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts derived from the susceptible mouse strain C3H/HeJ. When infectious virus production was measured, a strong decrease in virus titer was observed in IFN-treated cells at a multiplicity of infection (moi) of 1 and 0.5 pfu/cell. Analysis of virus-specified mRNAs by Northern blot assay revealed that IFN-alpha had a significant effect on the expression of viral mRNAs at 48h. In particular, the mRNAs of the major immediate early (IE) transcription units, IE1, IE2, and IE3, were impaired by IFN-alpha. In addition, decrease of IE1 mRNA synthesis was accompanied by a reduction of the major IE product (pp89), as revealed by Western blot assay. These results suggest that IFN-alpha may inhibit MCMV replication by directly impairing IE gene transcription.

[Specific IgG for viral capsid antigen (VCA) of Epstein-Barr virus: evaluation of several commercial kits]. / IgG specifiche per l'antigene VCA del virus di Epstein-Barr: valutazione di alcuni "kits" commerciali.

Four methods to detect IgG antibodies to Epstein-Barr virus in serum samples of aspirate in renal transplant patients were evaluated. Further to technical characteristics, the assays were compared as to sensitivity, specificity and positive and negative predictive value. 100% of specificity was found for all methods whereas sensibility and negative predictive value were different among the different methods.

Production of monoclonal antibodies against the carbohydrate of group A streptococci.

Mouse hybridomas were isolated by fusing P3.X63.Ag8-653 myeloma cells with spleen cells from BDF1 mice repeatedly immunized with Streptococcus pyogenes type 3 ATCC 12384. Two out of 480 growing cultures secreted antibodies against the bacterial strain used as immunogen, as evaluated by an ELISA assay. After cloning the most representative continuously growing clone, 1-181, the binding specificity of these monoclonal antibodies was determined using either Streptococci with carbohydrate of different group or different bacteria strains. Binding properties suggest that the 1.181 monoclonal antibodies specifically recognize the carbohydrate moiety of Streptococci group A.