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PubMed2006

Molecular interactions between porcine and human gammaherpesviruses: implications for xenografts?

BACKGROUND: Reactivation of latent herpesviruses is an important cause of morbidity and mortality in human transplantation. This issue might be further complicated in the case of xenotransplantation. Zoonotic viruses could reactivate and replicate in the transplanted tissue, and interactions with homologous human viruses could take place. Since the pig is a favoured animal as donor of organs for human transplants, we analysed the possibility of interactions between porcine and human herpesviruses. Porcine lymphotropic herpesvirus 1 (PLHV-1) is a gammaherpesvirus homologous to Epstein-Barr virus (EBV) and to human herpesvirus 8 (HHV-8), is highly prevalent in pigs and is associated to lymphoproliferative disease in immunosuppressed and transplanted miniature swine. METHODS: The main viral transactivators of PLHV-1, ORF50, ORF57, ORFA6/BZLF1(h), were cloned and tested for their transactivating ability on several EBV and HHV-8 promoters using reporter assays. Also the effects of HHV-8 ORF50, ORF57 and ORFK8 and EBV BRLF1/ R-transactivator (Rta) and BZLF1/ Z-transactivator (Zta) on PLHV-1 lytic promoters were analysed. RESULTS: Porcine lymphotropic herpesvirus 1 ORF50 upregulated all HHV-8 promoters and PLHV-1 ORFA6/BZLF1(h) transactivated EBV promoters. Furthermore, transfection of PLHV-1 ORF50 into BC-3 cells, latently infected with HHV-8, resulted in HHV-8 reactivation. Likewise, HHV-8 ORF50 and EBV BRLF1/Rta had a strong transactivating effect on PLHV-1 promoters. Also EBV BZLF1/Zta and HHV-8 ORF57 induced PLHV-1 transactivation, but at lower levels. CONCLUSION: The results suggest that reciprocal molecular interactions between human and porcine herpesviruses might occur in vivo, and support the hypothesis that PLHV-1 might have pathogenic relevance in the course of xenotransplantation.

Animals↗
PubMed1994

A modified plaque assay and infected cell hybridization assay for wild-type and recombinant LuIII autonomous parvovirus.

We describe a convenient infected cell hybridization assay for determining the infectious titer of a recombinant, replication-defective parvovirus. We previously generated recombinant derivatives of the autonomous parvovirus LuIII, transducing the luciferase reporter gene. Since luciferase expression is not readily detected at the single cell level, and since the recombinants cannot form plaques, we developed an alternative assay for infectious particles. Co-infection of NB324K cells with wild-type LuIII (multiplicity of infection ca. 5) and the recombinant virions allows amplification of the transducing DNA, which can be detected by hybridization with a probe for the reporter gene. Cell lysis and DNA transfer to a nylon membrane is performed in situ, in the culture dish, and hybridization is performed with a digoxigenin-labeled probe, using immunological detection. During this work we developed a conveniently modified plaque assay for wild-type LuIII that should be applicable to other lytic viruses. The modification employs a reduced volume of agarose overlay that is in turn overlaid with liquid medium, thus avoiding the need to maintain stocks of culture medium at higher than normal concentration.

Hybridization, Genetic↗
PubMed2000

Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages.

Recombinant phages are generated when Lactococcus lactis subsp. lactis harboring plasmids encoding the abortive type (Abi) of phage resistance mechanisms is infected with small isometric phages belonging to the P335 species. These phage variants are likely to be an important source of virulent new phages that appear in dairy fermentations. They are distinguished from their progenitors by resistance to Abi defenses and by altered genome organization, including regions of L. lactis chromosomal DNA. The objective of this study was to characterize four recombinant variants that arose from infection of L. lactis NCK203 (Abi(+)) with phage phi31. HindIII restriction maps of the variants (phi31.1, phi31.2, phi31.7, and phi31.8) were generated, and these maps revealed the regions containing recombinant DNA. The recombinant region of phage phi31.1, the variant that occurred most frequently, was sequenced and revealed 7.8 kb of new DNA compared with the parent phage, phi31. This region contained numerous instances of homology with various lactococcal temperate phages, as well as homologues of the lambda recombination protein BET and Escherichia coli Holliday junction resolvase Rus, factors which may contribute to efficient recombination processes. A sequence analysis and phenotypic tests revealed a new origin of replication in the phi31.1 DNA, which replaced the phi31 origin. Three separate HindIII fragments, accounting for most of the recombinant region of phi31.1, were separately cloned into gram-positive suicide vector pTRK333 and transformed into NCK203. Chromosomal insertions of each plasmid prevented the appearance of different combinations of recombinant phages. The chromosomal insertions did not affect an inducible prophage present in NCK203. Our results demonstrated that recombinant phages can acquire DNA cassettes from different regions of the chromosome in order to overcome Abi defenses. Disruption of these regions by insertion can alter the types and diversity of new phages that appear during phage-host interactions.

Bacteriolysis↗
PubMed2002

Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors.

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.

Adenoviruses, Human↗
PubMed1983

Involvement of Escherichia coli K-12 DNA polymerase I in the growth of bacteriophage Mu.

We examined several aspects of bacteriophage Mu development in Escherichia coli strains that carry mutations in the polA structural gene for DNA polymerase I (PolI). We found that polA mutants were markedly less efficient than PolI wild-type (PolI+) strains in their capacity to form stable Mu lysogens and to support normal lytic growth of phage Mu. The frequency of lysogenization was determined for polA mutants and their isogenic PolI+ derivatives, with the result that mutants were lysogenized 3 to 8 times less frequently than were PolI+ cells. In one-step growth experiments, we found that phage Mu grew less efficiently in polA cells than in PolI+ cells, as evidenced by a 50 to 100% increase in the latent period and a 20 to 40% decrease in mean burst size in mutant cells. A further difference noted in infected polA strains was a 10-fold reduction in the frequency of Mu-mediated transposition of chromosomal genes to an F plasmid. Pulse labeling and DNA-DNA hybridization assays to measure the rate of phage Mu DNA synthesis after the induction of thermosensitive prophages indicated that phage Mu replication began at about the same time in both polA and PolI+ strains, but proceeded at a slower rate in polA cells. We conclude that PolI is normally involved in the replication and integration of phage Mu. However, since phage Mu does not exhibit an absolute requirement for normal levels of PolI, it appears that residual PolI activity in the mutant strains, other cellular enzymes, or both can partially compensate for the absence of normal PolI activity.

Bacteriophage mu↗
PubMed2003

The role of the cytotoxic T-lymphocyte response and virus cytopathogenicity in the virus decline during antiviral therapy.

Although it is clear that HIV can lyse HIV-infected CD4 T cells, it is still controversial whether the depletion of CD4 T cells seen in HIV-infected patients after years of asymptomatic disease is caused by the direct cytopathic effects of the virus or is mediated by the immune response. Assuming the initial decline in viraemia during highly active antiretroviral therapy (HAART) is caused by the death of cells productively infected with HIV, I investigate how the rate of the virus decline is affected by the efficiency of the cytotoxic T-lymphocyte (CTL) response. I find that whether the stronger immune response causes a more rapid virus decline depends critically on how the virus is controlled by the CTL response (lytic versus non-lytic mechanisms). Moreover, variation in the efficiency of the immune response does not always cause variation in the rate of the virus decline (and, therefore, in the death rate of infected cells), implying that the constancy of the virus decline rate measured in different patients does not necessarily indicate that the virus is cytopathic. The potential problems associated with the model and the approach undertaken are also discussed.

Antiretroviral Therapy, Highly Active↗
PubMed2001

Effects of interaction between parvovirus minute virus of mice NS1 and coactivator CBP on NS1- and p53-transactivation.

The non-structural protein NS1, encoded by the parvovirus minute virus of mice (MVM), is a potent regulator of viral gene expression in addition to prominent roles in viral replication and cytopathic effects associated with parvoviral infection. Although NS1 involves the modulation of viral and cellular transcription, the primary activation mechanism of MVM NS1 remains unclear. In the present study, we show here that the coactivator CREB binding protein, CBP, could potentiate NS1-mediated transcription as measured on the P38 promoter, which drives expression of the MVM capsid genes. NS1 bound to the two related cysteine-histidine-rich regions of CBP, referred to as C/H1 and C/H3, the former of which has an antagonistic function to CBP upon the NS1-transactivation. Furthermore, NS1 inhibited the synergistic transactivation by CBP and p53. These findings suggested that CBP as a transcriptional coactivator is required for NS1-mediated viral and cellular transcription in parvovirus-infected cells, resulting in cell proliferation and differentiation to achieve its lytic cycle.

3T3 Cells↗
PubMed2006

Increased frequency and broadened specificity of latent EBV nuclear antigen-1-specific T cells in multiple sclerosis.

Epidemiological studies consistently demonstrate that patients with multiple sclerosis are almost universally infected with Epstein-Barr virus (EBV) and that the risk of developing the disease increases with the level of EBV-specific antibody titers. The EBV-encoded nuclear antigen-1 (EBNA1) maintains the viral episome in replicating infected human B cells, and EBNA1-specific CD4+ T cells have been identified as a crucial part of the EBV-specific immune control in healthy individuals. We studied 20 untreated EBV seropositive patients with multiple sclerosis and 20 healthy EBV carriers matched demographically and for the expression of multiple sclerosis-associated HLA-DR alleles for their immunological control of EBV latency at the level of EBNA1-specific T cells. Using 51 overlapping peptides covering the C-terminal of EBNA1 domain of EBNA1 (amino acids 400-641), peptide-specific CD4+ memory T cells in patients with multiple sclerosis were found to be strikingly elevated in frequency, showed increased proliferative capacity and an enhanced interferon-gamma production. In contrast to EBNA1, T-cell responses to three other latent and three other lytic immunodominant EBV antigens and human cytomegalovirus (HCMV) epitopes did not differ between patients and controls, indicating a distinct role for EBNA1-specific T-cell responses in multiple sclerosis. CD4+ T cells from healthy virus carriers preferentially recognized multiple epitopes within the centre part of the C-terminal, whereas the stimulatory epitopes in multiple sclerosis patients covered the entire sequence of this domain of EBNA1. Quantification of EBV viral loads in peripheral blood mononuclear cells (PBMC) by real-time polymerase chain reaction (PCR) showed higher levels of EBV copy numbers in some patients with multiple sclerosis, although the overall difference in viral loads was not statistically significant compared with healthy virus carriers. We suggest that the accumulation of highly antigen-sensitive EBNA1-specific Th1 cells in multiple sclerosis is capable of sustaining autoimmunity by cross-recognition of autoantigens or by TCR-independent bystander mechanisms.

Adult↗
PubMed1992

Lytic infection of primary rhesus kidney cells by simian virus 40.

In an attempt to analyze the persistent infection of rhesus monkey cells with Simian virus 40 (SV40) in vitro, as described previously (reviewed in L. C. Norkin, Microbiol. Rev. 46, 384-425, 1982), we infected primary rhesus cell cultures (PRK), derived from a SV40-free monkey colony with SV40. Surprisingly, SV40 infected PRK cell cultures released as much infectious virus as cultures of the permissive African green monkey kidney cell line TC7. Infected PRK cells exhibited typical symptoms of a lytic infection, and the bulk of infected PRK cells died within 8 days postinfection (p.i.). A considerable proportion of infected PRK cells exhibited distinct SV40-caused cytopathic effects (CPE), similar to CPE in infected TC7 cells. We conclude that the in vivo persistence of SV40 in rhesus monkeys is not determined by cellular host factors, but by the immune system of the infected animals.

Animals↗
PubMed2006

Selection and characterization of canine, swine and rabbit cell lines resistant to bovine viral diarrhea virus.

Contamination of cell cultures with adventitious viruses may pose serious risks for virology diagnosis, research and vaccine production. This article reports the selection and characterization of three cell lines resistant to bovine viral diarrhea virus (BVDV), a major contaminant of cell cultures. The resistant cells were obtained from canine (MDCK), swine (PK-15) and rabbit (RK-13) parental cell lines by expanding and cloning cells that survived lytic infection with BVDV. All three selected cell lines were resistant to three standard BVDV strains and to 10 field isolates as demonstrated by immunofluorescence for viral antigens and by co-cultivation with susceptible cells. Inoculation of selected cells with BVDV (10 TCID50/cell) resulted in frequencies of infection of <10(-5) (MDCK-R, PK-15R) and 3.3 x 10(-4) (RK-13R). Comparing to the parental cell lines, these cells were >10,000-, >20,000- and 600-fold less susceptible to BVDV, respectively. Inoculation of resistant cells with BVDV in the presence of polyethylene-glycol increased the frequency of infection in the order of >437-, >346- and 87-fold, respectively, indicating that resistance is probably due to a block in viral entry. Nevertheless, each resistant cell line retained the susceptibility to three other viruses that replicate in the parental cells. Thus, these cells may be useful for many virology purposes, with a very low risk of BVDV contamination.

Animals↗
PubMed1992

Second-site homologous recombination in Epstein-Barr virus: insertion of type 1 EBNA 3 genes in place of type 2 has no effect on in vitro infection.

This study was undertaken to develop a general strategy for the introduction of mutations into specific sites in the Epstein-Barr virus (EBV) genome. Previous approaches were limited by the need for physical linkage of the transfected EBV DNA fragment to a positive selection marker. In our experiments, a positive selection marker was introduced into one site in the EBV genome and a distant, nonlinked, marker was introduced into another site. Each marker was on a large EBV DNA fragment and was inserted into the genome by transfection into cells carrying a resident EBV genome. The resident EBV genome was simultaneously induced to replicate by using a cotransfected expression plasmid for the EBV immediate-early transactivator, Z (J. Countryman, H. Jenson, R. Seibl, H. Wolf, and G. Miller, J. Virol. 61:3672-3679, 1987; G. Miller, M. Rabson, and L. Heston, J. Virol. 50:174-182, 1984). Eleven percent of the resultant EBV genomes which incorporated the positive selection marker also incorporated the nonlinked marker. Both markers uniformly targeted the homologous EBV genome site. In this way novel EBV recombinants were constructed in which the EBV type 1 EBNA 3A, EBV type 1 EBNA 3A and 3B, or EBV type 1 EBNA 3A, 3B, and 3C genes were introduced into a largely type 2 EBV genome, replacing the corresponding type 2 gene(s). No difference was observed in primary B-lymphocyte growth transformation, in latent EBV gene expression, or in spontaneous lytic EBV gene expression. These new recombinants should be useful for ongoing analyses of the type specificity of the immune response.

Amino Acid Sequence↗
PubMed2004

Protein arginine methylation during lytic adenovirus infection.

Arginine methylation of proteins affects major processes in the cell, including transcriptional regulation, mRNA metabolism, signal transduction and protein sorting. Arginine methylation of Ad (adenovirus) E1B 55-kDa-associated protein E1B-AP5 was recently described by us [Kzhyshkowska, Schutt, Liss, Kremmer, Stauber, Wolf and Dobner (2001) Biochem. J. 358, 305-314]. In this first example of protein arginine methylation analysis in Ad-infected cells, we investigated methylation of the E1B-AP5 and the viral L4-100 kDa protein. We demonstrate that E1B-AP5 methylation is enhanced during the course of infection in a cell-type-specific manner. We also show that L4-100 kDa is efficiently methylated in Ad-infected cells. L4-100 kDa formed complex with methyltransferase in vivo during productive infection, and can be methylated by HRMT1L2 (human protein arginine methyltransferase 1) in vitro. Comparative analysis of E1B-AP5 and L4-100 kDa protein methylation in Ad-infected HeLa, MCF-7 and H1299 cells revealed that the profile of protein arginine methylation correlates with the efficiency of Ad proteins production. Our results suggest that protein arginine methylation is an important host-cell function required for efficient Ad replication.

Adenoviridae↗
PubMed1984

Properties of Streptomyces phage SLE111.

Phage SLE111 infecting Streptomyces levoris 1331 was morphological different from most actinophages described and yielded very high titres (10(11) p.f.u/ml) after lytic growth. Typical morphological changes of infected hyphae were observed by phase contrast and electron microscopy of ultrathin sections. The double-stranded, linear DNA (44.2 +/- 1.3 kb) was characterized, according to electron microscopic analysis, by absence of cohesive ends, of terminal redundance, and circular permutation. A stem-loop structure containing inverted repeats of about 200 bp was identified by electron microscopy in two alternative positions in the SLE111 genome.

Bacteriophages↗
PubMed1998

Limited transmission of Kaposi's sarcoma-associated herpesvirus in cultured cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is a novel gammaherpesvirus strongly implicated in the pathogenesis of Kaposi's sarcoma. Although virions can be produced in high yield from latently infected B-cell lines treated with phorbol esters, little is known about the infectivity of such virus, and efficient serial propagation of KSHV has been problematic. Here we report on the infectivity of KSHV produced from phorbol-induced BCBL-1 cells, employing an assay based on the detection of a spliced late mRNA by a sensitive reverse transcriptase PCR (RT-PCR) method. The results of this study confirm previous observations that 293 cells are susceptible to viral infection; however, infection with BCBL-1-derived virus is inefficient and the pattern of viral gene expression in infected cells may not fully reproduce that of authentic lytic infection. In keeping with this finding, serial propagation of BCBL-1-derived virus could not be demonstrated on 293 cells. Eleven of 38 other cell lines tested also supported KSHV infection, as judged by this RT-PCR assay, including cells of B-cell, endothelial, epithelial, and fibroblastic origin; however, in all cases, infection proceeded at or below the levels observed in 293 cells.

Gene Expression Regulation, Viral↗
PubMedDate not supplied

Interferon induction by cell-cell interaction of HIV-infected monocytes/macrophages with lymphocytes.

An in vitro model of interferon (IFN) induction in HIV infection was established. IFN are induced by a cooperative mechanism, involving monocytes/macrophages (M/M) as well as lymphocytes. M/M non-lytically infected with HTLV-IIIB did not produce IFN, but were able to induce high titres of IFN activity when cocultured with peripheral blood lymphocytes, cell-cell contact being required. IFN alpha as well as IFN gamma were induced, as shown by neutralization with specific antisera. The antiviral activity of HIV-induced IFN was shown by infectivity reduction assays, and the immune-modulating capacity was demonstrated by activation of M/M leading to neopterin release.

Biopterins↗
PubMed1995

Molecular biology of lactococcal bacteriophage c2.

The 22163 bp genome of the lytic prolate-headed lactococcal phage c2 was fully sequenced. Mapping of restriction sites and RNA transcripts demonstrated the presence of early and late genes. Early and late promoters were identified. The early region contained 21 ORFs, with predicted protein products of 4.4-32.9 kDA, all reading right to left. Significant similarity was found between a putative protein encoded by an early region ORF and the erf (essential recombination function) gene product of Salmonella phage P22. The late genes for which a function has been identified, all of which read from left to right, included a possible holin gene, and genes encoding three major and six minor phage structural proteins. Analysis of the cohesive termini revealed complementary, non-symmetrical, 9-base single-stranded 3' extended DNAs. The exploitation of phage sequence data and analysis of phage genomes to find ways of inhibiting phage replication is discussed.

Bacteriolysis↗
PubMed1975

Spontaneous interferon production and Epstein-Barr virus antigen expression in human lymphoid cell lines.

Established human lymphoid cell lines, many of which spontaneously produce interferon, differ in the efficiency by which they allow expression of Epstein-Barr virus (EBV) lytic functions. Six EBV carrying lymphoid cell lines, selected to either be extremely susceptible or very refractory to EBV superinfection, were tested for spontaneous interferon production. Only the three cell lines which were poorly superinfectable with EBV were found to produce interferon. These same three lines could not be induced to express EBV-specific early antigens from intrinsic EBV genomes. It is suggested that interferon acts as a negative control factor affecting a cell's susceptibility to EBV.

Antigens, Viral↗
PubMed2005

Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21.

Cultured melanoma cell lines despite exhibiting similar in vitro morphology, display significant phenotypic and growth rate differences when propagated as in vivo xenografts. Previously we have shown that Coxsackievirus A21 (CVA21) lytically infects in vitro cultures of malignant melanoma cells and is efficient at reducing the tumor burden of mice bearing slow-growing SK-Mel-28 melanoma xenografts. The oncolytic activity of CVA21 against in vivo melanoma xenografts, which possess rapid growth rates and more extensive vascular structure than SK-Mel-28 xenografts warrants further investigation. In the present study we evaluated the oncolytic action of CVA21 against rapidly growing melanoma xenografts (ME4405) which exhibit a highly vascular phenotype. Flow cytometric analysis indicated that in vitro cultures of ME4405 cells expressed comparable levels of the CVA21 cellular receptors, ICAM-1 (intercellular adhesion molecules-1) and DAF (decay accelerating factor) to SK-Mel-28 cells. Despite similar levels of CVA21 receptor expression, SK-Mel-28 cells appear to be more susceptible to viral lysis than ME4405 cells, even though the kinetics of virus replication in both lines was comparable. Intratumoral, intraperitoneal or intravenous administration of CVA21 were equally effective in reducing the tumor volume of ME4405 xenografts in immunodeficient mice, and provides further evidence for the use of CVA21 as a novel oncolytic agent against varying phenotypes of malignant melanoma.

Animals↗