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PubMed2005

Oncolysis of human ovarian cancers by echovirus type 1.

A small number of enteroviruses possess the capacity to induce rapid and marked lytic infections in cells of various human malignancies. During screening of representative human enteroviruses for their oncolytic capacity, we observed that echovirus type 1 (EV1) displayed a high level of tropism for human ovarian cancer cells. EV1 is an enterovirus which largely causes asymptomatic infections in humans and whose tissue tropism is primarily regulated via interactions with the I domain of the alpha subunit of cell surface-expressed integrin alpha2beta1. We evaluated the capacity of wild-type EV1 to act as an oncolytic agent of ovarian cancers propagated as cell monolayers, multicellular spheroids or xenografts in SCID mice. EV1 infection of in vitro propagated ovarian cell lines expressing high levels of integrin alpha2beta1 was assessed for specific viral attachment, antibody blockade, induction of cytopathic effect and production of progeny virions. EV1 lytically infected all 8 human ovarian cancer cell lines tested (2008, DOV13, JAM, OVCA-429, OVCAR-3, OVHS-1, OAW-42 and IGROV-1) but not the immortalized normal ovarian surface epithelial cell line (HOSE) or human PBMCs. EV1 challenge was equally effective in the oncolysis of human ovarian cancer cells whether in monolayer or spheroidal environments. The therapeutic efficacy of EV1 was demonstrated by rapid reduction of tumor burden by a single viral intratumoral injection in SCID mice bearing multiple preformed s.c. xenografts. Using an in vivo i.p. human ovarian cancer xenograft model, administration of EV1 was further shown to significantly inhibit the formation and burden of ascites tumors. These findings demonstrate an important proof of principle for employing wild-type EV1 as a potential oncolytic agent in the control of human ovarian cancers.

Animals↗
PubMed1981

Adherent spleen cells from mice acutely infected with cytomegalovirus suppress the primary antibody response in vitro.

In the present study, we have demonstrated that the hemolytic plaque response of spleen cells from mice acutely infected with murine cytomegalovirus (MCMV) is suppressed in vitro. Mishell-Dutton cultures established with spleen cells from C57BL/6 mice infected 4 days earlier with 4.2 X 10(3) plaque-forming units of MCMV were found to produce significantly fewer direct plaques upon stimulation with sheep red blood cells than corresponding control cultures. Suppression could not be attributed to a generalized lytic destruction of spleen cells, nor was it produced by addition of virus directly to uninfected spleen cell cultures. However, in cultures containing various mixtures of cells from control and infected mice, the direct plaque response was markedly below the predicted response, suggesting that the uninfected cells were actively suppressed by cells from infected mice. Suppression was dependent on the presence of intact spleen cells, since it was not found in the presence of lysed cells. The capacity of spleen cells from infected mice to mediate suppression was demonstrated in the plastic-adherent subpopulation. Treatment of these cells with anti-thymocyte serum (ATS) and complement did not abolish the capacity to suppress the immune response. Thus, adherent ATS-resistant cells from the spleens of mice acutely infected with MCMV, rather than virus alone, have the capacity to actively suppress the hemolytic plaque response in vitro.

Animals↗
PubMed1982

Characterization of a hamster brain cell line persistently infected with measles virus.

A persistent infection of measles virus in hamster brain cell cultures was established. Hamster brain cells were transformed with a human papovavirus strain BK and infected with a wild-type measles virus in order to get the cell line persistently infected with measles virus. About 75 per cent of these M-HB/MVB-cells were measles virus-infected. The cells released only small amount of infectious virus and produced low levels of interferon-like activity into the growth medium. During the first 50 passages no large syncytia typical of a lytic measles virus infection were seen. The products of measles virus infection in the cells and in cell culture fluids were followed at two temperatures. Another cell line persistently infected with measles virus (Lu-carrier-cells, 28) was investigated in parallel. In both cell lines measles antigens were cytoplasmic, but during the observation period large amounts of measles nucleocapsid accumulated in the nuclei of the M-HB/MVB-cells. The virus-specific protein synthesis in M-HB/MVB-cells was weak and the intracellular amount of immunoreactive measles nucleocapsid was only 50 per cent (600 ng/10(5) infected cells) of the (1200 ng/10(5) cells) found in Lu-carrier-cells. Also the release of nucleocapsid was minimal in hamster brain cells. The decreased temperature had no clear-cut effect on virus-specific protein synthesis or on the release of the virus-specific products.

Animals↗
PubMed1995

Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus-infected mice.

We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV-infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages.

Animals↗
PubMed1994

Construction of a recombinant cDNA of echovirus 6 that established a persistent in vitro infection.

cDNA clones of lytic acute and nonlytic persistent strains of echovirus 6 were used to construct a recombinant cDNA. The 3' region of the infectious wild-type cDNA genome, which extended from VPg to the end of the noncoding region, was exchanged with the cDNA fragment representing the same region of the persistent viral genome. Sequence analyses indicated that there was one mutation in the 3C protease and eight mutations in the 3D polymerase. Transfection of the recombinant cDNA into WISH cells resulted in cellular survival and synthesis of viral RNA. The viral RNA was retained in the transfected cell line after cultivation for 7 months. Supernates, collected from cell cultures at 1, 3, and 7 months after transfection with the recombinant cDNA, transmitted the viral RNA to uninfected cells. The results indicated that the recombinant cDNA established a persistent echovirus 6 infection that was transmissible by nonlytic virus particles.

Base Sequence↗
PubMed2003

Complete nucleotide sequence and molecular characterization of two lytic Staphylococcus aureus phages: 44AHJD and P68.

The first complete nucleotide sequences of two lytic Staphylococcus aureus double stranded DNA phages, 44AHJD (16784 bp) and P68 (18227 bp), are reported. Both are small isometric phages, with short, non-contractile tails and a pre-neck appendage. Based on their morphology, their genome size, the similarity of the encoded gene products, the type of infection and on the possession of a type B DNA polymerase, 44AHJD and P68 are allocated to the order Caudovirales, family Podoviridae, genus 'phi29-like phages'. The genome of 44AHJD differs from that of P68 by a deletion spanning nucleotides 10091 to 11531 of the P68 genome. The electrophoretic analysis of the terminal DNA fragments of P68 DNA and P68 DNA protein complex suggested the presence of a terminal protein at either DNA end. In contrast to the lysis cassette of the phi29-like phages, which is located at the end of the late operon, the lysis cassette of 44AHJD and P68 is located within the structural genes.

Bacteriophages↗
PubMed2001

Activators of the Epstein-Barr virus lytic program concomitantly induce apoptosis, but lytic gene expression protects from cell death.

Expression of the lytic cycle genes of Epstain-Barr virus (EBV) is induced in type I Burkitt's lymphoma-derived cells by treatment with phorbol esters (e.g., phorbol myristate acetate [PMA]), anti-immunoglobulin, or the cytokine transforming growth factor beta (TGF-beta). Concomitantly, all these agents induce apoptosis as judged by a sub-G1 fluorescence-activated cell sorter (FACS) profile, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. However, caspase activation is not required for induction of the lytic cycle since the latter is not blocked by the caspase inhibitor ZVAD. Furthermore, not all agents that induce apoptosis in these cultures (for example, cisplatin and ceramide) induce the EBV lytic programme. Although it is closely associated with the lytic cycle, apoptosis is neither necessary nor sufficient for its activation. Multiparameter FACS analysis of cultures treated with PMA, anti-Ig, or TGF-beta revealed BZLF1-expressing cells distributed in different phases of the cell cycle according to which inducer was used. However, BZLF1-positive cells did not appear to undergo apoptosis and accumulate with a sub-G1 DNA content, irrespective of the inducer used. This result, which suggests that lytic gene expression is protective, was confirmed and extended by immunofluorescence staining doubled with TUNEL analysis. BZLF1- and also gp350-expressing cells were almost always shown to be negative for TUNEL staining. Similar experiments using EBV-positive and -negative subclones of Akata BL cells carrying an episomal BZLF1 reporter plasmid confirmed that protection from apoptosis was associated with the presence of the EBV genome. Finally, treatment with phosphonoacetic acid or acyclovir prior to induction with PMA, anti-Ig, or TGF-beta blocked the protective effect in Mutu-I cells. These data suggest that a late gene product(s) may be particularly important for protection against caspase activity and cell death.

Apoptosis↗
PubMed1985

Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus.

Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789-4793, 1983). This observation was based on our inability to propagate an adenovirus type 5 mutant lacking functional VAI and VAII genes. Subsequently, it was found that this mutant was viable and able to grow to a low titer. Therefore, we examined the complementation of the VAI RNA function by the Epstein-Barr virus-encoded RNAs by constructing additional adenovirus type 5 substitution mutants containing multiple copies of the Epstein-Barr virus-encoded RNA genes in nonessential early transcriptional region III. The new substitution mutants synthesized viral polypeptides at late times at levels comparable to those observed in wild type-infected cells. Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells.

Adenoviruses, Human↗
PubMed2006

Herpes simplex virus regulatory proteins VP16 and ICP0 counteract an innate intranuclear barrier to viral gene expression.

HSV regulatory proteins VP16 and ICP0 play key roles in launching the lytic program of viral gene expression in most cell types. However, these activation functions are dispensable in U2OS osteosarcoma cells, suggesting that this cell line either expresses an endogenous activator of HSV gene expression or lacks inhibitory mechanisms that are inactivated by VP16 and ICP0 in other cells. To distinguish between these possibilities, we examined the phenotypes of somatic cell hybrids formed between U2OS cells and highly restrictive HEL fibroblasts. The U2OS-HEL heterokarya were as non-permissive as HEL cells, a phenotype that could be overcome by providing either VP16 or ICP0 in trans. Our data indicate that human fibroblasts contain one or more inhibitory factors that act within the nucleus to limit HSV gene expression and argue that VP16 and ICP0 stimulate viral gene expression at least in part by counteracting this innate antiviral defence mechanism.

Cell Line↗
PubMed1994

Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv. campestris.

We have isolated and characterized a lytic double-stranded DNA Xanthomonas campestris pv. campestris bacteriophage (XTP1) capable of mediating generalized transduction. The phage transduces chromosomal markers at frequencies of 10(-5) to 10(-6) transductants per PFU. We demonstrated its genetic utility by the isolation and cotransduction of linked transposon insertions to a nonselectable locus, xgl, required for the cleavage of 5-bromo-3-chloro-indoyl-beta-D-galactoside and showed that rif and str alleles in X. campestris are 75% linked. One-step growth experiments showed that the latent and rise periods were each 2 h and the average burst size was 35. The DNA genome is approximately 180 kb, presumably modified in a sequence-specific manner, and may be covalently attached to protein(s). Electron micrographs show the phage particle to have an icosahedral head and contractile tail with tail fibers uniquely attached to a location 40 nm proximal from the end of the tail.

Bacteriophages↗
PubMed2002

Differential receptor usage of small ruminant lentiviruses in ovine and caprine cells: host range but not cytopathic phenotype is determined by receptor usage.

The ovine maedi-visna (MVV) and caprine arthritis-encephalitis (CAEV) small ruminant lentiviruses (SRLV) exhibit differential species tropism and cytopathic effects in vitro. Icelandic MVV-K1514 is a lytic SRLV which can infect cells from many species in addition to ruminants, whereas a lytic North American MVV strain (85/34) as well as nonlytic MVV strain S93 and CAEV can infect only ruminant cells. In the present study, we determined if differential receptor usage in sheep and goat cells is the basis of differential species tropism or cytopathic phenotype of SRLV. Infection interference assays in sheep and goat synovial membrane cells using pseudotyped CAEV vectors showed that North American MVV strains 85/34 and S93 and CAEV use a common receptor (SRLV receptor A), whereas MVV-K1514 uses a different receptor (SRLV receptor B). In addition, human 293T cells expressing CAEV but not MVV-K1514 envelope glycoproteins fused with a goat cell line persistently infected with MVV-K1514, indicating that MVV-K1514 does not use SRLV receptor A for cell-to-cell fusion. Therefore, our results indicate that the differential species tropism of SRLV is determined by receptor usage. However, receptor usage is unrelated to cytopathic phenotype.

Animals↗
PubMed2002

Transcriptional regulation of the interleukin-6 gene of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus).

Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus is linked to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), all of which are viewed as cytokine-driven malignancies. In particular, interleukin-6 (IL-6) has been found to promote the growth and proliferation of cells from KS and PEL. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]), which functions similarly to the cellular IL-6. Therefore, vIL-6 has been proposed to play an important role in tumor progression. Several groups have reported that vIL-6 is expressed from the HHV-8 genome at higher levels in PEL and MCD lesions than in KS lesions. However, it is not clear how vIL-6 expression is regulated. We characterized the transcription at the vIL-6 gene locus by Northern blot analysis and, in contrast to previous reports, we observed two distinct transcripts from induced PEL cell lines. This observation was confirmed by primer extension, as well as 5' and 3' rapid amplification of cDNA ends. Two transcription initiation sites and putative TATA boxes were mapped. A luciferase reporter system was used to show that each of the two putative TATA boxes contributed to vIL-6 promoter activity. Since virally encoded transcriptional activator Rta potently activates the viral lytic gene expression cascade, we examined the role of Rta in controlling vIL-6 gene expression and found that Rta activated the vIL-6 promoter. The Rta-responsive element was further mapped through a series of deletion constructs. Electrophoretic mobility shift assays demonstrated that Rta binds directly to the vIL-6 Rta-responsive element, and the core Rta-responsive element was mapped to a 26-bp region spanning from nucleotide 18315 to 18290 on the viral genome. We propose that the existence of two vIL-6 promoters offers opportunities for differential regulation of vIL-6 gene expression in different tissue types and may account for the variable vIL-6 levels observed in KS, PEL, and MCD.

Amino Acid Sequence↗
PubMed1992

Expression of nef, vpu, CA and CD4 during the infection of lymphoid and monocytic cell lines with HIV-1.

The expression of the capsid antigen (CA) and the two regulatory proteins nef and vpu as well as the CD4 cell surface receptor was followed in HIV-infected lymphoid and promonocytic cells. In the lytic phase of infection all three viral proteins were expressed; production of these proteins coincided with the increase of CA antigen and infectious virus in culture supernatants and with prominent cytopathic effects. After selection of persistently infected cells, the number of lymphoid cells expressing detectable levels of nef decreased to zero; the number of cells positive for CA ranged between 40 to 70%. In chronically infected promonocytic cells nef and vpu expression was reduced to undetectable levels, whereas most of the cells accumulated CA intracellularly. Infectious cell free virus and CA in the supernatant of promonocytic cells had low titers. CD4 surface expression declined in all cell lines investigated before cell free virus was detectable.

Amino Acid Sequence↗
PubMed1994

Measles virus infects mouse fibroblast cell lines, but its multiplication is severely restricted in the absence of CD46.

Mouse cell lines (L, NIH3T3, and RMA cells) infected with the Edmonston strain of measles virus (MV) did not exhibit cytopathic effects (CPE), consistent with the finding that mice are not susceptible to MV. Northern blot analysis, however, revealed that MV genes were transcribed in infected L and NIH3T3 cells, although expression levels were much lower than those in lytically infected Vero cells. Expression of MV genes was not detected in infected RMA cells. L and NIH3T3 cells were found to synthesize viral proteins and produce infectious virions after infection. These cell lines did not express on the surface the molecule detectable by antibodies directed against human CD46, the recently identified MV receptor. L cell transfectants expressing human CD46 exhibited CPE after MV infection, and produced 50 times more viral transcripts and 20 times more infectious virions than the parental L cells. The lowest titer of MV that induced viral multiplication in L cells as detected by cocultivation with Vero cells was larger than that in CD46+ L cells by two orders of magnitude. Our results indicate that MV can infect some mouse cells in the absence of CD46, yet the presence of CD46 facilitates multiplication and cytopathogenicity of MV in mouse cells.

3T3 Cells↗
PubMed2006

Characterization of the lytic-lysogenic switch of the lactococcal bacteriophage Tuc2009.

Tuc2009 is a temperate bacteriophage of Lactococcus lactis subsp. cremoris UC509 which encodes a CI- and Cro-type lysogenic-lytic switch region. A helix-swap of the alpha3 helices of the closely related CI-type proteins from the lactococcal phages r1t and Tuc2009 revealed the crucial elements involved in DNA recognition while also pointing to conserved functional properties of phage CI proteins infecting different hosts. CI-type proteins have been shown to bind to specific sequences located in the intergenic switch region, but to date, no detailed binding studies have been performed on lactococcal Cro analogues. Experiments shown here demonstrate alternative binding sites for these two proteins of Tuc2009. CI2009 binds to three inverted repeats, two within the intergenic region and one within the cro2009 gene. This DNA-binding pattern appears to be conserved among repressors of lactococcal and streptococcal phages. The Cro2009 protein appears to bind to three direct repeats within the intergenic region causing distortion of the bound DNA.

DNA, Viral↗
PubMed1997

Integrin alpha v beta 6 enhances coxsackievirus B1 lytic infection of human colon cancer cells.

Viral entry into host cells depends upon specific interactions between virus attachment proteins and cell surface receptors that enable virus binding and internalization of virus and/or the virus-receptor complex. We have recently reported that the ubiquitous cell surface molecule, decay-accelerating factor (DAF), is a major cell attachment receptor for Coxsackieviruses B1, B3, and B5. However, DAF permits only virus binding and not virus internalization, invoking the presence of secondary or accessory receptors. Among the known receptors for enteroviruses are members of the cell adhesion molecule family known as integrins. In the present study, we found that expression of the epithelial-restricted integrin, alpha v beta 6, on colonic epithelial cells significantly enhanced Coxsackievirus B1-mediated cell lysis. Importantly, the viral-mediated cell killing required the presence of the 11-amino-acid C-terminal cytoplasmic extension unique to the beta 6 subunit, providing the first evidence of regulation of viral infectivity by integrin cytoplasmic domains. These results indicate that alpha v beta 6 expression on intestinal epithelial cells critically affects Coxsackievirus B1 infectivity. This may be essential in the conversion of asymptomatic enterovirus infection into clinically apparent disease.

Antigens, Neoplasm↗
PubMed1999

In vitro infection and pathogenesis of Chlamydia pneumoniae in endovascular cells.

The strength of the epidemiologic and clinical associations of Chlamydia pneumoniae with atherosclerosis can be increased by the demonstration that C pneumoniae can initiate and sustain growth in human vascular cells as well as in animal models. To investigate the biological basis for the dissemination and proliferation of this organism in vascular cells, the in vitro growth of C pneumoniae was studied in 2 macrophage cell lines, peripheral blood monocyte (PBMC)-derived macrophages, human bronchoalveolar lavage (BAL) macrophages, several endothelial cell lines, and aortic artery smooth muscle cells. Five of 5 strains of C pneumoniae were capable of 3 passages in human U-937 macrophages and in murine RAW 246.7 macrophages. Titers were suppressed in both macrophage types with each passage as compared with growth in HEp-2 cells. Both human BAL macrophages and PBMC-derived macrophages were able to inhibit C pneumonia eafter 96 hours' growth. Eleven C pneumoniae strains were capable of replicating in normal human aortic artery-derived endothelial cells, umbilical vein-derived endothelial cells, and pulmonary artery endothelial cells. Infection in human aortic artery smooth muscle cells was also established for 13 strains of C pneumoniae. C pneumoniae was also capable of growing in endothelial cells derived from human cadaver coronary artery endothelial cells (CAEC). U-937 human macrophages that were infected with C pneumoniae were capable of transmitting the infection to CAEC when they were brought into contact with the endothelial cells by centrifugation, rocking overnight, and direct layering overnight, with and without using artificial laboratory tissue culture enhancements, such as centrifugation of the inoculum and cycloheximide in the growth media. The in vitro ability of C pneumoniae to maintain infections in macrophages, endothelial cells, and aortic smooth muscle cells may provide support for the hypothesis that C pneumoniae can infect such cells, which when followed by an immune response may contribute to atheroma formation in vivo. Stimulation of cytokine responses by infection with C pneumoniae has indicated that this organism is capable of interacting with the immune system. In vitro infection by C pneumoniae of U-937 macrophages stimulated the production of IL-1beta, IFN-gamma, and TNF-alpha in tissue culture. Human CAEC that are infected with C pneumoniae produce more IL-8 compared with those inoculated with killed C pneumoniae or negative control cells, indicating a chemokine response to infection that may play a role in recruitment of inflammatory cells to sites of infection in vascular cells. When IFN-gamma was used to up regulate HEp-2 and U-937 cells before infection by C pneumoniae, inhibition of a lytic growth cycle occurred in a dose related response. However, removal of the IFN-gamma after 24 to 48 hours' exposure allowed subsequent productive growth in the cells, perhaps indicating the prior induction of a persistent infection. More studies are needed to study the complex relationship between lytic infection and persistence, the ability of C pneumoniae to affect the immune response of vascular cells, and the potential for C pneumoniae to influence the initiation of or progression of atheromatous lesions.

Cell Division↗
PubMed1996

Identification of a phage-coded DNA-binding protein that regulates transcription from late promoters in bacteriophage P4.

The genetic element P4 can propagate as a temperate phage or as a multicopy plasmid in its host Escherichia coli. Late in the lytic cycle and in the plasmid condition, transcription of the P4 essential genes depends on the activation of the late promoters P(LL) and P(sid), which control the transcription of the left and right operons, respectively. Both P4 late promoters are positively regulated by the product of the P4 delta gene, which is transcribed from P(sid). We have identified a new P4 gene, vis, that appears to play a relevant role in P4 late transcription control. vis is the first gene downstream of P(LL) and codes for a basic 88 amino acid protein with a potential helix-turn-helix motif. Expression of the cloned vis gene suppresses all the phenotypic traits exhibited by P4 vir1, a mutant that carries a promoter-up mutation in the late promoter P(LL). By Northern hybridization analysis we showed that vis negatively regulates transcription from P(LL) and enhances transcription from P(sid). Thus, vis auto-regulates its expression by repressing its own promoter and enhancing transcription of delta, which is required for P(LL) activation. The vis gene was fused with the glutathione S-transferase gene and the GST-Vis fusion protein was partially purified. By gel retardation assays and DNA footprinting we demonstrated that GST-Vis binds to a 32 bp long region immediately downstream of P(LL). We also showed, by gel retardation, that GST-Vis binds to the P sid region. A sequence present in both P(LL) and P(sid) regions may represent the Vis binding consensus sequence. The dual role of Vis on the control of P4 late transcription may be required for a regulated expression of the replication functions when P4 propagates in the plasmid state.

Amino Acid Sequence↗