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Replication of bovine papilloma virus (BPV) DNA requres two virus-encoded proteins, El and E2, while afl other proteins are supplied by the host cell. Here, we describe the isolation of the El protein and show that it is a mult nal protein. antigen, which plays a pivotal role in SV40 DNA replication. However, unlike the SV40 large tumor antigen, the helicase activity of El was stimulated 5-fold by the presence of a fork structure at the junction between single-stranded and doublestranded DNA and was supported efficiently by all eight nudeoside triphosphates. The El-catalyzed ATPase activity required the presence of single-stranded or double-stranded DNAs.In cells transformed with bovine papilloma virus (BPV), the viral DNA exists as a free replicating plasmid at a constant copy number (1). The replication of BPV DNA in vivo was shown to depend completely upon two BPV-encoded proteins, the 48-kDa E2 protein and the 68-kDa El protein (2). In addition, the minimal origin sequence required to support BPV DNA replication has been identified (nt 7911-22 of BPV DNA) (3,4). This sequence contains a binding site for the El protein and part of a sequence that acts as a binding site for the E2 protein.The in vitro replication of BPV origin-containing DNA (ori+ DNA), using extracts of a mouse mammary tumor cell line (FM3A), was established in Botchan's laboratory (4). They demonstrated that the in vitro synthesis of DNA absolutely required the BPV minimal origin and the viral El protein, whereas all other required proteins were supplied by the uninfected mouse cell extracts. At low concentrations of El protein, the replication reaction was stimulated markedly by the BPV E2 protein (4).We have isolated and characterized the two viral-encoded proteins required for this replication pathway, El and E2. In this report, we describe the biochemical properties of the purified BPV El protein. We show that this protein supports the replication of BPV oril DNA in vitro, as reported (4). We also demonstrate that this protein possesses a number of different activities required for the replication of the BPV viral genome. These include a DNA helicase activity that moves in the 3' -* 5' direction, a BPV ori+ DNA-binding activity that is stimulated by ATP and MgCl2, and the ability to unwind superhelical DNA leading to the production of highly unwound superhelical DNA (form U DNA). Thus the role of El in the BPV system is analogous to the role of large tumor antigen (T antigen) in the replication of simian virus 40 (SV40) DNA (5-7). MATERIALS AND METHODSCell Cultures and Recombinant Baculoviruses. Growth conditions of the mouse FM3A cell line and the preparation of extracts were as described (4). Recombinant baculoviruses containing wild-type BPV El and E2 genes have been described (8). The El protein used here contained the hemagglutinin epitope of influenza virus (9) followed by a thrombin cleavage site. An oligonucleotide encoding the amino acid residues MYPYDVPDYASLGGPLPRGS was ligated in the Nia III site (nt 848) at the first ...

The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, El and E2, while all other proteins are derived from the host. We described previously the isolation of the El protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The El protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maxal effi'ciency. The El protein also catalyzed a BPV origindependent DNA unwinding reaction. In this report, we show that at low levels of El protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by El, but it had no effect on the nonspecific El-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the El protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing El binding to the BPV or4ign via DNA-protein and protein-protein interactions.Bovine papilloma virus (BPV) provides an attractive model system to study the regulation of eukaryotic DNA replication. The viral DNA is maintained in BPV transformed cells as a nuclear plasmid with a constant copy number (1). Recent studies indicate that in vivo BPV DNA replication requires two BPV viral-encoded proteins-the 68-kDa El protein and the 48-kDa E2 protein (2). In addition, the minimal origin sequence that supports BPV DNA replication has been identified (nt 7911-22 of the BPV type 1 genome) (3,4). This sequence contains a binding site for the El protein (El BS) and part of a sequence that acts as a binding site for the E2 protein (E2 BS).The replication of BPV oril DNA in vitro, using extracts of a mouse mammary tumor cell line (FM3A), was recently established in Botchan's laboratory (4). They demonstrated that DNA synthesis required the BPV minimal origin and the El protein, whereas all other required proteins were supplied by uninfected mouse cell extracts. At low concentrations of El protein, the replication reaction was stimulated markedly by the E2 protein (4).We have described the isolation of the El protein and showed that it supported BPV ori+ DNA replication in vitro (5). We also demonstrated that this protein possessed a number of different activities required for BPV DNA replication. These include (i) a DNA helicase activity, for which the protein translocates in the 3' to 5' direction, (ii) a BPV oril DNA binding activity that is stimulated by ATP and MgCl2, and (iii) the capacity to unwind covalently closed circular ori+ DNA leading to highly unwound DNA products (5). Thus, the role of El in the BPV system is analogous to that of large tumor antigen...

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Bovine papilloma virus (BPV) contains a cis-acting DNA element which can enhance transcription of distal promotors. Utilizing both direct and indirect transient transfection assays, we showed that a 59-base-pair DNA sequence from the BPV genome could activate the simian virus 40 promoter from distances exceeding 2.5 kilobases and in an orientation-independent manner. In contrast to the promoter 5'-proximal localization of other known viral activators, this element was located immediately 3' to the early polyadenylation signal in the BPV genome. Deletion of these sequences from the BPV genome inactivated the transforming ability of BPV recombinant plasmids. Orientation-independent reinsertion of this 59-base-pair sequence, or alternatively of activator DNA sequences from simian virus 40 or polyoma virus, restored the transforming activity of the BPV recombinant plasmids. Furthermore, the stable transformation frequency of the herpes simplex virus type 1 thymidine kinase gene was enhanced when linked to restriction fragments of BPV DNA which included the defined activator element. This enhancement was orientation independent with respect to the thymidine kinase promoter. The enhancement also appeared to be unrelated to the establishment of the recombinant plasmids as episomes, since in transformed cells these sequences are found linked to high-molecular-weight DNA Bovine papilloma virus type 1 (BPV) provides a model system in mammalian cells for testing the assumptions implicit in the arguments described above. BPV viral DNA, as well as a 69% subgenomic fragment (69T), can transform rodent cells in culture (26); the viral genome in these cells is maintained as a multicopy nuclear episome (2a, 24). We reasoned that linking a selectable genetic marker to the BPV genome might substantially increase the transformation efficiency of that marker, by bypassing the need for integration of the foreign DNA. In this way we could test whether the establishment of the transforming DNA is a limiting step in transformation.We have reported previously (27) that linking BPV DNA to the herpes simplex virus (HSV) thymidine kinase (TK) gene can enhance the transformation frequency of TK-cells to TK+ more than 100-fold. This effect is independent of the position and orientation of the BPV sequences relative to the direction of HSV TK transcription. However, the enhanced transfor-1108 on May 7, 2018 by guest

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Initiation of bovine papillomavirus (BPV) DNA synthesis in vivo and in vitro depends on the interaction of the viral initiator protein El with the replication origin (ori+ DNA). The viral E2 protein assists this interaction, resulting in a cooperative assembly of both proteins on the replication origin. Using gel mobility-shift experiments, we demonstrate that in the presence of both El and E2 proteins two classes of ori+ DNA complexes were formed: complex 1 (cl) and complex 2 (c2). Formation of ci depended on both the El and E2 proteins and both proteins were contained within ci. The generation of c2 was dependent on the El protein and could be enhanced by E2, but the E2 protein was not detected within c2. At high E2/El ratios, ci was the dominant complex formed. Under these conditions, El-dependent BPV DNA synthesis in vitro was inhibited. At low E2/El ratios, the stimulation of c2 was correlated with the stimulation of BPV DNA replication by E2 in vitro. These data suggest that E2 assists El in the formation of an intermediate ci complex, which is replication inactive. The ci complex is converted in turn to the replicationactive c2 complex, which contains El but lacks E2. We propose that the ratios of ci and c2 formed in response to the levels of El and E2 proteins determine the potential for BPV DNA synthesis in vitro and in vivo and may contribute to copy number regulation of BPV plasmids within the cell.Studies of prokaryotic replication systems and small DNA viruses in eukaryotic cells have shown that initiation of DNA synthesis requires the interaction of a specific initiator protein with its cognate replication origin (see ref. 1 for review). In addition, initiator proteins such as the simian virus 40 large tumor antigen facilitate the recruitment of cellular proteins and DNA polymerases to generate an active replication complex at the origin (2-4).Papillomaviruses, such as bovine papillomaviruses (BPV), are maintained in latently infected cells as nuclear plasmids (see ref. 5 for review) and thus provide an additional eukaryotic system to investigate the assembly of a replication complex and to study the control of copy number.BPV DNA replication in vivo requires two viral proteinsthe 68-kDa El and the 48-kDa E2 polypeptides (6). The minimal in vivo origin see also Fig. 5) contains an A+T-rich region (ATR), an El binding site (BS) that includes a region of dyad symmetry (DSR), and the E2 BS12 (nt 16-27). El-dependent BPV DNA synthesis in vitro does not require the E2 protein at high levels of El (9-11). At low levels of El, E2 enhances in vitro DNA synthesis (9) even in the absence of a well-defined E2 BS (12).The El polypeptide specifically binds to the BPV replication origin (ori+) (7, 9, 13) and this process can be stimulated and stabilized in the presence ofATP (14, 15). In addition, the El-associated DNA-dependent NTPase and NTP-dependent DNA helicase activities lead to the specific unwinding of BPV origin-containing plasmid DNAs (10,14).In support of a direct role for the 48-kDa E2 tra...

Pexastimogene devacirepvec (Pexa-Vec) is a vaccinia virus-based oncolytic immunotherapy designed to preferentially replicate in and destroy tumor cells while stimulating anti-tumor immunity by expressing GM-CSF. An earlier randomized Phase IIa trial in predominantly sorafenib-naïve hepatocellular carcinoma (HCC) demonstrated an overall survival (OS) benefit. This randomized, open-label Phase IIb trial investigated whether Pexa-Vec plus Best Supportive Care (BSC) improved OS over BSC alone in HCC patients who failed sorafenib therapy (TRAVERSE). 129 patients were randomly assigned 2:1 to Pexa-Vec plus BSC vs. BSC alone. Pexa-Vec was given as a single intravenous (IV) infusion followed by up to 5 IT injections. The primary endpoint was OS. Secondary endpoints included overall response rate (RR), time to progression (TTP) and safety. A high drop-out rate in the control arm (63%) confounded assessment of response-based endpoints. Median OS (ITT) for Pexa-Vec plus BSC vs. BSC alone was 4.2 and 4.4 months, respectively (HR, 1.19, 95% CI: 0.78-1.80; p = .428). There was no difference between the two treatment arms in RR or TTP. Pexa-Vec was generally well-tolerated. The most frequent Grade 3 included pyrexia (8%) and hypotension (8%). Induction of immune responses to vaccinia antigens and HCC associated antigens were observed. Despite a tolerable safety profile and induction of T cell responses, Pexa-Vec did not improve OS as second-line therapy after sorafenib failure. The true potential of oncolytic viruses may lie in the treatment of patients with earlier disease stages which should be addressed in future studies. ClinicalTrials.gov: NCT01387555 ARTICLE HISTORY