Parvovirus NS1 stimulates P4 expression by interaction with the terminal repeats and through DNA amplification (original) (raw)
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Journal of Virology
Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in trans, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G 1 is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G 1 were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.
trans-Activation of parvovirus P38 promoter by the 76K noncapsid protein
Journal of Virology, 1985
The autonomously replicating parvoviruses contain a 5-kilobase linear single-stranded DNA genome that produces two noncapsid proteins, Ni and N2, and two overlapping capsid proteins, VP1 and VP2. To characterize the regulation of viral transcription, we began with a study of the promoter for the coat proteins (P38) at map unit 38. Various constructions containing the P38 promoter were fused to the bacterial gene for chloramphenicol acetyltransferase (cat), and the relative efficiency of expression was determined in the presence and absence of parvovirus gene products. Our results show that the P38 promoter is a weak promoter This work was supported by grant LB 506 85-56 from the state of Nebraska; Public Health Service grants RO1, CA26801, and CA36727, from the National Institutes of Health; and grant DBM-8444778 from the National Science Foundation. I thank L. Laimins for plasmids pAlocat and pSV2cat, Carol Wilson for technical assistance, and R. Hines, A. Rizzino, and E. Bresnick for reviewing the manuscript.
Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter
Journal of Virology, 1987
The parvovirus early protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene. We have examined the trans-activation of P38 by NS1 by using fusions of P38 to the reporter gene, chloramphenicol acetyltransferase (cat). Maximal trans-activation requires a small 5' cis element (tar) between-137 and-116. The tar element has activity in both orientations when 5' to the P38 promoter, but no activity has been detected 3' to the promoter. The wild-type P38 has a biphasic response to NS1 depending on the dosage of the NS1-expressing plasmid. Promoters lacking the tar also have a biphasic response that is reduced about 10-fold, and they can be inhibited by larger doses of the NS1 plasmid. Heterologous promoters from other viruses and the Harvey-ras oncogene promoter are inhibited by NS1. Truncated and internally deleted versions of NS1 lose the trans-activation, but some of them retain the inhibitory properties. Thus transactivation can be uncoupled from inhibition. The tar element has shown no activity with the heterologous simian virus 40 early promoter. In contrast, the P38 promoter responds to a heterologous enhancer, but the enhanced promoter loses activity to trans-activation by NS1. In summary, the P38 tar element has some of the properties of an enhancer with a high preference for a 5' position and a stringent requirement for the P38 promoter. * Corresponding author. promoter specificity and has some activity in the absence of the proximal promoter elements. The inhibition of expression of P38 or heterologous promoters by NS1 and truncated or deleted mutants of NS1 will also be described.
Both excision and replication of cloned autonomous parvovirus DNA require the NS1 (rep) protein
Journal of Virology, 1989
When a bacterial plasmid containing the entire genome of LuIII virus except for the terminal 18 nucleotides from the right end is transfected into HeLa cells, the viral DNA is rescued and replicated, with production of infectious virus. This experimental system was used to examine the viral proteins and cis elements required for the excision and replication of viral DNA. The deletion of the entire NS1 gene provided a viral genome that was excised from the plasmid and replicated only when an NS1 gene was provided in trans. A frameshift mutation in the NS2 intron that truncates NS1 prevented excision and replication. Deletion of the left-end terminal inverted repeat or the right-end inverted repeat prevented excision of viral DNA from that end but not from the wild-type terminus. The viral terminus excised from the plasmid was protected from a processive degradation process, which began on the vector portion of the plasmid. The inhibitor of DNA polymerases alpha and delta, aphidicolin...
Journal of Virology, 1998
Parvovirus B19 infections are associated with diverse clinical manifestations, ranging from no symptoms to severe symptoms. The virus shows an extreme tropism for replication in erythroid progenitor cells, possibly due to the activity of the only functional promoter (p6) of the B19 virus genome in combination with both cell- and cell cycle-specific factors and the trans -activator protein NS1. As presented here, p6 promoter sequences derived from several B19 virus isolates proved to be highly conserved. Furthermore, mutations did not affect any of the potential binding sites for transcription factors. One variation of the base at position 223 was identified only in B19 virus isolates derived from patients with persistent infection or chronic arthritis. To determine promoter activity and to characterize regulatory elements, sequences spanning the total p6 promoter and subfragments of them were introduced into a eukaryotic expression vector upstream of the luciferase gene (from Photin...
Virology, 2002
The nonstructural proteins of parvovirus exert a variety of disparate functions during viral infection ranging from promoter regulation, involvement in DNA replication, and induction of apoptosis. Our interest was focused on the possible mechanism by which the NS1 protein mediates its effects on the p6 promoter of parvovirus B19. It is known that the p6 promoter is highly active in different cell lines and interaction with the viral NS1 protein results in a further increase of the activity. The protein may function by binding directly to the viral DNA or via an indirect binding through interaction with cellular transcription factors bound to the promoter. We examined the interaction of the NS1 protein with cellular transcription factors which are involved in regulating the promoter activity. After purified baculovirus-expressed NS1 protein in gel retardation assays was added, an altered complex formation was observed, indicating that NS1 protein interacts with Sp1/Sp3 transcription factors. Enzyme-linked immunosorbent assays verified these findings. The direct interaction of NS1 protein with p6 promoter elements was analyzed by a coprecipitation assay whereby labeled oligonucleotides spanning the entire promoter region were incubated with NS1 protein followed by an immunoprecipitation with NS1-specific antibodies. An eight-nucleotide-long, almost palindromic sequence (AGGGCGGA) was found as potential NS1-binding motif. Footprint analysis with oligonucleotides containing this DNA motif confirmed this result. Thus, transcriptional regulation by the NS1 protein may involve both the interaction with Sp1/Sp3 that binds to the promoter region and direct binding of NS1 to the promoter DNA.
Journal of Virology
The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.
Journal of Virology, 1978
A temperature-sensitive mutant of H-1, ts14, that is partially defective in replicative-forn (RF) DNA synthesis has been isolated. ts14 H-1 is characterized by a decrease in plaque-forming ability and production of infectious virus at the restrictive temperature of 39.50C. RF DNA synthesis of ts14 is reduced to 3 to 7% of that of wild-type H-1 at either the restrictive or the permissive temperature. A complementation analysis of RF synthesis of ts14 and a viable defective H-1 virus, DI-1, or wild-type H-3 indicates that the defective RF DNA synthesis of ts14 is cis-acting. ts14, unlike wild-type H-1, causes a multiplicity-dependent inhibition of DI-1 or H-3, but not LuII, RF DNA synthesis. Mixed infections of cells with two parvoviruses also exhibited a cross-interference for viral protein synthesis that was multiplicity dependent. ts14 inhibited infectious virus production of H-1 or H-3, but not LuIlI. LuIIIor H-3-pseudotype particles were produced by coinfection with H-1. H-3 and H-1 showed similar interactions with ts14, and H-3 DNA was more homologous to H-1 than was LuIII by comparative physical mapping studies. The results suggest that ts14 is a mutant with a defect in a regulatory sequence of its DNA that influences RF DNA replication.
The Journal of general virology, 2001
Nonstructural protein 1 (NS1) of minute virus of mice is involved in viral DNA replication, transcriptional regulation and cytotoxic action in the host cell. Viral DNA replication is dependent on the ability of NS1 to form homo-oligomers. To investigate whether oligomerization is required for NS1 transcriptional activities, a functionally impaired mutant derivative of NS1 that was able to interact with the wild-type (wt) protein and inhibit its activity in a dominant-negative manner was designed. This mutant provided evidence that transactivation of the parvoviral P38 promoter and transinhibition of a heterologous promoter by NS1 were both affected by the co-expression of the wt and the dominant-negative mutant form of NS1. These results indicate that additional functions of NS1, involved in promoter regulation, require oligomer formation.
Journal of Virology, 2000
The phosphoproteins (P proteins) of paramyxoviruses play a central role in transcription and replication of the viruses by forming the RNA polymerase complex L-P and encapsidation complex (N-P) with nucleocapsid protein (N) and binding to N protein-encapsidated genome RNA template (N-RNA template). We have analyzed the human parainfluenza virus type 3 (HPIV3) P protein and deletion mutants thereof in an in vitro transcription and in vivo replication system. The in vitro system utilizes purified N-RNA template and cell extract containing L and P proteins coexpressed via plasmids using a recombinant vaccinia virus expression system. The in vivo system takes advantage of minigenome replication, which measures luciferase reporter gene expression from HPIV3 minigenomes by viral proteins in a recombinant vaccinia virus expression system.