Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter

Abstract

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.

Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins.

“…Two plasmids were constructed in which the P4 promoter of MVMp is replaced by the MMTV-LTR promoter: pULB3238 has a 594 bp deletion in the capsid genes whereas pULB3232 contains intact VP genes (see Materials and methods). In order to determine the basal and induced levels of expression of the NS genes under control of the MMTV-LTR promoter in both plasmids, we have taken advantage of the ability of NS-1 proteins to transactivate the late parvoviral P38 promoter (Rhode and Richard, 1987;Doerig et al, 1988). Thus, NB-E cells were cotransfected with pMMTV-LTR-NS (pULB3232, pULB3238) and pP38cat (pULB3562) plasmids.…”

“…NS proteins are known to regulate the synthesis of parvoviral DNA replicative intermediates and capsid proteins positively (Cotmore and Tattersall, 1987;Rhode and Richard, 1987). However, the particular MVMp molecular clone used for the present constructions does not replicate, due to a small deletion in its non-coding 5' terminal part (Boissy and Astell, 1985;F.Dupont, personal communication).…”

“…The mechanism by which NS proteins disturb target cells is presently unknown. It is well established that non-structural proteins participate in parvoviral DNA replication (Tullis et al, 1988) and exert both positive and negative controls over homologous and heterologous transcription promoters (Rhode and Richard, 1987). Thus, NS proteins may conceivably interfere with the on-going replication and/or expression of cellular DNA, resulting in cytostatic and cytocidal effects.…”

“…Construction of new plasmids pULB3545 (pP38). The HaeIlI (1853)-PstI (2129) fragment of MVMp DNA (Astell et al, 1986), containing the P38 promoter and its transactivation region (tar) (Rhode and Richard, 1987), was inserted between the BamHI and PstI sites of the polylinker of pUC 18 (Yammisch-Perron et al, 1985). The BamHI sticky end was filled in with the Klenow fragment of DNA polymerase I. pULB3562 (pP38cat).The EcoRI-XAoI fragment, comprising the P38 promoter of MVMp, was recovered from pULB3545 and inserted between the SailI and XhoI sites of pBGU7 (Cornelis et al, 1988b).…”

“…However, recent results indicate that the so-called non-structural proteins of parvoviruses, designated NS or Rep and involved in the regulation of viral DNA replication and expression (Cotmore and Tattersall, 1987;Berns and Bohenzky, 1987), may disturb host cells. Thus, NS/Rep proteins appear to interfere with a series of non-parvoviral processes, including gene expression programmed by heterologous promoters (Labow et al, 1987;Rhode and Richard, 1987), initiator-induced DNA amplification (Heilbronn et al, 1989), in vitro neoplastic transformation (Hermonat, 1989) and stable cell transformation with exogenous DNA (Labow et al, 1987;Ozawa et al, 1988;Brandenburger et al, 1990). These data suggest that the NS gene products encoded at least by the three studied viruses H1, MVM and B19, may have a cytotoxic activity.…”