Specific transcriptional initiation in vitro on murine type C retrovirus promoters.

Abstract

We have investigated the ability of molecularly cloned murine type C retroviral DNA to direct accurate initiation of RNA synthesis when added to cell-free extracts. Two different cloned proviruses were used. The first was derived from an integrated molecule of AKR murine leukemia virus and contains adjacent host information. The origin of the second was an unintegrated permuted copy of Harvey murine sarcoma virus. We found that the leukemia virus cloned provirus, as predicted by structural considerations, contained two functional RNA polymerase II promoters located in the U3 region present at either end of the molecule. These promoters initiate transcription at equal rates in vitro. We also found that the permuted sarcoma virus clone contained an RNA polymerase II promoter in the U3 region. Removal of viral sequences 49 bases upstream of the in vitro sarcoma virus initiation site by restriction cleavage results in loss of specific transcription, indicating a role for this information in in vitro promotion. The 5' ends ofin vitro and in vivo viral RNA were compared by nuclease mapping techniques and found to be identical. Based on this evidence, we conclude that murine retroviral genomes contain sufficient information to initiate transcription independent of any host information in vitro and that these viral promoters are probably also active in vivo. In addition to the promoter in U3, Harvey murine sarcoma virus contains a second promoter in vitro that initiates nean the 5' boundary of the transformation-specific (src) region ofthe virus. Initiation by this promoter was insensitive to low levels of a-amanitin, and the RNA transcript could be terminated to yield a 340-nucleotide product. The retroviruses are a group of single-strand RNA viruses that integrate a double-strand DNA copy of their genome into host cell DNA as an obligatory intermediate oftheir replication cycle (1). Although it has been established that cellular RNA polymerase II (pol II) is responsible for transcription of the integrated viral genome (2, 3), it remains uncertain whether information necessary for the initiation of viral transcription is encoded by the viral genome or by host DNA adjacent to the site of integration. The discovery that proviruses contain a directly repeated sequence located at either end of the molecule (4-8) raised the possibility that proviral DNA contains the control elements necessary to function as an independent transcriptional unit. Sequence analysis of these DNA repeats (the long terminal repeat or LTR) for several retroviruses (9-12) confirmed that they contain sequences associated with both initiation and termination of eukaryotic pol II-dependent transcription.Recently, cell-free systems that have the ability to accurately initiate transcription on cloned promoters in vitro have been described (13,14). Subsequently, transcription initiation on various genes (15-17), including a-cloned cDNA fragment of an avian retrovirus (18), have been reported. We have studied the initiation of transcription in vit...

Nucleotide Sequence of the p21 Transforming Protein of Harvey Murine Sarcoma Virus

“…This would place the 5' end of a putative RNA polymerase III transcript around position 45 to 47 (Fig. 2), a location consistent with published data (15). We still cannot assess the physiological significance of this sequence in determining transcripts within the Ha-MuSV genome in vivo.…”

Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design

“…Region 3 consists of about 65 bp between region 2 and the CCAAT box, and region 4 consists of 100 bp at the extreme 3' end of U3 that contains the viral promoter ( Fig. 1) (28,56). We determined the base that appears most frequently at each position in the alignment ( Fig.…”

Permissive Sense and Antisense Transcription from the 5′ and 3′ Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1

“…Recent studies have shown that LTRs contain bidirectional promoters that are able to initiate transcription in both the sense and antisense orientations (2)(3)(4). With respect to expression of viral genes, a promoter within the 5= LTR of the provirus regulates sense transcription and, therefore, production of the viral gene products necessary for viral particle synthesis (gag, pro, pol, and env) as well as regulatory and accessory genes (5). For all human T-cell leukemia virus (HTLV) family members (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), as well as for the human immunodeficiency virus type 1 (HIV-1) (16-18), a separate promoter or promoters within the 3= LTR regulate antisense transcription.…”