Human foamy virus genome possesses an internal, Bel-1-dependent and functional promoter.

Abstract

The human foamy or spumaretrovirus (HSRV) is a complex retrovirus that encodes the three retroviral genes gag, pol, and env and, in addition, at least three bel genes. The HSRV Bel-1 protein was identified as a transcriptional trans-activator. HSRV transcription starts in the 5' long terminal repeat at a defined guanine residue. We report here that a second efficiently utilized start site of transcription is contained within a HSRV env DNA sequence upstream of the bel genes. Bel-specific transcripts that initiate at the internal transcriptional start site at nt 9196 were identified in HSRVinfected cells by primer extension and Si nuclease analysis, and the intragenic promoter was shown to be constitutively activated by Bel-1 in the HSRV provirus. In transient expression assays with indicator gene constructs, expression by the HSRV intragenic promoter/enhancer is Bel-1 dependent. The data provide evidence for an intragenic start site of transcription in the genome of a complex, exogenous human retrovirus and are discussed in terms of a model for regulating spumaretroviral gene expression.The human foamy or spumaretrovirus (HSRV) is an exogenous retrovirus that was isolated from the lymphoblastoid cells of a nasopharyngeal carcinoma patient (1). The HSRV genome encodes accessory bel genes located 3' of env (2, 3). The pronounced complexity of the HSRV genome was revealed in the peculiar HSRV splicing pattern: about a dozen different HSRV transcripts were identified that included singly and multiply spliced mRNA species ofthe different bel genes (4). The bel-J gene product was identified as a transcriptional trans-activator for the long terminal repeat (LTR)-directed transcription (5-7) and shown to be absolutely required for viral replication and gene expression (8). The results so far indicated that HSRV gene expression is complex and comparable in structural and functional complexity to other human retroviruses (9). It is still unknown how expression of the various accessory transcripts and gene products in comparison with HSRV gag, pol, and env mRNAs and proteins is regulated. Despite repeated attempts to identify complex retroviral rev and rex analoga in HSRV, corresponding genes and/or RNA response elements have not been reported for any foamy virus (10).In this study, defined indicator gene constructs and RNA from HSRV-infected cells were used to identify the internal start site of HSRV transcription located near the 3' end of the env gene. The internal HSRV promoter depends on Bel-1 for efficient gene expression. These results point to, as far as we know, a unique mode of retroviral gene expression. MATERIALS AND METHODSPlasmids and Construction of Recombinant Clones. Infectious HSRV DNA clone pHSRV13 (8) served as source ofviralThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.DNAs; nucleotide numbering starts at the first base of the 5' LTR....

Foamy virus vector integration sites in normal human cells

“…FVs are found in several mammalian species (7) but are not endemic in human populations (8), and the prototype ''human FV'' isolate (9) is now thought to be a chimpanzee virus [SFVcpz(hu)] (10,11). FVs have several distinguishing properties, including a transcriptional transactivator expressed from an internal promoter (12)(13)(14), a DNA-based genome that forms by reverse transcription in virion-producing cells (15,16), and an essential cis-acting region overlapping the pol and env genes (17)(18)(19).…”

Bel1-mediated Transactivation of the Spumaretroviral Internal Promoter Is Repressed by Nuclear Factor I

“…This report is the first that describes the identification of a cellular transcription factor that negatively regulates Bel1-mediated transactivation of the core IP.BRE in vitro and in cell cultures. This promoter is transactivated by Bel1, the key regulator of viral expression (7,(11)(12)(13)(14). In transiently transfected 293T cells, different NFI proteins specifically repressed the level of Bel1-driven transactivation as assessed by luc expression assays.…”

“…Sense primer was: IPs (Ϫ192 to Ϫ173), 5Ј-CGTCATGCTTTGG-ACTGGAC-3Ј; the antisense primer was: IPas (Ϫ1 to Ϫ22), 5Ј-GATA-GATCTCAGCTTTTGCTCTTTCAATCTG-3Ј. PCR reactions using sense primer IPs and antisense primer IPas were done with Pfu polymerase (Stratagene) and pHSRV13 (12). PCR reactions were carried out with the buffer recommended by supplier supplemented for 1 min at 94°C, 1 min at 56°C, 2 min at 72°C for 35 cycles.…”

“…A, the internal promoter is shown from position Ϫ192 to the Ϫ1 cap site determined previously (12). The core IP.BRE from position Ϫ166 to Ϫ116 is in BOLD FACE and underlined; the minimal IP.BRE (from Ϫ166 to Ϫ140) of 27 bp is marked by broken underlining, and the IP.NFI from Ϫ142 to Ϫ116 by solid underlining; six predicted NFI half-sites are marked by overlining and numbered; NFI half-sites s1, s2, and s7 (identified here) are boxed and were analyzed in more detail (see "Results").…”

“…A key regulator of viral gene expression, the Bel1 transactivator of the human foamy virus (HFV), recently renamed primate foamy virus) binds directly to DNA target sites with no or low sequence conservation that are located in both the 5Ј-long terminal repeat (LTR) and the internal promoter (IP) (8 -11). The IP is a strong early promoter that is crucial for viral replication and critically dependent on Bel1 (11)(12)(13). Expression of Bel1 is initiated at the IP until threshold levels of the Bel1 transactivator are reached which subsequently turn on the 5Ј-LTR promoter to direct synthesis of virus structural proteins (13)(14)(15).…”