The proviral genome is transcribed by the cellular RNA polymerase
n.
By definition, transcription starts at the 5’ end of the upstream R region, reading through the entire viral genome into the cellular DNA before being processed. Transcriptional initiation is also possible from the 3* LTR (reading in the same direction as that from the 5’ LTR), but the absolute level of transcription is much lower (Klaver & Berkhout, 1994). This is presumably a result of the difficulty in forming a transcription initiation complex on a stretch of DNA that is being actively transcribed from the 5’ LTR. The 3* LTR has an efficient poly-A signal, again by definition at the junction of the 3’ R region and the 3* U5 region. The poly-A signal in the 5’ LTR does not function efficiently (Bohnlein et al. y 1989), as in vitro 3’ poly-A processing has been shown to require an upstream element and an upstream TAR sequence (Gilmartin et al., 1992).The viral promoter and enhancer elements lie in the upstream U3 region of the 5’ LTR (fîgure 5), and can be divided into several regions. The first is known as the promoter core region. It is located in the 3* region of the U3 sequence and is crucial for the initiation of transcription. This region contains a TATA box element at position -28 to -24 (relative to the first base transcribed) (Rosen et al. y 1985). This motif is present in many eukaryotic gene promoters and is known to bind the cellular transcription factor TFIID. A transcription initiation complex can form around this element, which will ultimately include the cellular RNA polymerase n. The presence of a number of other cellular factors, bound to elements on the DNA adjacent to the TATA box, facilitate the formation of the initiation complex. The transcription factor Spl is one such factor. Three Spl binding sites have been located in the core region of the HIV LTR at position -78 to -47 (Rosen et al. y 1985).
Upstream of the core region is the promoter modulatory region. This contains several DNA sequence motifs that can bind cellular transcription factors, facilitating the formation of the transcription initiation complex. Factors can also bind that block the formation of this complex, decreasing the rate of transcription initiation. An important feature of the factors that bind to elements of the modulatory region is their responsiveness to cellular stimuli such as cellular activation. Therefore, proviral transcription can be linked to and respond to a specific cellular environment for the maximal production of virus.
The modulatory region of the HIV-1 LTR contains the following sites; AP I limited homology sequences at -347 to -329 (Franza et al., 1988); NF-AT sites at -292 to -255 (Siekevitz et al. y 1987); USF site at -173 to -159 (Giacca et al. y 1992); TCF-la site at -139 to -124 (Waterman et al. y 1991); and 2 NFxB sites at -104 to -81 (Nabel et al. y 1987).
Figure
5 The HIV-1 LTR promoter.This figure illustrates the array of transcription factor binding sites located in the core, modulatory, and TAR regions of the HIV-1 LTR, (Adapted from Rosen et û/., 1985).
H IV -1 5 -LTR P rom oter
V ira l T ra n scrip tio n
AP-1 N F - A T USF TCF-la N F - K B S P l T A T A INT UBPl/LBPl UBP-2 CTF/NFl P B S S D l Y
□-□□■II □_____□ □□
M o d u la to ry C ore 4-1 T A R U 3 R U 5 gag leader (not to sc a ld Core region T A T A ( - 2 8 1 0 - 2 4 ) M odulatory region N F -K B (-104 t o-81)T A R region gag leader
U B P l / L B P l (4-1 to 4-59) P B S - prim er binding site
S P l (-78 t o -47) T C F - la (-139 t o -124) U B P -2 (4-1 to 4-59) S D1 - major 5’splice donor
INT (-5 t o -15) U S F (-159 to -173) C T F /N F l (4-1 to 4-59) Y — packaging signal
N F - A T (-292 t o -255)
The AP-1 like sequences allow the binding of the Fos transcription factor, which has been shown to increase after cellular activation. These sequences may also bind a transcription factor that has been shown to be a member of the steroid/thyroid hormone rec^tor subfamily (Orchard et al., 1990). The transcription factor NFxB is also important in the transcription of genes that respond to cell activation stimuli. In resting cells, NFxB is completed with the inhibitor IxB (Baeuerle & Baltimore, 1988). In response to external stimuli such as phorbol esters or the cytokine TNFck, protein kinase A or protein kinase C can phosphorylate IxB. Phosphorylated IxB dissociates from NFicB, allowing NFicB to enter the nucleus and activate transcription of a range of host genes such as IL-2, as well as the HIV-1 LTR.
The modulatory region of the HIV-1 LTR between bases -420 and -154 is also known as the negative response element (NRE) (Siekevitz et al., 1987, Rosen et al., 1985). Sequences contained in this region are thought to exert a negative influence on HIV-mediated transcription, as deletion of the NRE resulted in a 3-fold increase in both HIV transcription and replication (Lu et al., 1989). However, the significance of this NRE element and its role in the viral cycle are still unclear.
The third region of the LTR is known as the TAR region. It is located from position +1 to +59 in the R region of the LTR, and is the first sequence to be transcribed. This element has been shown to confer transcriptional transactivating potential to the core promoter (Sodroski et al., 1985b). As the TAR region is transcribed it forms an RNA stem loop secondary structure. This structure has been shown to interact with the virally encoded Tat protein in association with cellular factors (Selby et al., 1989). In the absence of the Tat gene, the vast majority of transcripts terminate soon after reaching the TAR region (Kao et al., 1987, Feinberg et al.,
1991). However, in the presence of a Tat/TAR interaction, a dramatic overall increase in the rate of transcription of full length genomic transcripts is observed (Laspia et al., 1989).