All HIV-dependent expression vectors in common use are based on the HIV long terminal repeat promoter (LTR). An early HIV gene product, Tat, increases the level of transcript that is initiated at the LTR. The placement of reporter genes downstream of the LTR results in a responsiveness to the synthesis of Tat, a measure of HIV replication. The earliest indicator lines made use of reporter enzymes, such as luciferase and β-galactosidase [1-4], permitting a direct measurement of reporter gene induction. Tat appears to increase HIV transcriptional activity by two mechanisms. The first identified Tat activity is not directed towards the proviral DNA promoter, but rather through direct association with the growing nascent RNA chain. Tat associates with a 5' RNA loop structure [5-7], the transactivation response element (TAR), to promote completion of the initiated transcript [8-10], an activity also defined as processivity or anti-termination. More recent work has provided evidence that Tat also stimulates assembly of transcription factors to the DNA promoter ; that is, Tat promotes initiation, as well as elongation . However, the LTR as a promoter is inherently leaky. Following integration of the HIV DNA into the host chromatin, the LTR transcribes the early gene products Tat, Rev, and Nef. That is, there is a required basal level of transcription that is Tat-independent. In addition, the site of integration or insertion of the LTR-based expression vector can mediate high levels of transcriptional activity in some cells , leading to expression from the reporter LTR in the absence of HIV. For example, in the generation of HIV indicator cells with LTR-based reporters, it has been necessary to remove 25% or more of the stably transfected cells [4,14] since they generate reporter transcript in the absence of HIV. While high expression cells can be removed in the generation of reporter clones, this inherent leakiness prevents the use of viral vectors to deliver the LTR-based reporter construct to detect the presence of existing HIV-positive cells in a mixed population.
By eliminating this non-specific activity, that is, non-HIV induction of signal, from an HIV expression vector, a wider use of this convenient and efficient tool would be possible. In addition to Tat, HIV transcriptional activity is also affected by an early gene product, Rev. Rev binds to a 3' loop structure, the Rev response element (RRE), present in unspliced and singly-spliced HIV transcripts, to permit nuclear export and translation of these mRNAs [15-17]. This viral specific activity exploits an essential cellular process. The removal of non-coding regions of transcripts (introns) prior to translation is critical to all cells. Introns are operationally defined by the presence of strong splicing sites, and HIV exploits this cellular activity by including multiple splice sites with varying activity [18,19] to generate multiple coding regions within the same stretch of HIV RNA. The existence of these sites results in the generation of fully spliced transcripts in the early phase of HIV infection [20,21]. Once Rev is expressed, RRE-containing HIV transcripts can be delivered to the cytosolic translational machinery.
It is of interest that in HIV infection protein expression from singly or non-spliced HIV transcripts appears absolutely dependent on Rev expression [15,22,23]. This dependency is lost with seemingly minor modifications to HIV DNAs [24,25], and the earliest reported RRE-containing expression constructs [26,27] can display varied Rev-independent expression. These were designed to elucidate Rev function, and the focus was not to eliminate background signal. In this report, by inclusion of multiple components of the HIV genome, we have constructed a lentiviral expression vector that displays a full dependency on the presence of HIV. As it is silent in HIV-negative cells, it differs dramatically from LTR-based systems.