Human immunodeficiency virus (HIV) is the primary etiological agent of acquired immunodeficiency syndrome (AIDS). Although there is depletion of most of the T4 lymphocytes which are preferentially infected by HIV, some cells survive and can remain latently infected for protracted periods. Infection of HIV is dependent upon the transactivator tat protein which increases expression of viral genes transcribed from the long terminal repeat (LTR) by interacting with cis-responsive sequences (TAR) present in the LTR. Reactivation from latency may be mediated by tat itself or by the """"""""immediate early"""""""" proteins produced by one of several heterologous DNA viruses which frequently accompanies HIV infection. Different DNA elements of the HIV LTR may be involved in transactivation by these immediate early proteins and tat. Interception of HIV transactivation by a dominant inhibitory protein represents a novel approach aimed at blocking HIV infection at the level of gene regulation. The ability of a mutated protein to inhibit the wild type (wt) protein from transactivating its a target promoter is referred to as squelching. Squelch mutant proteins of three diverse transactivating genes, including E1A of adenovirus (Ad), are now known which strongly supports the prediction that tat squelch mutants can be generated. Site-directed mutagenesis will be used to mutate the tat coding region. Conservative mutations will be made from the Cys-rich region through the highly basic region (codons 22 - 57). To test for transactivation, the mutant tat gene under the control of a foreign promoter, will be cotransfected with an HIV LTR-CAT reporter plasmid in HeLa and Jurkat cells. Mutant tat plasmids which fail to transactivate will be cotransfected with the wt tat plasmid and the HIV LTR-CAT plasmid. A squelch tat mutant will be identified by its ability to inhibit CAT activity in a concentration dependent manner. Random mutations will also be made using degenerate oligonucleotides and a biological screening method will be employed which directly assays for the squelch phenotype in HeLa cells and allows an individual plasmid containing the mutant tat gene to be rescued and sequenced. The E1A protein of Ad5 is known to transactivate the HIV LTR. ElA mutants from Ad3, Ad5 and Adl2 which squelch transactivation of Ad5 early promoters, will be tested for squelching transactivation of the HIV LTR by wt E1A and tat, respectively. ElA from Ad35, virus found in 10% of AIDS patients, will be cloned and first tested for transactivation of the HIV LTR. Ad35 ElA will then be tested for squelching the HIV LTR by generating a mutation in a region that is conserved in other E1As and which produces the squelch phenotype. Mutants of tat or ElA that squelch transactivation of the HIV LTR will be stably expressed in human cell lines and tested for their ability to inhibit infection by HIV. In the long term, understanding the details of both transactivation and squelching of the HIV LTR may lead to methods of gene therapy and help in the design of therapeutic agents.
| Agulnick, A D; Thompson, J R; Ricciardi, R P (1994) An ATF/CREB site is the major regulatory element in the human herpesvirus 6 DNA polymerase promoter. J Virol 68:2970-7 |
| Thompson, J R; Agulnick, A D; Ricciardi, R P (1994) A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6. J Virol 68:4478-85 |
| Agulnick, A D; Thompson, J R; Iyengar, S et al. (1993) Identification of a DNA-binding protein of human herpesvirus 6, a putative DNA polymerase stimulatory factor. J Gen Virol 74 ( Pt 6):1003-9 |
