Adenovirus Ela has two different transcriptional regulatory activities. It activates a number of viral and cellular genes and also represses the activity of certain enhancers. We have observed that the Ela proteins coded by a trans-activation defective dominant negative mutant (hr5) of Ad5 suppress basal HIV-1 LTR activity and also inhibit trans-activation of the LTR mediated by a variety of agents including the tat gene of HIV. Hr5 also has a strong inhibitory effect on HIV replication in transient assays. The negative regulatory effect of hr5 appears to be at least partly mediated by the repression of the HIV enhancer. In addition, an effect on other LTR promoter elements is also possible. We propose to investigate the mechanism of enhancer repression in detail. The HIV enhancer region consists of two core enhancers. We will identify which of the two core enhancers is the target for hr5-mediated repression. To elucidate the mechanism of repression, the ability of nuclear factors from cells infected with hr5 to bind to the individual enhancers will be determined by electrophoretic mobility shift assays. Possible association of E1a proteins with known enhancer binding proteins will be investigated by a DNA affinity precipitation assay and by immunoprecipitation analysis using E1a antibodies. The hr5-mediated repression can be relieved by the tax gene of HTLV-II and phorbol esters. The enhancer sequence wherein relief of hr5- mediated repression occurs and nuclear factors whose enhancer binding activity may be affected as a consequence thereof will be identified. We will determine whether herpes simplex virus-1 infection or the hepatitis B virus X gene could also relieve the inhibitory effect of hr5 on LTR expression. In addition to enhancer repression we will also investigate the effect on interaction of various transcription and regulatory factors with promoter elements, including the trans-activation region (TAR) and the negative regulatory element (NRE). To investigate the effect of hr5 on HIV replication in detail, the effect of hr5 viral infection on HIV-1 replication in primary human peripheral blood lymphocytes will be studied. To study the effect of hr5 on the expression LTR of other immunodeficiency viruses and to extend the in vitro results to an animal model int he future, the effect of hr5 on simian immunodeficiency virus (SIV) LTR expression and SIV replication in primary human blood lymphocytes will be examined. We will design a derivative of hr5 specifically suited for inhibiting HIV replication. Such a variant will be constructed by substituting the V1 domain of CD4 for part of adenovirus fiber. The hr5:CD4 recombinant virus is expected to have restricted tropism for only those cells expressing HIV Env protein (gp120) and should also be valuable in specific targeting of Ela and other inhibitory agents to HIV-infected cells.
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