Human immunodeficiency virus-1 tat- and tat/nef-defective genomes containing HIV-regulated diphtheria toxin A chain gene inhibit HIV replication

To assess the ability of human immunodeficiency virus-1 (HIV-1) tat- and tat/nef-defective genomes containing diphtheria toxin A chain gene (DTA) to inhibit replication of HIV in human cells. Plasmids were constructed to contain the HIV-1 genome disabled by tat and tat/nef deletions, and sequences coding for the A subunit of diphtheria toxin gene were inserted into one of these deletions. An infectious clone of HIV-1 (pBRU-3) was cotransfected into HeLa-CD4 cells, together with plasmids carrying the modified DTA-containing genomes. Cell culture supernatants were collected and titrated for the virus by multinuclear activation of beta-galactosidase (MAGI) assay. Each of the DTA-containing plasmids suppressed HIV production by no less than 96%, whereas the defective non-DTA containing plasmids did not interfere with the virus growth. Plasmids containing wild-type DTA inhibited HIV replication slightly more than its moderately attenuated mutant form, probably by limiting the synthesis of viral proteins. These modified DTA-containing HIV constructs gave no evidence of virus growth in HIV susceptible cells that supported the multiplication of the parent plasmid. None of the modified DTA-containing plasmids was toxic to cells cotransfected with a selectable marker, as shown by the ability of cotransfectants to multiply and form colonies at rates identical to controls exposed to non-specific DNA. This suggested that DTA was probably not expressed in the absence of activating wild-type HIV plasmid. HIV-regulated DTA in the background of a HIV replication and expression of defective provirus may be taken into consideration as a therapy approach to the treatment of HIV infection, based on its selective and specific toxicity only to HIV infected CD4-positive cells.