Recombinant vaccinia viruses expressing the HIV-1 protease, p55 GAG, p41 GAG, complete GAG-POL and truncated GAG-POL precursor proteins were constructed. Protein expression was confirmed by immunological procedures. Using the above recombinants, we demonstrated that self-assembly of p55 GAG protein is sufficient to form the framework of the nascent human immunodeficiency virus (HIV-1) particle. The particles which budded from the cells infected with a vaccinia-GAG construct were mostly spheres with a concentric ring of electron dense material. Expression of the GAG and POL proteins resulted in mature particles containing a condensed core which assumed the nucleoid structure characteristic of lentiviruses. When the POL frame in the GAG-POL ORF was truncated at the end of the protease domain, p55 GAG processing was markedly reduced and the maturation of the resultant particles was defective. We have extended these findings to show that HIV-1 protease expressed from the vaccinia vector can process exogenously substrates such as denatured GAG protein expressed in E. coli or synthetic peptide substrates. However, this protease failed to process particle associated GAG protein whether in the form of purified HIV particles or GAG particles expressed from the vaccinia recombinant. We have expressed protein cleavage mutants of GAG using the vaccinia vector. Mutations that eliminate the myristoylation or the nucleic acid binding CYS containing motif (NBCys) of the p9 subunit of GAG protein have been introduced into GAG-POL expression vector to examine whether they interfere with capsid formation and RNA packaging. We have developed a simple in vivo model for HIV-1 RNA packaging. A subgenomic HIV RNA encompassing R, U5 and U3 elements surrounding the minimal packaging sequence was expressed from the T7 promoter using vaccinia T7 polymerase vector. Co-infection with GAG or GAG-POL recombinant vaccinia viruses lead to HIV particles that selectively packaged the mini HIV RNA. This system is being used to define the molecular parameters of RNA packaging. It is our eventual goal to develop molecular strategies to interfere with RNA packaging and capsidation. Finally, to understand the biochemical processes of HIV provirus synthesis and its subsequent integration, we have over-expressed and partially purified the HIV-1 integrase and the RNAse H associated with the 15 kDa C-terminal cleavage product of the p64 RT.
