ABSTRACT
In previous studies, we demonstrated that the substitution of amino acid triplets for alanines in the carboxy-terminal portion (amino acids 341-352: ATL EEM MTA CQC) of the capsid protein domain (p24) of human immunodeficiency virus type 1 (HIV-1) partly led to an inhibitory effect on the capacity to form virus-like particles (VLPs). In these experiments, the uncleaved Pr55gag precursor protein was expressed by recombinant vaccinia viruses. We have now investigated the effects of these mutations with respect to a replication-competent HI-provirus system. Substitution of amino acids 344-346 (EEM) for alanines, which was previously shown to lead to an inhibition of VLP formation, completely blocked assembly and release of HIV. A substantial reduction of HIV synthesis was also observed in the proviral system after exchange of amino acids 347-348 [MT(A)] which, in contrast, was formerly shown to result in an increased formation of VLPs. Western blot analysis of lysates of cells transfected with these mutated proviral constructs revealed an abnormal intracellular processing pattern of the Pr55gag precursor molecules. Further analyses suggest a structural aberration of these altered polyproteins as the basis for the observed block of virus formation.
Subject(s)
Capsid/chemistry , HIV Core Protein p24/physiology , HIV-1/growth & development , Virus Replication , Amino Acid Substitution , Animals , COS Cells , Gene Products, gag/metabolism , Gene Products, gag/ultrastructure , HIV Core Protein p24/chemistry , HIV-1/ultrastructure , Morphogenesis , Protein Conformation , Protein Precursors/metabolism , Protein Precursors/ultrastructure , Protein Processing, Post-Translational , Structure-Activity RelationshipABSTRACT
A structurally highly ordered arrangement of the polyprotein precursor, Pr55gag is a necessary prerequisite for assembly, budding and maturation of the human immunodeficiency virus type 1 (HIV-1). In particular, distinct regions of the matrix protein (p17) and the capsid protein (p24) contained within Pr55gag are functionally active during these processes. In order to determine such regions we exchanged amino acid triplets within p17 (amino acids 46-61) and p24 (amino acids 341-352) for alanine residues and deleted the whole regions. Synthetic peptides derived from these regions had been shown previously to inhibit the production of infectious virus. The effect of the mutations on the release of viral particles was investigated by using recombinant baculoviruses for the expression of mutated Pr55gag as virus-like particles and by use of the respective HI proviruses for monitoring the production of infectious particles.
