Proline residues in the HIV-1NH(2)-terminal capsid domain: Structure determinants for proper core assembly and subsequent steps of early replication

Recent analyses suggest that the p24 capsid (p24(CA)) domain of the HIV-1 g roup-specific antigen (Gag) may be divided into two structurally and functi onally distinct moieties: (i) an amino-terminal portion, previously shown t o bind the cellular chaperone cyclophilin A, and (ii) a carboxy-terminal do main, known to contribute to the interaction of the Gag and Gag-Pol precurs ors during the early assembly process. In order to gain deeper insight into the role of the amino-terminal domain of the p24(CA) protein during viral replication, eight highly conserved proline residues known to promote turns and to terminate alpha-helices within the p24 tertiary structure were repl aced by a leucine residue (P-position-L). Following transfection of the pro viral constructs in COS7 cells, the majority of the mutants resembled wild- type viruses with respect to the assembly and release of virions. However, although the released particles contained wild-type levels of genomic viral RNA, the mature products of the Gag and Gag-Pol polyproteins as well as th e Env glycoproteins-all of them, except mutant P225L-were either noninfecti ous or severely affected in their replicative capacity. Entry assays monito ring the process of viral DNA synthesis led to the classification of select ed provirus mutants into four different phenotypes: (i) mutant P225L was in fectious and allowed complete reverse transcription including formation of 2-LTR circles; (ii) mutants P149L, P170L, and P217L failed to form 2-LTR ci rcles; (iii) mutant P222L displayed a severe defect in binding and incorpor ating cyclophilin A into virions, was delayed with respect to DNA polymeriz ation, and failed to form a 2-LTR replication intermediate: and (iv) mutant P133L was unable even to synthesize a first-strand cDNA product. All repli cation-defective mutants were characterized by severe alterations in the st ability of virion cores, which were in two cases reflected by visible chang es in the core morphology. These results suggest that proline residues in t he NH2-terminal capsid domain represent critical structure determinants for proper formation of functional Virion cores and subsequent stages of early replication, (C) 2000 Academic Press.