Proline Residues in the HIV-1 NH2-Terminal Capsid Domain: Structure Determinants for Proper Core Assembly and Subsequent Steps of Early Replication

Abstract

Recent analyses suggest that the p24 capsid (p24(CA)) domain of the HIV-1 group-specific antigen (Gag) may be divided into two structurally and functionally distinct moieties: (i) an amino-terminal portion, previously shown to bind the cellular chaperone cyclophilin A, and (ii) a carboxy-terminal domain, known to contribute to the interaction of the Gag and Gag-Pol precursors 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 replaced by a leucine residue (P-position-L). Following transfection of the proviral 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 the Env glycoproteins-all of them, except mutant P225L-were either noninfectious or severely affected in their replicative capacity. Entry assays monitoring the process of viral DNA synthesis led to the classification of selected provirus mutants into four different phenotypes: (i) mutant P225L was infectious and allowed complete reverse transcription including formation of 2-LTR circles; (ii) mutants P149L, P170L, and P217L failed to form 2-LTR circles; (iii) mutant P222L displayed a severe defect in binding and incorporating cyclophilin A into virions, was delayed with respect to DNA polymerization, and failed to form a 2-LTR replication intermediate; and (iv) mutant P133L was unable even to synthesize a first-strand cDNA product. All replication-defective mutants were characterized by severe alterations in the stability of virion cores, which were in two cases reflected by visible changes in the core morphology. These results suggest that proline residues in the NH(2)-terminal capsid domain represent critical structure determinants for proper formation of functional virion cores and subsequent stages of early replication.

Formation of a Human Immunodeficiency Virus Type 1 Core of Optimal Stability Is Crucial for Viral Replication

“…However, the relevance of these findings to HIV remains unclear; there are significant differences in the architectures of HIV and RSV cores, suggesting that these capsids may mediate different functions during infection or may interact differently with host cell factors following delivery into the cytosol. In a recent study by Fitzon et al (14), mutagenesis of proline residues in HIV-1 CA led to defects in core stability, which correlated with reduced infectivity, similar to the effects observed in our study.…”

“…Alternatively, the mutants may be defective for nuclear transport or integration of the provirus. Consistent with the latter hypothesis, other groups have reported CA mutations that are competent for second-strand transfer yet show defects in two-LTR circle formation, a marker of HIV-1 nuclear import (8,14). Further analysis of the unusual phenotype of the P38A and Q63A/Q67A mutants may reveal additional roles for CA during HIV-1 infection.…”

Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A

“…The detailed mechanism of how cyclophilin A promotes HIV-1 replication is not understood, although several mechanisms have been discussed in recent years (5,6,22,23,28,30,(43)(44)(45). In light of the CA N ͞CypA cocrystal structure showing CA G89-P90 bound exclusively in the trans conformation (22), models depicting CypA as having a chaperone role in HIV-1 virulence have been suggested (28).…”

Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

“…It has been suggested that the N-terminal β-hairpin of CA destabilizes the immature lattice and facilitates the formation of mature capsid (39,40). To explore the formation of the N-terminal β-hairpin of CA in real-time and to probe other conformational changes that take place in ΔGag upon ordered proteolytic cleavage by HIV-1 protease, 1 H-15 N TROSY spectra of ΔGag were recorded at a series of time points in the presence of a minute amount of HIV-1 protease (Fig.…”