Human Immunodeficiency Virus Type 1 N-Terminal Capsid Mutants That Exhibit Aberrant Core Morphology and Are Blocked in Initiation of Reverse Transcription in Infected Cells

Abstract

A group of conserved hydrophobic residues faces the interior of the coiled-coil-like structure within the N-terminal domain of the human immunodeficiency virus type 1 (HIV-1) capsid protein (CA). It has been suggested that these residues are important for maintaining stable structure and functional activity. To investigate this possibility, we constructed two HIV-1 clones, in which Trp23 or Phe40 was changed to Ala. We also constructed a third mutant, D51A, which has a mutation that destroys a salt bridge between Pro1 and Asp51. All three mutants are replication defective but produce virus particles. Mutant virions contain all of the viral proteins, although the amount and stability of CA are decreased and levels of virion-associated integrase are reduced. The mutations do not affect endogenous reverse transcriptase activity; however, the mutants are blocked in their ability to initiate reverse transcription in infected cells and no minus-strand strong-stop DNA is detected. The defect in reverse transcription is associated with striking defects in the morphology of mutant virus cores, as determined by transmission electron microscopy. Our data indicate that the mutations made in this study disrupt CA structure and prevent proper maturation of virus cores. We propose that this results in a defect in core stability or in an early postentry event preceding reverse transcription.

Hydrogen Bonding at a Conserved Threonine in Lentivirus Capsid Is Required for Virus Replication

“…The results for each substitution mutant were consistent in both assays. In keeping with the phenotypes of mutants of the HIV salt bridge participant residues, HIV D(51)A CA and HIV P(1)L CA , D(50)A CA SIV was noninfectious and P(1)A CA SIV exhibited greatly reduced infectivity relative to wild-type virus (13,51,55) (Fig. 4).…”

“…Interestingly, in all three virological analyses of the threonine substitution mutants, the T(47)A CA mutant bore a strong resemblance to previously characterized mutants with substitutions of the two residues that participate in the salt bridge in HIV CA, P(1)L CA and D(51)A CA . Like T(47)A CA SIV, P(1)L CA and D(51)A CA HIV do not replicate well, are noninfectious, and have largely acentric mature cores (13,51,55). The similarities between these mutants, in conjunction with the fact that T(47) CA is highly conserved (suggesting its functional importance), prompted us to analyze this CA threonine residue in additional experiments.…”

“…A single role has been proposed for the N-terminal ␤-hairpin in mature CA: interactions between the ␤-hairpins of CA monomers may facilitate dimerization and core assembly (15,20,23,55). Amino acid substitutions in HIV CA that disrupt the salt bridge, thereby destabilizing the ␤-hairpin, cause a variety of viral defects, including abnormal mature core morphology and loss of infectivity (13,51,55). Because the conformational change in the CA N terminus is essential for proper CA function in the virus life cycle, the interactions that facilitate the formation of the ␤-hairpin and the salt bridge in mature CA are important potential targets for antiviral drug design (50; reviewed in reference 29).…”

“…rangement, the N-terminal proline of CA [HIV P(1) CA ] folds back into the core of the protein to form a buried salt bridge (a hydrogen bond between two oppositely charged groups [35]) with the side chain of an aspartate in helix 3 [HIV D (51) CA ], forming a ␤-hairpin structure in the first 13 amino acids of CA (15,20,37). As formation of the salt bridge and the ␤-hairpin requires the free amino group of P(1) CA that is generated upon Pr55…”

Kinetic Analysis of the Role of Intersubunit Interactions in Human Immunodeficiency Virus Type 1 Capsid Protein Assembly In Vitro

“…However, cleavage alone is not sufficient for function. Mutations that allow maturation and yet result in the formation of cores with aberrant morphology inhibit infectivity apparently by blocking the initiation of reverse transcription (9,13,32,33,38,40,41).…”