The effect of capsid mutations on HIV-1 uncoating

Abstract

Efficient uncoating requires not only an optimal cellular environment, but also some intrinsic properties of the viral capsid protein itself. Using an in vitro uncoating model, we demonstrated that substitution of each serine residue with alanine at the three major phosphorylation sites of HIV-1 capsid protein, i.e. Ser-109, Ser-149 and Ser-178, could significantly reduce uncoating activity of purified core particles. We also showed that the core stability of mutant viruses was lower than that of the wild-type virus so that the lack of efficient uncoating of each mutant could not be due to an increase in capsid physical stability. However, serine-to-aspartic acid mutation to mimic the negative charge of phosphor-serine could not restore either uncoating activity or infectivity, and treatment of purified core particles with a phosphatase did not alter the uncoating activity. Our data indicated that mutations at phosphoacceptor sites of capsid disturbed the uncoating mechanism, but the defect may not be directly caused by the lack of phosphate on the core particles undergoing uncoating.

Role of Human Immunodeficiency Virus Type 1 Integrase in Uncoating of the Viral Core

“…2A), which is consistent with published results (64,69). For isolating viral cores, previous studies have employed protocols using different concentrations of sucrose and nonionic detergents to separate the cores based on density (1,5,8,19,20,22,30,55,59,60). In this study, virions were exposed to 1% Triton X-100 to remove the viral envelope, and intact cores (densities, 1.20 to 1.26 g/ml) were separated from intact virions (densities, 1.16 to 1.18 g/ml) and from non-particle-associated proteins using a continuous sucrose gradient (20)(21)(22).…”

“…Compared to the wild type (WT), a hyperstable CA mutant has an increased core yield and slower core disassembly, whereas an unstable CA mutant has a decreased core yield and more rapid core disassembly. These results indicate that core stability is a key determinant in the uncoating process and that changes in stability result in a block to infection (19,22,31,46,55,59). The CA protein and core stability also play critical roles in the ability of HIV-1 to infect nondividing cells (66) and have also been suggested to be important for nuclear import of the PIC (15).…”

“…Currently, a model of biphasic CA disassembly has been suggested to account for the initial CA release after entry and the presence of CA persisting near the nuclear periphery (15). Defects in core stability can lead to a block at other steps, including reverse transcription of the viral RNA by reverse transcriptase (RT) (19,22,31,46,54,55,59). Uncoating of the viral core and synthesis of the viral cDNA generate the preintegration complex (PIC), a subviral structure whose composition has been only partially characterized (52).…”

“…Since CA surrounds the viral ribonucleoprotein complex, various CA mutants have been studied to better understand the uncoating process, and certain mutations in CA result in decreased infectivity and loss of reverse transcription products (19,22,31,46,54,55,59). Examining events upstream of reverse transcription indicates that uncoating of the cores of these CA mutants, as measured by core stability and yield, is defective (22,59). HIV-1 core stability defects, as determined using an in vitro core disassembly assay, fall into two categories, unstable and hyperstable, with both leading to a block in infectivity (22).…”

Analysis of the Viral Elements Required in the Nuclear Import of HIV-1 DNA

“…Recent evidence indicates that CA may be involved in nuclear import (14,25,90). CA plays a pleiotropic role during the early steps of viral infection, and mutations in CA can affect reverse transcription, uncoating, and postnuclear entry events (74,87,88,92). However, it is true that one of the major differences between complex and simple retroviruses is the relative paucity of CA in viral nucleoprotein complexes derived from the former.…”