Requirement for Integrase during Reverse Transcription of Human Immunodeficiency Virus Type 1 and the Effect of Cysteine Mutations of Integrase on Its Interactions with Reverse Transcriptase

Abstract

Retroviral integrase catalyzes the essential step of integrating a double-stranded DNA copy of the viral genome into a host cell chromosome. Mutational studies have revealed that integrase is involved in additional steps of viral replication, but the mechanism for the pleiotropic effect is not well characterized. Since Cys residues generally play crucial roles in protein structure and function, we introduced Cys-to-Ser substitutions at positions 56, 65, and 130 of human immunodeficiency virus type 1 (HIV-1) integrase to determine their effects on integration activity and viral replication. None of the substitutions significantly affected the enzymatic activities in vitro. When introduced into the NL4-3 molecular clone of HIV-1, mutant viruses encoding Cys mutations at positions 56 and 65 of integrase replicated similarly to the wild-type virus in CD4؉ -T-cell lines, whereas the C130S-containing virus was noninfectious. The entry and postintegration steps of the viral life cycle for all mutant viruses were normal, and all had particle-associated reverse transcriptase (RT) activity. However, early reverse-transcribed DNA products were absent in the lysate of cells infected with the C130S mutant virus, indicating that the mutation abolished the ability of the virus to initiate endogenous reverse transcription. Coimmunoprecipitation using purified integrase and RT showed that the C-terminal domain of wild-type HIV-1 integrase interacted with RT. The interaction between integrase and RT was not affected in the presence of a reducing or alkylating agent, suggesting that the interaction did not involve a disulfide linkage. The C130S substitution within the core region may disrupt the protein recognition interface of the C-terminal domain and abolish its ability to interact with RT. Our results indicate that integrase plays an important role during the reverse-transcription step of the viral life cycle, possibly through physical interactions with RT.

Single amino acid substitution in HIV‐1 integrase catalytic core causes a dramatic shift in inhibitor selectivity

“…The non-infectivity of viruses harboring the C130S IN protein was attributed to a viral failure to initiate the reverse transcription process [7]. This provided evidence that the IN-RT interaction is functionally critical for viral replication, and the C130S substitution indirectly abolished the contacts between the IN C-terminal domain and RT [7]. Another substitution at this position, C130A, exhibited wild-type replication kinetics [19].…”

“…Coimmunoprecipitation studies revealed that WT IN interacted with reverse transcriptase (RT) via its C-terminal domain, but the interaction was abolished in C130S mutant IN proteins. The non-infectivity of viruses harboring the C130S IN protein was attributed to a viral failure to initiate the reverse transcription process [7]. This provided evidence that the IN-RT interaction is functionally critical for viral replication, and the C130S substitution indirectly abolished the contacts between the IN C-terminal domain and RT [7].…”

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site

“…The C130S and nonconservative W132 substitutions create structural perturbations that disrupt multimer formation and therefore the binding interactions between these inhibitors and the peptide region 128 AAC-WWAGIK 136 in the context of a multimeric IN structure. Structural perturbations caused by C130S have been documented and proposed to disrupt other processes, but not inhibitor binding (22)(23)(24). Substitutions made at amino acid residue W131 (W131D and W131E) have been shown to increase the solubility of IN fragments, enhancing their ability to crystallize, without effecting protein structure (25,26).…”

Eukaryotic elongation factor 1 complex subunits are critical HIV-1 reverse transcription cofactors

“…After entry into the host cell cytoplasm, the HIV-1 core, containing the viral genomic RNA, RT, and integrase (IN), reorganizes to form the reverse transcription complex (RTC). The RTC requires both IN and RT to be active (5), and they are believed to recruit cellular factors to facilitate DNA synthesis (6). Although two-hybrid library and genomewide RNAi screening methods have implicated more than 50 cellular proteins as important for reverse transcription (6,7), whether any of these cellular proteins are RTC components, or whether they direct the RTC in other ways, is unclear.…”