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Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase.

Dar MJ, Monel B, Krishnan L, Shun MC, Di Nunzio F, Helland DE, Engelman A - Retrovirology (2009)

Bottom Line: We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect.The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%.Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA, USA. mjd82+@pitt.edu

ABSTRACT

Background: The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants.

Results: Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-1(1-276) and HIV-1(1-273), that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-1(1-270) and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal beta strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription.

Conclusion: HIV-1(1-270), HIV-1(1-266), and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect. The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%. Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.

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IN mutant viral infectivity. Normalized levels of IN mutant infectivities are shown relative to WT HIV-1 (set at 100%). Each experiment amassed duplicate luciferase assays of duplicate infections. Shown is the mean ± SEM of five independent experiments. RLU, relative light units.
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Figure 6: IN mutant viral infectivity. Normalized levels of IN mutant infectivities are shown relative to WT HIV-1 (set at 100%). Each experiment amassed duplicate luciferase assays of duplicate infections. Shown is the mean ± SEM of five independent experiments. RLU, relative light units.

Mentions: To assess HIV-1 infectivity, HeLa-T4 cells were infected with normalized levels of single-round viruses that carry the luciferase reporter gene in place of nef. Two days post-infection, cells were harvested and resulting luciferase activities were normalized to the levels of total protein in the different cell extracts [42,47]. Deletion of up to 9 amino acids from the IN C-terminus failed to affect HIV-1 infectivity (Figure 6). IN mutants HIV-11-276 and HIV-11-273 supported about 50% and 20% of the level of WT infection, respectively, whereas HIV-11-270, HIV-11-266, and the HIV-1/2 tail chimera were non-infectious (Figure 6).


Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase.

Dar MJ, Monel B, Krishnan L, Shun MC, Di Nunzio F, Helland DE, Engelman A - Retrovirology (2009)

IN mutant viral infectivity. Normalized levels of IN mutant infectivities are shown relative to WT HIV-1 (set at 100%). Each experiment amassed duplicate luciferase assays of duplicate infections. Shown is the mean ± SEM of five independent experiments. RLU, relative light units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2770994&req=5

Figure 6: IN mutant viral infectivity. Normalized levels of IN mutant infectivities are shown relative to WT HIV-1 (set at 100%). Each experiment amassed duplicate luciferase assays of duplicate infections. Shown is the mean ± SEM of five independent experiments. RLU, relative light units.
Mentions: To assess HIV-1 infectivity, HeLa-T4 cells were infected with normalized levels of single-round viruses that carry the luciferase reporter gene in place of nef. Two days post-infection, cells were harvested and resulting luciferase activities were normalized to the levels of total protein in the different cell extracts [42,47]. Deletion of up to 9 amino acids from the IN C-terminus failed to affect HIV-1 infectivity (Figure 6). IN mutants HIV-11-276 and HIV-11-273 supported about 50% and 20% of the level of WT infection, respectively, whereas HIV-11-270, HIV-11-266, and the HIV-1/2 tail chimera were non-infectious (Figure 6).

Bottom Line: We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect.The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%.Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA, USA. mjd82+@pitt.edu

ABSTRACT

Background: The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants.

Results: Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-1(1-276) and HIV-1(1-273), that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-1(1-270) and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal beta strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription.

Conclusion: HIV-1(1-270), HIV-1(1-266), and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as their removal unveiled a reverse transcription defect. The F185K mutation reduced the in vitro activities of 1-279 and 1-276 integrases by about 25%. Mutant proteins 1-279/F185K and 1-276/F185K are therefore highlighted as potential structural biology candidates, whereas further deleted tail variants (1-273/F185K or 1-270/F185K) are less desirable due to marginal or undetectable levels of integrase function.

Show MeSH
Related in: MedlinePlus