Limits...
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.

Show MeSH

Related in: MedlinePlus

LEDGFp75-dependent concerted integration activities of WT and IN mutant proteins. (A) The scanned ethidium-stained agarose gels reveal the migration positions of donor, supercoiled (s.c.), and open circular (o.c.) substrate DNAs, as well as half-site and concerted integration reaction products. Donor DNA was omitted from the reactions analyzed in lanes 1 and 25, whereas IN was omitted from lanes 2 and 26. The remaining lanes contained the indicated IN proteins and, at times, LEDGFp75. The concentration of HIV-1/2 IN in lanes 29 and 30 was 1.6 μM, whereas all other IN concentrations were 0.8 μM. The migration positions of molecular mass standards in kb are shown to the left of the gel. (B and C) Levels of overall and concerted DNA strand transfer activities, respectively, normalized to INWT (set to 100%). Results are mean ± SEM for two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2770994&req=5

Figure 5: LEDGFp75-dependent concerted integration activities of WT and IN mutant proteins. (A) The scanned ethidium-stained agarose gels reveal the migration positions of donor, supercoiled (s.c.), and open circular (o.c.) substrate DNAs, as well as half-site and concerted integration reaction products. Donor DNA was omitted from the reactions analyzed in lanes 1 and 25, whereas IN was omitted from lanes 2 and 26. The remaining lanes contained the indicated IN proteins and, at times, LEDGFp75. The concentration of HIV-1/2 IN in lanes 29 and 30 was 1.6 μM, whereas all other IN concentrations were 0.8 μM. The migration positions of molecular mass standards in kb are shown to the left of the gel. (B and C) Levels of overall and concerted DNA strand transfer activities, respectively, normalized to INWT (set to 100%). Results are mean ± SEM for two independent experiments.

Mentions: Supercoiled pGEM-3 plasmid DNA was incorporated into the reaction mixture to help identify concerted integration reaction products (Figure 2D and 5A). Integration of only one donor DNA end into one plasmid DNA strand yields a tagged circle whose mobility through agarose matches that of starting relaxed circular plasmid (Figure 5A). Pairwise integration of two oligonucleotides by contrast yields a linearized product whose size is slightly larger than linear plasmid (Figure 2D). IN DNA strand transfer activity was barely detectable in the absence of LEDGFp75, yielding slight increases in the nicked or open circular plasmid population (Figure 5A, compare lanes 3 and 27 to lanes 2 and 26, respectively) [31]. LEDGFp75 greatly stimulated IN activity such that the supercoiled target DNA was largely consumed, yielding a mixture of half-site and concerted integration products (Figure 5A, lanes 4 and 28). IN mutant product formation was quantified to reflect overall levels (half-site plus concerted, Figure 5B) of DNA strand transfer activities or just concerted integration (Figure 5C). The overall activities of the various deletion mutant proteins in large part mirrored their oligonucleotide-based DNA strand transfer activities (compare Figure 5B to 4B). Though 0.49 μM INHIV1/2 supported about 40% of INWT activity in the oligonucleotide-based assay (Figure 4B), 0.8 μM protein failed to support appreciable product formation in the concerted assay format (Figure 5A, lane 31). Doubling the amount of input INHIV1/2 to 1.6 μM yielded significant half-site product formation (about 66% of INWT, Figure 5A, lane 30 and Figure 5B) in the absence of detectable concerted integration activity (Figure 5C). Taken together, our data indicate that the C-terminal tail does not play a specific role in concerted DNA integration, though the introduction of a foreign sequence for the HIV-1 tail can uncouple pairwise from single end integration activity. Though others noted that the F185K substitution ablated Mg2+-dependent integration of preprocessed oligonucleotide donor DNA into heterologous target DNA [61], our reaction conditions failed to reveal an affect of the solubilizing mutation on full-length IN activity in the presence of LEDGFp75 (Figure 5A, lane 6; panels B and C). We furthermore conclude that the C-terminal 9 amino acids of HIV-1 IN can be removed without dramatically effecting Mg2+-based single end or concerted DNA integration activities (Figures 3, 4, 5)., We highlight these derivatives as potential candidates for structural biology studies despite the approximate 20-25% reductions in IN1-279 and IN1-276 activities brought on by the F185K change. We would by contrast advise against extensive analysis of tailless IN1-270, due to its lack of detectable DNA strand transfer activity under these assay conditions (Figure 4 and 5).


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)

LEDGFp75-dependent concerted integration activities of WT and IN mutant proteins. (A) The scanned ethidium-stained agarose gels reveal the migration positions of donor, supercoiled (s.c.), and open circular (o.c.) substrate DNAs, as well as half-site and concerted integration reaction products. Donor DNA was omitted from the reactions analyzed in lanes 1 and 25, whereas IN was omitted from lanes 2 and 26. The remaining lanes contained the indicated IN proteins and, at times, LEDGFp75. The concentration of HIV-1/2 IN in lanes 29 and 30 was 1.6 μM, whereas all other IN concentrations were 0.8 μM. The migration positions of molecular mass standards in kb are shown to the left of the gel. (B and C) Levels of overall and concerted DNA strand transfer activities, respectively, normalized to INWT (set to 100%). Results are mean ± SEM for two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: LEDGFp75-dependent concerted integration activities of WT and IN mutant proteins. (A) The scanned ethidium-stained agarose gels reveal the migration positions of donor, supercoiled (s.c.), and open circular (o.c.) substrate DNAs, as well as half-site and concerted integration reaction products. Donor DNA was omitted from the reactions analyzed in lanes 1 and 25, whereas IN was omitted from lanes 2 and 26. The remaining lanes contained the indicated IN proteins and, at times, LEDGFp75. The concentration of HIV-1/2 IN in lanes 29 and 30 was 1.6 μM, whereas all other IN concentrations were 0.8 μM. The migration positions of molecular mass standards in kb are shown to the left of the gel. (B and C) Levels of overall and concerted DNA strand transfer activities, respectively, normalized to INWT (set to 100%). Results are mean ± SEM for two independent experiments.
Mentions: Supercoiled pGEM-3 plasmid DNA was incorporated into the reaction mixture to help identify concerted integration reaction products (Figure 2D and 5A). Integration of only one donor DNA end into one plasmid DNA strand yields a tagged circle whose mobility through agarose matches that of starting relaxed circular plasmid (Figure 5A). Pairwise integration of two oligonucleotides by contrast yields a linearized product whose size is slightly larger than linear plasmid (Figure 2D). IN DNA strand transfer activity was barely detectable in the absence of LEDGFp75, yielding slight increases in the nicked or open circular plasmid population (Figure 5A, compare lanes 3 and 27 to lanes 2 and 26, respectively) [31]. LEDGFp75 greatly stimulated IN activity such that the supercoiled target DNA was largely consumed, yielding a mixture of half-site and concerted integration products (Figure 5A, lanes 4 and 28). IN mutant product formation was quantified to reflect overall levels (half-site plus concerted, Figure 5B) of DNA strand transfer activities or just concerted integration (Figure 5C). The overall activities of the various deletion mutant proteins in large part mirrored their oligonucleotide-based DNA strand transfer activities (compare Figure 5B to 4B). Though 0.49 μM INHIV1/2 supported about 40% of INWT activity in the oligonucleotide-based assay (Figure 4B), 0.8 μM protein failed to support appreciable product formation in the concerted assay format (Figure 5A, lane 31). Doubling the amount of input INHIV1/2 to 1.6 μM yielded significant half-site product formation (about 66% of INWT, Figure 5A, lane 30 and Figure 5B) in the absence of detectable concerted integration activity (Figure 5C). Taken together, our data indicate that the C-terminal tail does not play a specific role in concerted DNA integration, though the introduction of a foreign sequence for the HIV-1 tail can uncouple pairwise from single end integration activity. Though others noted that the F185K substitution ablated Mg2+-dependent integration of preprocessed oligonucleotide donor DNA into heterologous target DNA [61], our reaction conditions failed to reveal an affect of the solubilizing mutation on full-length IN activity in the presence of LEDGFp75 (Figure 5A, lane 6; panels B and C). We furthermore conclude that the C-terminal 9 amino acids of HIV-1 IN can be removed without dramatically effecting Mg2+-based single end or concerted DNA integration activities (Figures 3, 4, 5)., We highlight these derivatives as potential candidates for structural biology studies despite the approximate 20-25% reductions in IN1-279 and IN1-276 activities brought on by the F185K change. We would by contrast advise against extensive analysis of tailless IN1-270, due to its lack of detectable DNA strand transfer activity under these assay conditions (Figure 4 and 5).

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