HIV-1 Integrase

What is the primary function of HIV-1 integrase in the viral life cycle?

HIV-1 integrase (IN) serves dual critical functions in viral replication:

Primary function: Integration of reverse-transcribed viral DNA into the host chromosome, which involves two sequential catalytic steps: 3' processing (removal of terminal nucleotides from viral DNA) followed by strand transfer (insertion of processed viral DNA into host DNA) .

Secondary function: Binding to the viral RNA genome in virions, which is necessary for proper virion maturation and morphogenesis. When this function is disrupted, the viral genome becomes mislocalized within the virus particle and is prematurely degraded in target cells .

Both functions share common elements, including interaction with viral nucleic acids and assembly of higher-order IN multimers, but they represent distinct roles that are essential for productive viral infection .

How does HIV-1 integrase select integration sites in the host genome?

HIV-1 integration is not random but exhibits distinct preferences for specific genomic regions:

HIV-1 preferentially targets transcriptionally active genes in the nuclear periphery rather than distributing randomly across the genome . This targeting is primarily mediated by the cellular cofactor lens epithelium-derived growth factor (LEDGF/p75), which functions as a bimodal tether. LEDGF/p75 interacts with IN through its C-terminal integrase-binding domain while its N-terminal PWWP domain binds to nucleosomes trimethylated at Lys36 of histone H3 (H3K36me3), an epigenetic mark associated with actively transcribed genes .

Integration site preferences can be dramatically altered by specific mutations in integrase. For example, the K258R point mutation causes a more than 10-fold increase in integration into centromeric alpha satellite repeat sequences compared to wild-type virus . Such integrations into centromeric regions are known to be enriched in the latent reservoir of infected memory T cells and in elite controllers who limit viral replication without intervention .

Integration site distribution can be experimentally assessed using deep sequencing, quantitative PCR, and in situ immunofluorescence assays, while immunoprecipitation studies can identify host factors that influence targeting preferences .

What are the key residues in HIV-1 integrase that interact with viral DNA?

Several critical residues in HIV-1 integrase play essential roles in viral DNA interaction:

The catalytic triad (D64, D116, E152) forms the active site required for catalytic activity . Near this active site, lysine residues K156 and K159 are critical for functional interaction with viral DNA. Site-directed mutagenesis studies have shown that mutation of these residues to glutamate leads to loss of both 3′ processing and strand transfer activities in vitro . Viruses containing these mutations are replication-defective, specifically at the integration step, confirming their importance in viral DNA interaction .

Photo-crosslinking experiments using 5-iododeoxyuracil-substituted oligonucleotides demonstrate that K159 specifically interacts at the N7 position of the conserved deoxyadenosine adjacent to the scissile phosphodiester bond of viral DNA . K159 is positioned to interact with and orient viral DNA such that the scissile phosphodiester bond is placed close to the active site residues .

The impact of these residues varies between the isolated core domain and full-length integrase. In the context of the core domain (residues 50-212), individual mutations K156E and K159E dramatically reduce disintegration activity, while in full-length integrase, only the double mutant shows significant reduction in activity, suggesting functional redundancy in the complete protein .

What are the primary integrase strand transfer inhibitors (INSTIs) and how do they work?

Integrase strand transfer inhibitors (INSTIs) have become key components of antiretroviral therapy regimens:

Mechanism: INSTIs work by binding to the integrase-viral DNA complex (intasome) at the active site, displacing the reactive 3'-OH end of viral DNA and chelating the essential metal cofactors required for the strand transfer reaction .

FDA-approved INSTIs include raltegravir, elvitegravir, dolutegravir, and bictegravir, which are highly effective and well-tolerated components of current antiretroviral therapy regimens .

Resistance development: More than 40 substitutions have been associated with INSTI resistance in HIV-1, with the most prevalent mutations occurring at positions 66, 92, 143, 147, 148, and 155 . Some mutations show subtype-specific patterns - for example, G118R is rare in subtype B viruses but provides an alternative resistance pathway in non-B subtypes, while R263K is preferentially selected in subtype B viruses .

Beyond the active site, research has identified alternative binding sites for potential allosteric inhibitors, including the IN:IN dimer interface and regions behind the DNA binding domain . These sites represent promising targets for developing new inhibitors that might overcome resistance to current drugs .

How do mutations in HIV-1 integrase affect viral replication?

Mutations in HIV-1 integrase can have varied effects on viral replication:

Critical DNA-interacting residues: Mutation of lysine residues K156 and K159 to glutamate renders HIV-1 replication-defective . These mutant viruses are proficient for particle assembly, entry, and reverse transcription but specifically defective at the integration step, confirming their essential role in DNA binding and integration .

Integration site selection: The K258R point mutation substantially redirects integration site distribution, causing more than 10-fold increase in integration into centromeric alpha satellite repeat sequences . This mutation is present in databases of latent proviruses found in patients and may reflect an unappreciated aspect of viral latency establishment .

INSTI resistance: Mutations at positions 66, 92, 143, 147, 148, and 155 are primarily associated with resistance to integrase inhibitors . Different subtypes show differential selection of resistance mutations - G118R is rare in subtype B but provides an alternative pathway for dolutegravir resistance in non-B subtypes, while R263K is preferentially selected in subtype B viruses .

Virological evaluation through growth kinetics, reverse transcriptase activity assays, and integration site analysis provides comprehensive assessment of how specific mutations impact viral fitness and replication capacity .

What structural approaches are being used to overcome the challenges of resolving HIV-1 integrase structures?

Understanding HIV-1 integrase structure remains challenging due to the lack of a full-length crystal structure . Researchers have developed several innovative approaches:

Homology modeling: Quaternary HIV integrase models have been created using free software, based partially on available full-length prototype foamy virus integrase structures combined with truncated HIV integrase structures . These models provide valuable insights despite the absence of a complete experimental structure.

Modeling validation: These models have been tested through docking studies with potential inhibitors, including randomly selected molecules from the ZINC database and characterized inhibitors like FZ41 and HDS1 . Such studies have revealed potential binding sites at the IN:IN dimer interface and behind the DNA binding domain .

Structure-function correlation: Combining structural models with virological and biochemical studies helps validate the models and understand inhibitory mechanisms. For example, studies with HDS1 showed that it inhibits integrase at the DNA binding step rather than at strand transfer or 3' processing steps .

These modeling approaches are particularly valuable for screening integrase inhibitors and studying drug resistance mechanisms until more complete experimental structures become available .

What methods are most effective for studying HIV-1 integrase interactions with viral DNA?

Several complementary approaches provide insights into integrase-DNA interactions:

Site-directed mutagenesis and photo-crosslinking: These techniques have identified critical residues like K156 and K159 that interact with viral DNA . Photo-crosslinking using 5-iododeoxyuracil-substituted oligonucleotides demonstrated that K159 specifically interacts with the conserved deoxyadenosine adjacent to the cleavage site .

Functional assays: 3' processing, strand transfer, and disintegration assays assess how mutations affect different catalytic activities . These assays revealed that K156E and K159E mutations eliminate 3' processing and strand transfer activities while maintaining some disintegration activity in the context of full-length integrase .

DNA binding studies: Aggregation of fluorescently labeled substrates can measure DNA binding capacity . The K156E and K159E mutations in the core domain dramatically decreased interaction with disintegration substrate DNA .

Virological assays: Testing mutant viruses for replication capacity and analyzing specific stages of the viral life cycle confirms the relevance of identified interactions . Viruses with K156E or K159E mutations were proficient for particle assembly, entry, and reverse transcription but defective at the integration step .

The combined use of these techniques provides a comprehensive understanding of how integrase interacts with viral DNA during the integration process.

How do different HIV-1 subtypes affect integrase function and inhibitor susceptibility?

HIV-1 subtypes show important variations that impact integrase function and drug resistance:

Differential resistance pathways: Viral subtypes (especially B and C) have different mechanisms for selecting drug resistance mutations . G118R is rare in subtype B viruses but may provide an alternative pathway for dolutegravir resistance in non-B subtypes, while R263K is preferentially selected in subtype B viruses .

Natural polymorphisms: Variations in integrase sequences across subtypes may influence susceptibility to integrase strand transfer inhibitors (INSTIs) and the development of resistance . These natural polymorphisms create different genetic backgrounds that affect resistance pathways.

Clinical implications: Understanding subtype differences is crucial for optimizing treatment strategies in different geographic regions, particularly in Sub-Saharan Africa where non-B subtypes predominate . Monitoring integrase resistance-associated mutations in different populations is important as INSTI use increases globally .

Research approaches combining genotypic analysis, phenotypic resistance testing, and clinical outcomes data stratified by subtype provides the most comprehensive assessment of subtype effects on integrase function and inhibitor efficacy .

What approaches can identify novel binding sites in HIV-1 integrase for inhibitor development?

Several strategies can reveal potential new targets for HIV-1 integrase inhibitors:

Computational modeling and docking: Homology models of HIV-1 integrase can be used for virtual screening of compound libraries . Docking studies have identified potential binding sites at the IN:IN dimer interface and behind the DNA binding domain, distinct from the active site targeted by current drugs .

Experimental validation: Compounds identified through virtual screening can be tested in vitigation and biochemical assays to confirm binding and inhibitory activity . For example, studies with HDS1 and FZ41 compounds confirmed that they bind at similar locations behind the DNA binding domain with some overlap with the IN:IN dimer interface .

Mechanism determination: Detailed biochemical studies can determine how novel compounds inhibit integrase function . HDS1 was shown to inhibit integrase at the DNA binding step rather than at strand transfer or 3' processing steps, and did not directly interact with DNA .

These approaches have revealed at least two possible locations in integrase that could be targeted by allosteric inhibitors, distinct from current drug binding sites and from LEDGF inhibitor binding sites .