HIV1 Integrase

What is HIV-1 integrase and what is its primary role in viral replication?

HIV-1 integrase is one of three enzymes encoded by HIV-1, alongside protease and reverse transcriptase. Its canonical function is to catalyze the integration of reverse-transcribed viral DNA into the host chromosome, an essential step in the viral life cycle . This function makes integrase an attractive antiviral target because, unlike protease and reverse transcriptase, integrase has no human cellular counterpart . Recent research has revealed that integrase also plays a critical role in proper virion maturation and morphogenesis through interactions with the viral RNA genome .

What is the structural organization of HIV-1 integrase?

HIV-1 integrase consists of three distinct functional domains:

• N-terminal domain (NTD): Contains a zinc-binding HHCC motif important for protein multimerization

• Catalytic core domain (CCD): Houses the enzymatic active site with the conserved D,D,E motif that coordinates divalent metal ions (typically Mg²⁺) essential for catalysis

• C-terminal domain (CTD): Contains SH3-like fold involved in DNA binding and protein-protein interactions

Functionally, HIV-1 integrase operates as a multimer, typically assembling as a tetramer when performing its catalytic activities . The crystal structure of the catalytic domain complexed with inhibitors has been crucial for structure-based drug design efforts .

How does integrase facilitate the integration of viral DNA?

HIV-1 integrase catalyzes viral DNA integration through a two-step biochemical process:

• 3'-processing: Integrase cleaves a dinucleotide from each 3' end of the viral DNA, exposing reactive 3'-OH groups on conserved CA dinucleotides

• Strand transfer: Using these reactive 3'-OH groups, integrase catalyzes nucleophilic attack on phosphodiester bonds in target DNA, simultaneously joining the viral DNA ends to host chromosomal DNA

This process is guided by host factors, particularly LEDGF/p75, which interacts with both integrase and specific chromatin markers (H3K36me3) to direct integration preferentially into transcriptionally active genes in the nuclear periphery .

How does HIV-1 integrase interact with the viral RNA genome?

Recent research has revealed that HIV-1 integrase directly binds to the viral RNA genome in virions . Using cross-linking immunoprecipitation sequencing (CLIP-seq), scientists demonstrated that:

• IN exhibits distinct preferences for select viral RNA structural elements

• This interaction is specific and essential for proper virion morphogenesis

• IN substitutions that selectively impair RNA binding result in eccentric, non-infectious virions

• Allosteric integrase inhibitors (ALLINIs) impair IN binding to viral RNA in wild-type but not escape mutant viruses

These findings reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate a novel mechanism of action for ALLINIs beyond inhibition of integration .

What methodological approaches are used to study HIV-1 integrase-RNA interactions?

Researchers employ several complementary approaches to investigate integrase-RNA interactions:

• CLIP-seq (Cross-linking immunoprecipitation sequencing):

  • Allows identification of RNA molecules bound by IN in physiologically relevant settings

  • Provides near-nucleotide resolution of binding sites

  • Enables mapping of IN binding across the viral genome

• Virological assays:

  • Production of viruses with IN mutations affecting RNA binding

  • Analysis of virion morphology using electron microscopy

  • Assessment of viral infectivity in single-round infection assays

• Biochemical assays:

  • In vitro binding assays with recombinant IN and RNA transcripts

  • Size-exclusion chromatography to analyze IN multimerization

  • Mg²⁺-dependent HIV-1 IN 3′-processing and DNA-strand transfer activities

These techniques have collectively revealed the dual role of integrase in both integration and virion morphogenesis .

How do current classes of HIV-1 integrase inhibitors differ in their mechanisms of action?

Two major classes of integrase inhibitors target different functions of the enzyme:

The dual mechanism of action of ALLINIs, targeting both integration and virion maturation, makes them promising candidates for future antiviral therapies .

What experimental strategies are employed to discover novel HIV-1 integrase inhibitors?

The discovery of novel IN inhibitors employs multiple experimental approaches:

• High-throughput screening (HTS):

  • Homogeneous time-resolved fluorescence-based assays capable of identifying diverse inhibitors

  • Screening of large compound libraries (~370,000 compounds)

  • Identification of both diketo acid-containing scaffolds similar to INSTIs and novel chemical structures

• Structure-guided drug design:

  • Using crystal structures of IN catalytic domain with inhibitors (e.g., 5CITEP at 2.1-Å resolution)

  • Rational modification of lead compounds based on binding patterns

• Hit-to-lead optimization:

  • Structure-activity relationship (SAR) studies

  • Chemical modification to improve potency and selectivity

  • Example: Compound 12 optimization led to derivative 14e with improved potency against wild-type and mutant INs, and further to compound 22 with an antiviral EC₅₀ of ~58 μM and selectivity index >8500

• Resistance profiling:

  • Testing against INs containing archetypical INSTI- and ALLINI-derived resistant substitutions

  • Identifying compounds that maintain activity against resistant variants

These comprehensive approaches have yielded novel scaffolds that can overcome existing resistance mechanisms and potentially complement current HIV-1 therapies .

How do resistance mutations affect HIV-1 integrase inhibitor efficacy?

HIV-1 can develop resistance to integrase inhibitors through various mutations:

• INSTI resistance mutations:

  • Primary pathway mutations occur in the catalytic core domain near the active site

  • Common mutations include N155H, Q148H/R/K, Y143R/C/H

  • These mutations reduce inhibitor binding while preserving catalytic function

  • Second-generation INSTIs (dolutegravir, bictegravir) maintain efficacy against some first-generation INSTI-resistant viruses

• ALLINI resistance mutations:

  • Typically occur at the interface between catalytic core and C-terminal domains

  • Alter multimerization dynamics without compromising RNA binding

  • These mutations can confer cross-resistance to different ALLINIs

• RNA-binding affecting mutations:

  • Mutations that selectively impair IN binding to viral RNA result in non-infectious virions

  • Some of these mutations confer resistance to ALLINIs by altering multimerization patterns

Understanding resistance pathways is critical for developing inhibitors with higher genetic barriers to resistance and for designing effective combination therapies .

How is HIV-1 integrase strand transfer activity quantified in research settings?

Researchers employ several complementary methods to quantify integrase catalytic activities:

• In vitro biochemical assays:

  • Measurement of Mg²⁺-dependent 3′-processing activity

  • Quantification of DNA-strand transfer reactions

  • Concerted integration activity assays using purified recombinant integrase

• Cell-based assays:

  • Single-round infection assays using pseudotyped viruses (e.g., VSV-G pseudotyped HIV-Luc)

  • Measurement of luciferase reporter expression as an indicator of successful integration

  • Analysis of integration products by PCR-based methods

• Integration site analysis:

  • Isolation of integration products from in vitro concerted integration reactions

  • Subcloning and sequencing to determine integration site preferences

  • Next-generation sequencing approaches to generate comprehensive integration site libraries

These methods have collectively enabled the characterization of both wild-type integrase function and the effects of mutations or inhibitors on its catalytic activities .