HIV-1 adapts to lost IP6 coordination through second-site mutations that restore conical capsid assembly

Area of Science:

• Virology
• Structural Biology
• Molecular Biology

Background:

• The human immunodeficiency virus type 1 (HIV-1) capsid is a protein shell essential for viral replication.
• The capsid protein (CA) forms hexamers and pentamers (capsomers) with a central pore, hypothesized to regulate assembly and nucleotide import.
• Positively charged rings formed by CA residues Arg-18 (R18) and Lys-25 (K25) are believed to mediate these pore functions.

Purpose of the Study:

• To investigate the role of R18 and K25 in HIV-1 capsid function and replication through forced evolution.
• To identify compensatory mutations that restore viral fitness following mutations in the K25 residue.

Main Methods:

• Forced evolution of HIV-1 viruses with mutations in R18 and K25.
• Analysis of viral replication fitness, reverse transcription, and infection.
• In vitro and in virion capsid assembly assays.
• Mapping of second-site suppressor mutations.

Main Results:

• R18 mutants exhibited a complete failure to replicate.
• K25A mutants acquired compensatory mutations that restored near wild-type replication fitness.
• These suppressors rescued reverse transcription and infection without reintroducing lost pore charges.
• Compensatory mutations restored pentamer formation and enabled closed conical capsid assembly in vitro and in virions.

Conclusions:

• Lys-25 (K25) is not intrinsically required for HIV-1 nucleotide import or capsid assembly.
• HIV-1 requires a precise hexamer:pentamer equilibrium for proper capsid assembly.
• Compensatory mutations can restore viral fitness by tuning this equilibrium following pore mutations.