Ambrus, K. | Search bioRxiv

bioRxiv2026

Lenacapavir prevents production of infectious HIV-1 by abrogating immature virus assembly.

The HIV-1 capsid effector Lenacapavir (LEN) acts by disrupting the early (reverse transcription and nuclear entry) and late (assembly and maturation) stages of the viral lifecycle. The early stage requires an intact Capsid consisting of a lattice of capsid protein (CA) hexamers and pentamers. Phenylalanine-Glycine (FG) containing nuclear pore proteins interact with Capsid hexamers at their FG pockets, facilitating nuclear entry. Disruption of Capsid lattice stability, or competitive binding to the FG pocket, by LEN blocks infection. Here, we provide insight into the effects of LEN on the late stage. Using a combination of cryo-EM structure determination, in vitro assembly, and in situ viral assays, we determined that treatment of producer cells with LEN abrogates the production of infectious virus via multiple mechanisms. Previous studies have shown that HIV-1 produced from LEN treated cells have improperly formed Capsids. However, how LEN interacts with the CA domain of the Gag polyprotein during assembly, prior to maturation, is unclear. Using a viral protease (PR) defective HIV-1 clone, which traps the Gag lattice in an immature state, we found that LEN dramatically remodulates the CA domain. The resulting CA layer was mature-like despite the absence of PR cleavage. We dubbed this unnatural state as "premature" lattice. Proper immature and mature lattice formation requires inositol hexakisphosphate (IP6), but our cryo-EM work revealed a lack of IP6 in the premature lattice. These findings provide insight into the mechanisms by which LEN prevents infectious HIV-1 production.

molecular biology↗

bioRxiv2025

Translocation of HIV capsid core through the Nuclear Pore Complex by affinity gradient

The HIV capsid core encapsulates the viral genome for subsequent integration into host cellular DNA. Prior to nuclear entry, the core must translocate through the Nuclear Pore Complex (NPC). This transit involves interactions between the capsid core and phenylalanine-glycine (FG) repeats found in nucleoporins within the NPC. Despite this critical role in the viral replication cycle, the molecular mechanism of capsid core translocation remains unclear. FG repeats consist of three classes of canonical motifs: FG, GLFG, and FxFG motifs. These are segregated within the NPC to define distinct zones of the gating machinery. FG- and FxFG-type motifs are enriched in the cytoplasmic and nuclear ("nuclear basket") peripheries of the NPC while GLFG motifs are enriched in regions adjacent to the core of the NPC. To investigate the capsid core translocation, we use biochemical, biophysical, and structural approaches to study FG-capsid interactions in a quantitative manner. We show that the capsid (CA) interacts with a range of diverse FG repeats with varying affinities. GLFG motifs of core NUP98 exhibit increased affinity to CA proteins compared to other conventional FG/FxFG. However, the non-canonical FxFG motif of NUP153 at the "nuclear basket" significantly increases binding affinity to CA compared to canonical FxFG, therefore called FG super-motif. In addition, C-terminal motif of NUP153 consists of a stretch of basic residues, which enhances the affinity of this non-canonical FG super-motif to capsid core at the NPC nuclear periphery. We identified other binding enhancers of the NPC core FG-NUPs, NUP58 and POM121. The relationship between the binding strength of FG/FxFG binding enhancers of NUP58, POM121, NUP153 and their position within the NPC also shows capsid core binding affinity increases with increasing proximity to the "nuclear basket." Based on our data, the difference in binding affinities between the canonical FxFG motif and the enhancer-coupled FG super-motif of NUP153 to capsid cores at the "nuclear basket" is approximately 1,000-fold. Therefore, the diverse FG motifs and binding enhancers, which are naturally distributed within the NPC into distinct zones, create an avidity gradient--with changes in both concentration and binding affinity--along the cytoplasmic-nuclear axis. We suggest that HIV capsid translocation into the nucleus is potentiated by this gradient in a unidirectional manner (outside- to-inside) within the NPC.

microbiology↗