Effect of Lenacapavir and PF74 on HIV-1 capsid morphology: EM images

Lenacapavir (GS-6207; LEN) is a potent HIV-1 capsid inhibitor approved for
treating multidrug-resistant infection. LEN binds to a hydrophobic pocket
between neighboring capsid (CA) proteins in hexamers and stabilizes the
capsid lattice, but its effect on HIV-1 capsids is not fully understood.
Here, we labeled HIV-1 capsids with green fluorescent protein fused to CA
(GFP-CA) or a fluid-phase GFP content marker (cmGFP) to assess LEN's
impact on HIV-1 capsids. HIV-1 cores labeled with GFP-CA, but not cmGFP,
could be immunostained with an anti-GFP antibody and were less sensitive
to the capsid-binding host restriction factor MX2, demonstrating that
GFP-CA is incorporated into the capsid lattice and is a marker for capsid
lattice stability whereas cmGFP is an indicator of core integrity. LEN
treatment of isolated HIV-1 cores resulted in a dose-dependent loss of
cmGFP signal while preserving the GFP-CA signal, indicating that LEN
disrupts core integrity but stabilizes the capsid lattice. In contrast,
capsid inhibitor PF-3450074 (PF74) induced loss of core integrity and the
capsid lattice. Electron microscopy of LEN- or PF74-treated viral cores
revealed frequent breakage at the narrow end of the capsid and other
morphological changes. Our results suggest that LEN treatment does not
prevent nuclear envelope docking but inhibits nuclear import of cores with
or without loss of core integrity. In contrast, PF74 treatment blocks
nuclear import by inhibiting the nuclear envelope docking of viral cores,
highlighting their different mechanisms of nuclear import inhibition.