Agenda

HSV proteins orchestrate genome packaging into capsids and capsid stabilization. After nuclear egress, microtubule motors transport progeny capsids to the organelles mediating capsid envelopment. Only a couple of hours later, microtubule motors transport incoming capsids to the nuclear pores (NPCs), where they are destabilized and the genomes are released into the nucleoplasm for viral transcription and replication. We have developed cell-free assays to assemble functional capsid-host protein complexes from different capsid and cytosol fractions, and characterize them by quantitative mass spectrometry, immunoblotting, and electron microscopy.

Tegumented capsids recruited all subunits of dynein and kinesin motors, but preferred kinesin-1 with KIF5A and KLC2 over KIF5B, KIF5C, and KLC1. The tegument protein pUL36 depended on the 2453WE2454 motif but not on the 1766WD1767 or 1862WE1863 motifs to bind preferentially to KLC2 over KLC1. These data suggest that at equal availability, the capsids prefer the kinesin-1 KIF5A/KLC2.

Importin α and β mediate docking to NPCs and nuclear protein import. Purified human importin αs bound to tegumented but not naked capsids, with a preference for α1 over α5 and α7 over α3, α4, and α6. An NLS peptide from pUL36 inhibited the binding of α7 and, to some extent, of α5, but not others. These data suggest that capsids do not depend on a particular importin α for NPC targeting, while the pUL36-NLS is recognized by specific α isoforms.

Using cytosol from IFN-induced macrophages, we identified the IFN-inducible GTPase MxB that binds capsids, competes with capsid NPC targeting, and induces premature capsid destabilization in the cytosol. Interestingly, MxB binds to pUL25, which forms two capsid-stabilizing structures: the portal lid and the capsid-associated tegument complex.

Our data show that incoming capsids are stable containers for delivering genomes into the nucleoplasm, but that hosts also have means to intercept these capsid functions and thus infection.