# The varicella-zoster virus ORF16 protein promotes both the nuclear transport and the protein abundance of the viral DNA polymerase subunit ORF28

## TL;DR

This study shows how the varicella-zoster virus uses a protein called ORF16 to help another protein, ORF28, move into the nucleus and avoid being broken down, which is important for viral replication.

## Contribution

The study reveals that ORF16 promotes both the nuclear transport and protein stability of ORF28 in varicella-zoster virus.

## Key findings

- ORF16 mediates the nuclear import of the viral DNA polymerase subunit ORF28.
- ORF16 enhances the protein abundance of ORF28 by preventing its proteasome-mediated degradation.
- Radicicol disrupts the interaction between ORF16 and ORF28, affecting both nuclear entry and protein stability.

## Abstract

•Several VZV-encoded replication enzymes per se are localized in the cytoplasm.•The nuclear import of VZV ORF28 can be mediated by ORF16.•ORF16 can enhance the protein abundance of ORF28.•The low abundance of ORF28 in cells is due to rapid degradation by the proteasome.•Radicicol, a Hsp90 inhibitor, can disrupt the interaction between ORF16 and ORF28.

Several VZV-encoded replication enzymes per se are localized in the cytoplasm.

The nuclear import of VZV ORF28 can be mediated by ORF16.

ORF16 can enhance the protein abundance of ORF28.

The low abundance of ORF28 in cells is due to rapid degradation by the proteasome.

Radicicol, a Hsp90 inhibitor, can disrupt the interaction between ORF16 and ORF28.

Although all herpesviruses utilize a highly conserved replication machinery to amplify their viral genomes, different members may have unique strategies to modulate the assembly of their replication components. Herein, we characterize the subcellular localization of seven essential replication proteins of varicella-zoster virus (VZV) and show that several viral replication enzymes such as the DNA polymerase subunit ORF28, when expressed alone, are localized in the cytoplasm. The nuclear import of ORF28 can be mediated by the viral DNA polymerase processivity factor ORF16. Besides, ORF16 could markedly enhance the protein abundance of ORF28. Noteworthily, an ORF16 mutant that is defective in nuclear transport still retained the ability to enhance ORF28 abundance. The low abundance of ORF28 in transfected cells was due to its rapid degradation mediated by the ubiquitin-proteasome system. We additionally reveal that radicicol, an inhibitor of the chaperone Hsp90, could disrupt the interaction between ORF16 and ORF28, thereby affecting the nuclear entry and protein abundance of ORF28. Collectively, our findings imply that the cytoplasmic retention and rapid degradation of ORF28 may be a key regulatory mechanism for VZV to prevent untimely viral DNA replication, and suggest that Hsp90 is required for the interaction between ORF16 and ORF28.