Quantitative SUMO-1 Modification of a Vaccinia Virus Protein Is Required for Its Specific Localization and Prevents Its Self-Association

Abstract

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.

Regulation of Vaccinia Virus E3 Protein by Small Ubiquitin-Like Modifier Proteins

“…SUMO1 was reported previously to translocate to the viral factory in response to VACV infection (49). In addition, SUMO1 was previously isolated as an E3-interacting protein in a yeast two-hybrid assay (53).…”

Activation of the Double-stranded RNA-dependent Protein Kinase PKR by Small Ubiquitin-like Modifier (SUMO)

“…Our results demonstrate that VSV infection promotes PKR SUMOylation and that SUMO contributes to the antiviral activity of PKR, indicating that SUMOylation of PKR is an important antiviral response of the cell. Therefore, it is possible to speculate that recruitment of SUMO to viral factories (41,42) or targeting of the SUMOylation machinery by virus (43) may work as mechanisms to counteract PKR antiviral activity.…”

Small Ubiquitin-like Modifier (SUMO) Modification of the Androgen Receptor Attenuates Polyglutamine-mediated Aggregation

“…This is reminiscent of the role for SUMO in the function of A40R. This vaccinia virus protein is quantitatively SUMOylated, and SUMOylation is essential to prevent the protein from otherwise forming insoluble aggregates (69). The substoichiometric modification of AR coupled with the effects of non-modified AR, however, argues that additional in trans mechanisms may be at play.…”