Summary auto-generated
This study investigated whether differences in DNA binding properties between herpes simplex virus type 1 (HSV-1) Vmw175 and varicella-zoster virus (VZV) VZV140k proteins account for their distinct regulatory functions. Researchers created a hybrid protein by replacing Vmw175's DNA binding domain with the corresponding VZV140k domain. In cell culture experiments (transfection assays), the hybrid protein showed intermediate transactivation properties between the two parent proteins and lost Vmw175's ability to repress the IE3 promoter, instead resembling VZV140k. However, when incorporated into a recombinant HSV-1 virus (HSVR2DS), the hybrid protein resulted in approximately 10-fold reduced virus production compared to wild-type HSV-1, despite showing stronger gene activation in transfection assays. The hybrid virus produced higher levels of the modified Vmw175 protein and some other viral proteins. These results demonstrate that the DNA binding domain significantly influences the transcriptional regulatory properties of these alphaherpesvirus transactivators and contributes to viral fitness, though other protein domains outside the DNA binding region are also important for efficient virus replication.
Key findings
- The VZV140k DNA binding domain swap eliminated Vmw175's ability to autoregulate IE3 expression, converting it to properties similar to VZV140k
- The hybrid 175R2DS protein achieved intermediate transactivation levels (≈25% of VZV140k) in transfection assays, confirming proper protein folding
- Recombinant virus HSVR2DS expressing the hybrid protein exhibited ≈10-fold reduced growth compared to wild-type HSV-1 despite higher transactivation in transfection assays
- The hybrid virus expressed higher levels of the modified Vmw175 protein, particularly at late infection times
- Non-DNA-binding-domain sequences of Vmw175 are essential for optimal virus replication
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Abstract
The alphaherpesviruses encode major immediate early transactivator proteins that are essential for the expression of later classes of viral genes. We have previously shown that the extensive sequence similarity between the herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) members of the family (proteins Vmw175 and VZV140k) extends to function, since a virus which expresses VZV140k in place of Vmw175 is able to grow, albeit at reduced efficiency. We have also shown that the DNA binding characteristics of the isolated DNA binding domains of Vmw175 and VZV140k are related but distinct. In order to assess whether the different DNA binding properties of the two proteins are responsible for the differences in their individual transcriptional regulatory functions, we constructed a plasmid and an HSV-1 virus in which the VZV140k DNA binding domain coding sequences replace those of Vmw175. The characteristics of the resultant hybrid protein in transfection assays and during virus infection suggest that the nature of the DNA binding domain plays a significant role in the transactivation and repression properties of the Vmw1 75 family of proteins.