The Kaposi's-sarcoma-associated herpesvirus orf35 gene product is required for efficient lytic virus reactivation

- A recombinant viral clone that fails to express KSHV ORF35 (BAC16-ORF35-stop) was generated.
- BAC16-ORF35-stop preserves intact adjacent and overlapping reading frames.
- BAC16-ORF35-stop virus displayed significantly reduced lytic virus reactivation.
- Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency.
- ORF35 is important for efficient lytic reactivation of KSHV.

Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.