Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the gammaherpesvirinae subfamily. The present study describes the construction and characterization of a new dual fluorescent-tagged recombinant KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses GFP and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates, and that mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in cells undergoing lytic induction, and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress.
