CONSTRUCTION OF RECOMBINANT VIRUSES EXPRESSING FLUORESCENT MARKERS FOR THE STUDY OF KSHV INFECTION

Kaposi’s sarcoma-associated herpesvirus (KSHV) is causally linked to Kaposi’s sarcoma, primary effusion lymphoma, and plasmablastic multicentric Castleman’s disease. KSHV has two forms of infection: latent and lytic. Latent infection allows the virus to establish long-term persistent infection, whereas lytic infection is needed for the maintenance of viral reservoir and for virus spread. Primary infection with KSHV precedes lifelong latent infection which may reactivate and switch towards the lytic cycle. The infection cycle of KSHV has been extensively studied, yet different aspects related to the infection dynamics are still unknown.

Recombinant viruses encoding fluorescent genes help identify virus-infected cells, study the activity of viral promoters and provide a useful way to track the infection cycle. We used the full-length KSHV genome bacterial artificial chromosome 16 (BAC16) clone to construct new recombinant viruses encoding fluorescent proteins, mNeonGreen and mCherry (BAC16-mNeonGreen and BAC16-mCherry, respectively). These fluorescent proteins were cloned under the control of the constitutive cellular promoter EF1α and provide a way to identify infected cells. In addition, we constructed a dual fluorescent virus encoding GFP which is constitutively expressed and mCherry under the control of a strong viral lytic gene promoter (BAC16-GFP-pNut-mCherry). This virus enables identification of cells undergoing either latent or lytic infection. By using these recombinant viruses, we examined whether cells that are already KSHV-infected, in its latent form, will undergo secondary KSHV infection and to what extent. We found that primary infection of uninfected cells is less efficient as compared to secondary KSHV infection of latently infected cells. This suggests that latently-infected cells express viral or cellular gene product/s that promote KSHV-infection.