Bcl-xL is required to protect endothelial cells latently infected with KSHV from virus induced intrinsic apoptosis.

Kaposi's Sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS), a highly vascularized tumor common in AIDS patients and many countries in Africa. KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell, a cell expressing endothelial cell markers. To identify host genes important for KSHV latent infection of endothelial cells we previously used a global CRISPR/Cas9 screen to identify genes necessary for the survival or proliferation of latently infected cells. In this study we rescreened top hits and found that the highest scoring gene necessary for infected cell survival is the anti-apoptotic Bcl-2 family member Bcl-xL. Knockout of Bcl-xL or treatment with a Bcl-xL inhibitor leads to high levels of cell death in latently infected endothelial cells but not their mock counterparts. Cell death occurs through apoptosis as shown by increased PARP cleavage and activation of caspase-3/7. Knockout of the pro-apoptotic protein, Bax, eliminates the requirement for Bcl-xL. Interestingly, neither Bcl-2 nor Mcl-1, related and often redundant anti-apoptotic proteins of the Bcl-2 protein family, are necessary for the survival of latently infected endothelial cells, likely due to their lack of expression in all the endothelial cell types we have examined. Bcl-xL is not required for the survival of latently infected primary effusion lymphoma (PEL) cells or other cell types tested. Expression of the KSHV major latent locus alone in the absence of KSHV infection led to sensitivity to the absence of Bcl-xL, indicating that viral gene expression from the latent locus induces intrinsic apoptosis leading to the requirement for Bcl-xL in endothelial cells. The critical requirement of Bcl-xL during KSHV latency makes it an intriguing therapeutic target for KS tumors.

Zika virus triggers autophagy to exploit host lipid metabolism and drive viral replication.

BACKGROUND: Zika virus (ZIKV), an arbovirus of global concern, has been associated with neurological complications including microcephaly in newborns and Guillain-Barré syndrome in adults. Like other flaviviruses, ZIKV depends on cholesterol to facilitate its replication; thus, cholesterol has been proposed as a therapeutic target to treat the infection using FDA-approved statins. Cholesterol is stored in intracellular lipid droplets (LD) in the form of cholesterol esters and can be regulated by autophagy. We hypothesize that the virus hijacks autophagy machinery as an early step to increase the formation of LD and viral replication, and that interference with this pathway will limit reproduction of virus. METHODS: We pretreated MDCK cells with atorvastatin or other inhibitors of autophagy prior to infection with ZIKV. We measured viral expression by qPCR for NS1 RNA and immunofluorescence for Zika E protein. RESULTS: Autophagy increases in virus-infected cells as early as 6 h post infection (hpi). In the presence of atorvastatin, LD are decreased, and cholesterol is reduced, targeting key steps in viral replication, resulting in suppression of replication of ZIKV is suppressed. Other both early- and late-acting autophagy inhibitors decrease both the number of LD and viral replication. Bafilomycin renders cholesterol is inaccessible to ZIKV. We also confirm previous reports of a bystander effect, in which neighboring uninfected cells have higher LD counts compared to infected cells. CONCLUSIONS: We conclude that atorvastatin and inhibitors of autophagy lead to lower availability of LD, decreasing viral replication. We conclude that bafilomycin A1 inhibits viral expression by blocking cholesterol esterification to form LD. Video Abstract.