Host cell killing by the West Nile Virus NS2B–NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

Abstract

The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

Caspase 3-Dependent Cell Death of Neurons Contributes to the Pathogenesis of West Nile Virus Encephalitis

“…Although further study is warranted, we speculate that different neurons may undergo cell-specific death programs in response to WNV infection. Flaviviruses can induce cell death directly through viral replication and the production of proapoptotic proteins (41,43,44,52,57). Notably, pancaspase and caspase 3 inhibitors reduced the WNV-mediated death of primary neurons, which is consistent with studies showing that caspase inhibitors reduced apoptosis after transgenic expression of the flavivirus NS3 and E genes (41,42,44).…”

“…Several WNV proteins may contribute directly to this process. Ectopic expression of the WNV NS3 protein or its helicase or protease domain induced apoptosis and activation of caspase 3 and 8 (44). Expression of WNV capsid protein either in vitro or in the striata of mouse brains also triggered apoptosis downstream of caspase 3 and caspase 9 activation (63).…”

Induction of the Cellular MicroRNA, Hs_154, by West Nile Virus Contributes to Virus-Mediated Apoptosis through Repression of Antiapoptotic Factors

“…In addition, WNV infection initiates cellular apoptosis through a variety of caspase-dependent mechanisms, including induction of the unfolded protein response (UPR) (32), interaction of WNV proteins with apoptosis factors (42,57,58), and activation of inflammatory pathways (51). Cell death via these mechanisms contributes to the neuropathogenicity observed in WNV infection (44).…”

“…In general, cytoplasmic RNA viruses such as flaviviruses are not believed to express their own miRNAs (although small RNAs derived from the viral genome can be identified in infected cells [38]). However, recent studies have demonstrated that viral infection can result in changes in expression of cellular miRNAs (9,24,33,42,45,55,58). Several reports have demonstrated that specific miRNAs can exert positive or negative influences on viral replica-tion through direct interaction with viral nucleic acid sequences (25,28,37,40), while other reports have demonstrated that individual miRNAs can affect viral replication via targeting cellular proteins (24,50).…”

MKRN1 Induces Degradation of West Nile Virus Capsid Protein by Functioning as an E3 Ligase

“…For example, WNV is known to induce Baxdependent or -independent apoptosis in cancer and brain cells (28). Factors such as NS2B-NS3 and capsid also enforce the cytotoxic effects of WNV through the apoptotic process (31). On the other hand, cellular components that protect host cells from WNV are not well known.…”