Mitochondrial Import of Dengue Virus NS3 Protease and Cleavage of GrpEL1, a Cochaperone of Mitochondrial Hsp70

Abstract

Dengue virus infections pose a significant threat to human health at present, which is reported from nearly 140 countries. The genome of this virus encodes three structural and seven non-structural (NS) proteins along with two un-translated regions, one each on both ends. Among them, dengue protease (NS3) plays a pivotal role in polyprotein processing and virus multiplication. NS3 is also known to regulate several host proteins to induce and maintain pathogenesis.Certain viral proteins are known to interact with mitochondrial membrane proteins and interfere with their functions. But the association of a virus-coded protein with the mitochondrial matrix is not known. In this report, by using in silico analysis, we show that NS3pro alone is capable of mitochondrial import; however, dependent on its innate mitochondrial transport signal (MTS). Transient transfection and protein import studies confirm the import of NS3pro to the mitochondrial matrix. Similarly, NS3pro-helicase (1-464 amino acids of NS3) also targets the mitochondria. Intriguingly, reduced levels of the matrix localized GrpEL1, a co-chaperone of mtHsp70, were noticed in NS3pro, NS3pro-helicase expressing, and virus-infected cells. Upon using purified components, GrpEL1 undergoes cleavage, and the sites have been mapped to KR81A and QR 92S. Importantly, the levels of GrpEL1 are seriously compromised in severe dengue infected clinical samples. Our studies provide novel insights into the import of NS3 into host mitochondria and identify a hitherto unknown factor, GrpEL1 as a cleavage target, and thereby providing new avenues for the dengue research and the design of potential therapeutics. IMPORTANCE About 40% of the world's population is at the risk of dengue virus infections. There is no specific drug or potential vaccine for these infections until now. Lack of complete understanding about the pathogenesis is one of the hurdles for developing antivirals for this virus infection. In the present study, we show that the virus-coded protease imports to the mitochondrial matrix, which is the first-ever report with reference to the animal and human viruses. The analysis indicated that the observed mitochondrial import is due to the inherited mitochondrial transport signal. We also show that the matrix localized GrpEL1, a co-chaperone of mtHsp70, is also the substrate of dengue virus protease, as observed in in vitro, ex vivo, virus-infected cells, and dengue virus-infected clinical samples. Hence our studies reveal an essential aspect of the pathogenesis of dengue virus infections, which may aid in developing anti-dengue therapeutics.

Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection

“…It may be noted that clustering of the viral complex is a prerequisite for the fragmentation of mitochondrial network. Previous studies have indicated that NS2B3 complex-induced cleavage of GrpEL1, a matrix protein is a major cause of mitochondrial matrix fragmentation [31]. GrpEL1 cochaperones are essential for the activity of mtHsp70, which is involved in various functions such as protein import, folding, redox homeostasis, and Fe-S cluster biogenesis [33].…”

“…The GrpEL1 protein, a cochaperone of the Hsp-70 protein, cleavage led to the dysfunction of the Hsp-70 protein, hampering overall protein folding. Thus, GrpE1 cleavage leads to mitochondria and mitochondrial-associated membranes (MAMs) disruption and micro-fusion (MFN1 and 2), causing mitochondrial fragmentation [31]. NS2B3 proteases induce mitochondrial disruption responsible for inducing apoptosis in human medulloblastoma cells.…”

Respiratory illness virus infections with special emphasis on COVID-19

“…During the convalescent phase, exhaustion markers (NKG2A, CD4+ T cells, B cells and NK cells) on lymphocytes get back to normal levels [156]. Several viruses, including coronaviruses, apply different strategies to escape from the host defense mechanism [122,157,158]. In this direction, SARS-CoV2 appears to possess a few unique characteristics which aid in fast-spreading and cause severe illness.…”

Potential Role of Flavivirus NS2B-NS3 Proteases in Viral Pathogenesis and Anti-flavivirus Drug Discovery Employing Animal Cells and Models: A Review

“…GrpEL1 protein functions as a cochaperon of the Hsp-70 protein, which implies that the cleavage of the GrpEL1 protein may lead to the dysfunction of the Hsp-70 protein. The exact consequences of this dysfunction are yet to be elucidated; however, based on the correlation between the cellular level of the GrpEL1 protein and the platelet count, the possible dysfunction of the mitochondria was postulated, which leads to thrombocytopenia [141].…”