Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection.
Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca(2+) homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61-83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India.
Rahul
R
Bhowmick
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India.
Umesh Chandra
UC
Halder
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India.
Shiladitya
S
Chattopadhyay
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India.
Shampa
S
Chanda
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India.
Satabdi
S
Nandi
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India.
Parikshit
P
Bagchi
Department of Zoology, University of Calcutta, Ballygunge, Kolkata 700019, India.
Mukti Kant
MK
Nayak
Structural Genomics Section, Saha Institute of Nuclear Physics, Kolkata 700064, India.
Oishee
O
Chakrabarti
Department of Hygiene, Sapporo Medical University, Sapporo 060-8556, Japan.
Nobumichi
N
Kobayashi
Division of Virology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road Scheme-XM, Beliaghata, Kolkata 700010, India. Electronic address: chawlam70@gmail.com.
Mamta
M
Chawla-Sarkar
D000818:Animals; D017209:Apoptosis; D002118:Calcium; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D012400:Rotavirus Infections / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014118:Toxins, Biological / Q000096:biosynthesis* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis* / Q000235:genetics
D002118:Calcium; D020169:Caspases; D045304:Cytochromes c; D004768:Enterotoxins; D006023:Glycoproteins; D000226:Mitochondrial ADP, ATP Translocases; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; C081369:NS28 protein, rotavirus; C104282:nsp1 protein, Rotavirus