Tài liệu Báo cáo khoa học: Minor capsid proteins of mouse polyomavirus are inducers of apoptosis

Minor capsid proteins of mouse polyomavirus are inducers

of apoptosis when produced individually but are only

moderate contributors to cell death during the late phase

of viral infection

Sandra Huerfano, Vojteˇ ch Zˇ ı´la, Evzˇen Bourˇa, Hana Sˇ panielova´, Jitka Sˇ tokrova´ and Jitka Forstova´

Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic

Keywords

apoptosis; minor proteins; mouse

polyomavirus; VP2; VP3

Correspondence

J. Forstova´, Department of Genetics and

Microbiology, Charles University in Prague,

Vinicˇna´ 5, 128 44 Prague 2, Czech Republic

Fax: +420 2 21951729

Tel: +420 2 21951730

E-mail: [email protected]

(Received 4 November 2009, revised 15

December 2009, accepted 22 December

2009)

doi:10.1111/j.1742-4658.2010.07558.x

Minor structural proteins of mouse polyomavirus (MPyV) are essential for

virus infection. To study their properties and possible contributions to cell

death induction, fusion variants of these proteins, created by linking

enhanced green fluorescent protein (EGFP) to their C- or N-termini, were

prepared and tested in the absence of other MPyV gene products, namely the

tumor antigens and the major capsid protein, VP1. The minor proteins linked

to EGFP at their C-terminus (VP2–EGFP, VP3–EGFP) were found to dis￾play properties similar to their nonfused, wild-type versions: they killed

mouse 3T3 cells quickly when expressed individually. Carrying nuclear locali￾zation signals at their common C-terminus, the minor capsid proteins were

detected in the nucleus. However, a substantial subpopulation of both VP2

and VP3 proteins, as well as of the fusion proteins VP2–EGFP and VP3–

EGFP, was detected in the cytoplasm, co-localizing with intracellular mem￾branes. Truncated VP3 protein, composed of 103 C-terminal amino acids,

exhibited reduced affinity for intracellular membranes and cytotoxicity.

Biochemical studies proved each of the minor proteins to be a very potent

inducer of apoptosis, which was dependent on caspase activation. Immuno￾electron microscopy showed the minor proteins to be associated with

damaged membranes of the endoplasmic reticulum, nuclear envelope and

mitochondria as soon as 5 h post-transfection. Analysis of apoptotic markers

and cell death kinetics in cells transfected with the wild-type MPyV genome

and the genome mutated in both VP2 and VP3 translation start codons

revealed that the minor proteins contribute moderately to apoptotic pro￾cesses in the late phase of infection and both are dispensable for cell destruc￾tion at the end of the virus replication cycle.

Structured digital abstract

l MINT-7386399, MINT-7386463, MINT-7386515: VP3 (uniprotkb:P03096-2) and GRP94

(uniprotkb:P08113) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

l MINT-7386328, MINT-7386434, MINT-7386493: VP2 (uniprotkb:P03096-1) and GRP94

(uniprotkb:P08113) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

l MINT-7386294, MINT-7386413, MINT-7386482: VP2 (uniprotkb:P03096-1) and Lamin-B

(uniprotkb:P14733) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

l MINT-7386354, MINT-7386450, MINT-7386504: VP3 (uniprotkb:P12908-2) and Lamin-B

(uniprotkb:P14733) colocalize (MI:0403) by fluorescence microscopy (MI:0416)

Abbreviations

CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; FACS, fluorescence-activated cell sorting;

LDH, lactate dehydrogenase; MPyV, mouse polyomavirus; PARP, poly(ADP-ribose) polymerase; SV40, simian virus 40; tVP3, truncated VP3;

Z-VAD-FMK, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone.

1270 FEBS Journal 277 (2010) 1270–1283 ª 2010 The Authors Journal compilation ª 2010 FEBS