Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells

Abstract

Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER-Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261-3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

Fig. 1

Capsase-12 is activated earlier than caspase-8 and caspase-3. Vero E6 cell lysates following TULV infection on the indicated day p.i. were collected for immunoblotting and analyzed with antibodies against caspase-12, caspase-8, and caspase-3, respectively. The cleavage of caspase-12 took place on the fourth day p.i., while caspase-8 and caspase-3 were activated on the fifth day p.i.

Fig. 2

Activation of JNK pathway. (A) Mock-treated or Tula virus-infected cell lysates were resolved in SDS–PAGE and transferred onto nitrocellulose, the membranes were then treated (on the right) or untreated with CIP. (B) TULV-infected or mock Vero E6 cell lysates were collected at the indicated day p.i. and analyzed with antibodies against c-jun (rabbit monoclonal and rabbit polyclonal). Infection was monitored with antibody against nucleocapsid protein. β-actin was used as a loading control. (C) JNK inhibitor II SP600126 (50 μM, added every other day) blocked the cleavage of PARP in Vero E6 cells on the fifth day post-Tula virus infection.

Fig. 3

Gadd153/chop is induced during TULV infection. (A) Increased protein level of Gadd153/chop following infection. (B) Induction of Gadd153/chop at the late stage of TULV infection shown by conventional RT-PCR. (C) Real-time PCR confirms the transcriptional regulation of Gadd153/chop on the fifth day of TULV infection. β-actin (in A) and GAPDH (in B and C) were as controls.

Fig. 4

Changes in the integral ER membrane protein BAP31 shown by immunoblot. (A) Rabbit polyclonal antibody against BAP31 shows the decreased amount of the full-length and p20 fragment of Bap31 while a p10 fragment of Bap31 started to accumulate. (B) A specific monoclonal antibody against BAP31 confirms the down-regulation of the full-length of and p20 fragment BAP31.

Fig. 5

Increased protein and RNA expression of Grp78/BiP during Tula virus infection. (A) Grp78/BiP protein expression. (B) Increased mRNA of Grp78/BiP shown by semi-quantitive RT-PCR. (C) Increased mRNA of Grp78/BiP shown by real-time PCR. (D) Replication is required for ER stress. β-actin (in A and D) and GAPDH (in B and C) were as controls.

Fig. 6

The diagram shows a hypothesized model for Tula hantavirus-induced apoptosis.