Modulation of apoptosis by V protein mumps virus
© Rosas-Murrieta et al; licensee BioMed Central Ltd. 2011
Received: 17 December 2010
Accepted: 13 May 2011
Published: 13 May 2011
The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway.
We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process.
The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals.
The IFN type-I pathway is the major cellular mechanism of the antiviral response. The effect is the induction of gene expression to blocking the viral infection. The efficient antiviral cellular response promotes the development of viral strategies to antagonize the effect of IFN [1–4]. In the paramyxoviruses, inhibition of IFN type-I response occurs due to the activity of the nonstructural V protein [5, 6]. Activation of the JAK-STAT pathway by IFN simultaneously activates others processes regulated by IFN such as apoptosis, a physiological process where cells undergo morphological changes, activation of proteases, nuclear DNA fragmentation and cell death [1, 2]. The central component of the apoptotic machinery is a proteolytic system consisting of the family of cysteine proteases (caspases) [7–9]. Apoptosis can be initiated and executed through many different pathways, which can be categorized into two main groups: extrinsic and intrinsic [8, 9]. Sequential activation of a caspase by another creates an expansive cascade of proteolytic activity that produces digestion of structural proteins in the cytoplasm, DNA degradation and phagocytosis of apoptotic bodies . Because apoptotic cells are rapidly phagocytosed, apoptosis promotes development of an efficient immune response against viral antigens . Many viruses have evolved mechanisms to avoid or at least to control apoptosis . One of the mechanisms of pathogenicity of mumps virus is V protein expression required to blocking the expression of viral genes activated by IFN through cytoplasmic interaction with the STAT1-STAT2 heterodimer [13, 14]. What cellular process is the prime target to promote viral replication? Effects of the inactivation of the IFN pathway on apoptosis, in particular, are not known in detail. IFN-α induces apoptosis and stimulates the activity of caspases 1, 2, 3, 8 and 9 and promotes the extrinsic apoptotic pathway and the activation of caspase 8 as the initiator of the caspase cascade to execute apoptosis .
In this study we analyzed the effect of the VWT ( from the HN-A1081 population, neurovirulence associated) and VGly (from the HN-G1081 population) proteins of the Urabe AM9 strain vaccine of mumps virus [16–18], in order to determine whether, as the simian virus 5 V protein  and the C protein parainfluenza virus type I (HPIV-1) , they have the capacity of blocking IFN-α-induced apoptosis. VWT and VGly of the Urabe AM9 strain were expressed in cervical adenocarcinoma cells to analyze its effect on the activity of initiator and effector caspases. The cells were transfected with 10 μg of vector DNA (pCDNA4/His/Max-VA and VG) and TurboFect transfection reagent (Fermentas, Glen Burnie, MD, USA). 36 h after transfection, the cells were treated with 4000 IU/mL of IFN-α2b (Urifrón, Probiomed, Mexico) and 40 μM of MG-132 (Sigma, St. Louis, MO, USA). The activity of caspases 3, 7 and 8 was evaluated with the Caspase-Glo 3/7 kit and Caspase-Glo 8 kit (Promega, Madison, WI, USA) using the substrate Ac-DEVD-pNA for caspase 3/7 or a C-LETD-pNA for caspase 8 and incubated for 60 min at room temperature. The activity of caspases 3, 7 and 8 was measured by a GloMax 20/20 luminometer (Promega, Madison, WI, USA). The activity of caspase 9 was measured with the Caspase 9 Colorimetric Assay Kit (Biovision) using LEDH-pNA as substrate by absorbance at 420 nm, 2 h after adding substrate.
On the other hand, cytoplasmic replication of mumps virus produces dsRNA molecules that may activate the intrinsic apoptotic pathway by intracellular stress and the V proteins of mumps virus may have an effect on apoptosis that involves the intrinsic or mitochondrial pathway. In the system without IFN-α2b treatment, it was demonstrated that the enzyme activity increased 51% in cells that express the VWT and 8% in cells with VGly (Figure 1B). Treatment of the C33 cell line with IFN-α2b activates the mitochondrial apoptotic pathway detected by an increase of 107% in caspase 9 activity. In contrast, in the IFN-treated cells that express VWT and VGly proteins, the activity of caspase 9 decreased 35% and 38%, respectively (Figure 1B), suggesting the activation of the apoptotic mitochondrial pathway, which may be reduced by expression of V proteins of the mumps virus regulating the magnitude of apoptosis. Although data are obtained for detection of caspase 9, a high activation is not observed as with caspase 8. If there is strong activation of the intrinsic apoptotic pathway by another factor of mumps virus in combination with the V protein, this must be analyzed in further studies.
Caspases 8 and 9 are directly responsible for activation of the effector caspases 3 and 7. Their activity was analyzed in cells non-transfected and without stimulation with IFN-α2b, where we found that there is a low level of enzymatic activation probably due to the introduction of DNA plasmid (pCDNA4/HisMaX), whereas in cells that express VWT and VGly proteins there is an increase in the activity of caspase 3 and 7 of 457% and 526%, respectively (Figure 1C). When the system was stimulated with IFN-α2b, the activity of caspases 3 and 7 increased 77%, 91% y 76% in control cells, and with the VWT and VGly proteins, respectively, compared with to the system without treatment. A detailed comparison of caspases 3 and 7 activity with or without IFN shows that V proteins partially inhibit the activity, although there was no statistically significant difference on enzymatic activity in both proteins. These data suggest a partial inhibitory activity of 61% and 49%, respectively, on the IFN-α-induced apoptosis by the VWT and VGly proteins of mumps virus (Figure 1C). Despite activation of the IFN pathway, activity of caspases 3 and 7 decreased compared to cells that did not express the viral protein, suggesting a role for the V protein in the disruption of the antiviral response controlling the apoptosis. However, V proteins of Urabe AM9 vaccine strain did not completely inhibit caspase activity in our study and, therefore, the apoptosis. Despite the partial inhibition of caspases 8, 3 and 7, the reported residual activity suggests that nuclear DNA fragmentation, a key process in apoptosis, should be carried out.
Finally, our data provide evidence with regard to the VWT protein of the Urabe AM9 vaccine strain of mumps virus, which seems to exert a best modulatory role on apoptosis and cell proliferation with regard to the VGly protein. This suggests that, in viral pathogenesis, the regulation of apoptosis potentially mediates the damage. The detailed mechanism for the implementation of such activity should be studied in a future investigation. Information of the identity of caspases induced or inhibited by some factor in the mumps virus infection is important in order to establish the molecular mechanism of the virus in the intervention of apoptosis. This knowledge may suggest likely targets for antiviral development.
This work was supported by SEP-PROMEP (Grant 103.5/07/2594). We thank QFB Nelby Ivonne Cruz Saviñón and QFB William Toledo Rueda for their technical assistance.
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