Morbillivirus Canis Infection Induces Activation of Three Branches of Unfolded Protein Response, MAPK and Apoptosis

Abstract

Morbillivirus canis, commonly named Canine distemper virus (CDV), is a morbillivirus implicated in several signs in the Canidae family. In dogs (Canis lupus familiaris), common signs of infection include conjunctivitis, digital hyperkeratosis and neuropathologies. Even with vaccination, the canine distemper disease persists worldwide so the molecular pathways implicated in the infection processes have been an interesting and promising area in new therapeutic drugs research in recent years. It is known that in the process of virus infection, the endoplasmic reticulum (ER) loses its homeostasis, inducing stress and the subsequent unfolded protein response or UPR in which three ER-trans-membrane proteins are implicated: PERK, IRE1 and ATF6. Moreover, in prolonged ER stress, the apoptosis is induced through the CHOP, as a final step of viral infection. Cell culture and molecular techniques such as RT-qPCR and RT-PCR were used in the present study. We demonstrate the activation in vitro of the three UPR pathways after infection with an attenuated strain of CDV. Also, the implication of a MAPK pathway through the p38 protein and the apoptotic CHOP was demonstrated to contribute to the process of infection. Even more, our study suggested that CDV replication occurs in a PERK-dependent manner.

Keywords: ER stress; UPR pathways; apoptosis; canine distemper virus.

Figure 1

Activation of BIP in VERO CCL-81 cells. An RT-qPCR assay was conducted to evaluate the expression of the GRP78/BIP transcript following the infection with CDVOnd. The induction of ER stress was detected at 8 hpi, reaching its maximum value at 16 hpi. The levels of BIP mRNA were relativised to the mock experiment and represented as the mean ± standard deviation (SD) of three independent experiments. For statistical significance, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 2

Activation of the PERK pathway in CDVOnd infection. The relative levels of the ATF4 transcript indicate the activation of the PERK pathway following 16 hpi with the CDVOnd strain. The data were relativised to the mock well and are presented as means ± SD from three independent experiments. Statistical significance * p < 0.05, *** p < 0.001.

Figure 3

The ATF6 pathway is activated by the CDVOnd strain. The ATF6 mRNA levels were detected as early as 8 hpi. All values were relativised to the mock experiment. The values are presented as means ± SD of three independent experiments. Statistical significance values are indicated by * p ˂ 0.05 and ** p ˂ 0.01.

Figure 4

XBP1 spliced after CDVOnd infection. (a) The images displayed are representative photographs of 2% agarose gels with GAPDH (left) and XBP1 (right) bands for each condition from the same experiment. The triangles indicate the XBP1 transcripts, XBP1u (green) and XBP1s (blue) with a size of approximately 440 bp and 414 bp, respectively. The star symbol denotes a hybrid between the two forms of XBP1 (XBP1H), with an apparent size of 466 base pairs. (b,c) Band densitometry assay. The expression levels of XBP1 species were relativised using a value of 1 for the GAPDH bands. In (b), a comparison was made between the levels of XBP1u and XBP1s. In (c), the ratio XBP1s/XBP1u relativised to the mock condition was evaluated in each condition. Levels are represented as means ± SD of three independent experiments. In all cases, statistical significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 5

p38⍺ and CHOP induced by CDVOnd infection. The figure illustrates the relative levels of mRNA transcripts for two transcription factors involved in the activation of apoptotic genes. (a) Levels of the p38α mRNA transcript were found to be upregulated following CDV infection. (b) Levels of the CHOP transcript in response to CDV infection, illustrating the upregulation of this gene. The data were relativised to the mock experiment and expressed as means ± SD from three independent experiments. Statistical significance was determined using the following criteria: * p < 0.05, ** p < 0.01.

Figure 6

DNA laddering experiment after CDV infection. The study of apoptosis was conducted through the generation of DNA ladder-like patterns over a period of 16, 24, 48 and 72 h following the infection of CDVOnd. A 100 bp ladder (MW) was employed as a weight molecular marker. (a) Photographs taken at 4X of the mock and 48 hpi (up) and 72 hpi (down) wells. (b) Electrophoretic analysis of genomic DNA in a 1% agarose gel stained with GelGreen.

Figure 7

Production of viral RNA following CDV infection. The relative RNA level of the CDVOnd strain was investigated using primers to the N gene at different time points. The levels of the viral genome were relativised to the mock experiment and data are presented as means ± SD of three independent experiments. For a statistical significance: * p ˂ 0.05, *** p ˂ 0.001.

Figure 8

Inhibition of the PERK pathway affects viral progeny. Both experiments were conducted in duplicate in two conditions: without the PERK inhibitor (wo/GSK) and with the PERK inhibitor (w/GSK). (a) The TCID₅₀ assay was performed to quantify CDVOnd progeny at 48, 72 and 96 hpi. (b) Viral load performed by qPCR targeted the N gene of CDV. The mRNA levels were relativised to mock conditions.