The SAT Protein of Porcine Parvovirus Accelerates Viral Spreading through Induction of Irreversible Endoplasmic Reticulum Stress

Abstract

The SAT protein (SATp) of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER), and SAT deletion induces the slow-spreading phenotype. The in vitro comparison of the wild-type Kresse strain and its SAT knockout (SAT^-) mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SAT^- virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences in the quantity and the localization of CHOP were detected, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of the induction of irreversible ER stress in porcine testis (PT) cell necrosis and viral egress was confirmed by treatment of infected cells by ER stress-inducing chemicals (MG132, dithiothreitol, and thapsigargin), which accelerated the egress and spreading of both the wild-type and the SAT^- viruses. UV stress induction had no beneficial effect on PPV infection, underscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a coexpression experiment.IMPORTANCE SATp is encoded by an alternative open reading frame of the PPV genome. Earlier we showed that SATp of the attenuated PPV NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that slow spreading is a general feature of SAT^- PPVs and is the consequence of prolonged cell integrity. PPV infection induced ER stress in infected cells regardless of the presence of SATp, as demonstrated by the morphological changes of the ER and expression of the stress response proteins Xbp1 and CHOP. However, the presence of SATp made the ER stress more severe and accelerated cell death during infection, as shown by the higher rate of expression of CHOP and alteration of the localization of CHOP. The beneficial effect of irreversible ER stress on PPV spread was confirmed by treatment of infected cells with ER stress-inducing chemicals.

Keywords: CHOP; ER stress; SAT; Xbp1; alternative ORF; parvovirus; protoparvovirus; viral egress.

FIG 1

DNA and amino acid sequences of the wild-type (A) and the SAT⁻ mutant (B) Kresse strains (based on the U44978.1 sequence). The 2842nd and 2845th nucleotides were changed (T → A and T → C). These modifications did not change the amino acid sequence of VP1; however, they led to a stop codon in the SAT ORF and to the substitution of the nearest methionine.

FIG 2

Spread of the wild-type and the SAT⁻ mutant viruses in PT cells at a low multiplicity of infection (MOI, 0.01). Infected cells (red) were visualized with the assembled capsid-specific 3C9 primary antibody and the CF594 secondary antibody. (A) Cells were fixed at the indicated time points, and their nuclei were labeled with Hoechst 33342. (B) 3C9 antibody was added to Kresse-infected cells to monitor the inhibition of the secondary infections. The times of the beginning of the treatments are indicated. The cells were fixed at 24 h p.i.

FIG 3

Change of the viral copy numbers during infection in the medium of PT cells. (A) Copy numbers and infectious titers (triangles and columns, respectively) at a low multiplicity of infection (MOI, 0.01). (B) Total copy numbers and copy numbers measured after DNase treatment (triangles and columns, respectively) at a high multiplicity of infection (MOI, 3). Error bars indicate standard deviations.

FIG 4

Different forms of cell death during PPV Kresse infection in porcine testis cells. The error bars represent 1 standard deviation. (A) LDH activity in the supernatant of infected and control cells as an indicator of total cell death. The maximum absorption value (lysed control uninfected cells) was 1.79 at 18 h p.i. (B) Attached cell count as an indicator of viability of cells. The number of uninfected cells at 24 h represents 100%. (C) Proportion of attached propidium iodide-positive (PI⁺) cells (the values for wt virus-infected cells were calculated until 48 h p.i.). (D) Proportion of attached pyknotic and karyorrhectic cells, calculated by Hoechst staining (values for wt virus-infected cells were calculated until 48 h p.i.). (E) Swelling of nuclei at 22 h p.i.

FIG 5

Spreading of the PPV wild-type and SAT⁻ strains after ER stress inducer treatments at a low MOI (MOI, 0.01). Infected PT cells were fixed at 20 h p.i. The 3C9 anticapsid monoclonal antibody was used for the detection of infected cells (red). Cell nuclei (blue) were visualized by Hoechst staining. The concentrations of chemicals and the duration of treatments (for the indicated times [in hours] postinfection) are indicated.

FIG 6

Changes of viral copy numbers in the medium at a high multiplicity of infection (MOI, 3) after stress inducer treatments. All supernatants were harvested at 24 h p.i. (A) Cells were infected with the wild-type and SAT⁻ strains and treated with ER stress inducers (20 μM MG132 and 10 mM DTT) for different times. (B) Cells were infected with the wild-type and SAT⁻ strains and treated with UV-C light (sublethal dose) at different times p.i. for 5 min. (C). Infectious titer of supernatants of differently treated infected cells.

FIG 7

Morphological changes of the ER in PT cells during PPV infection. To visualize the ER and the viral particles, anticalreticulin antibody (red) and anticapsid (green) antibodies, respectively, were used.

FIG 8

Detection of ER stress protein markers during PPV infection. Cell nuclei (blue) were visualized by Hoechst staining. (A) Activation of the Xbp1 reversible ER stress marker after wt and SAT⁻ strain infection. Xbp1 (green) and viral capsid (red) were labeled with anti-Xbp1 and anticapsid antibodies, respectively, at 18 h p.i. (B) Activation of the CHOP irreversible ER stress marker after wt and SAT⁻ strain infection. CHOP (green) and viral capsid (red) were labeled with anti-CHOP and anticapsid antibodies, respectively, at 24 h p.i. (C) Activation of Xbp1 in SAT⁻ virus-infected and noninfected cells at 60 h p.i. (D) Induction of Xbp1 and CHOP after 20 μM MG132 treatment and sublethal UV treatment at 12 h.

FIG 9

Morphological changes of the ER in SAT-DsRed fusion protein-expressing PT cells. Cells were transfected with a SAT-DsRed-expressing plasmid and, as a control, with a DsRed (red)-expressing plasmid. The calreticulin was labeled by anticalreticulin monoclonal antibody (green), and the cell nuclei (blue) were visualized by Hoechst staining. The cells were fixed at different times after transfection. Arrows, apoptotic nuclei.

FIG 10

Localization of porcine CHOP and its effect on the spreading of the SAT⁻ virus strain. The viral capsid was labeled with anticapsid antibodies (green), while the nuclei (blue) were visualized by Hoechst staining. (A) Nuclear localization of the CHOP-DsRed fusion protein (red) in infected and noninfected cells. CHOP-DsRed-expressing plasmids were transfected with wild-type and pSAT⁻ infectious clones or alone into PT cells and fixed at 24 h posttransfection. (B) Cytopathic effect of CHOP-DsRed at 18 h posttransfection. Arrow, fragmented nuclei. (C) Spreading of the SAT⁻ strain after cotransfection of the pSAT⁻ infectious clone with CHOP-DsRed- and SAT-DsRed-expressing plasmids. As a positive control, the wild-type clone was transfected with DsRed-expressing plasmid. Cells were fixed at 96 h posttransfection, and the blue (nucleus) and green (virus-positive cells) channels were merged.