The effect of PRRSV and <i>HDAC6</i> overexpression on α-tubulin acetylation.

<p>Kinetic analysis of JXA1-induced α-tubulin acetylation in MARC-145 cells (A) and in untreated cells (B). Kinetic analysis of JXA1-induced α-tubulin acetylation in PAMs (C) and in untreated cells (D). Kinetic analysis of JXA1-induced α-tubulin acetylation in HDAC6-transfected MARC-145 cells (E) and in untreated cells (F). The α-tubulin acetylation or α-tubulin content was quantified and is presented as a ratio relative to the total amount of GAPDH (Actub/GAPDH or tub/GAPDH).</p

Overexpression of Histone Deacetylase 6 Enhances Resistance to Porcine Reproductive and Respiratory Syndrome Virus in Pigs

<div><p>Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically relevant viral pathogens in pigs and causes substantial losses in the pig industry worldwide each year. At present, PRRSV vaccines do not effectively prevent and control this disease. Consequently, it is necessary to develop new antiviral strategies to compensate for the inefficacy of the available vaccines. Histone deacetylase 6 (HDAC6) is an important member of the histone deacetylase family that is responsible for regulating many important biological processes. Studies have shown that HDAC6 has anti-viral activities during the viral life cycle. However, whether <i>HDAC6</i> overexpression enhances resistance to PRRSV in pigs remains unknown. In this study, we used a somatic cell cloning method to produce transgenic (TG) pigs that constitutively overexpress porcine <i>HDAC6</i>. These TG pigs showed germ line transmission with continued overexpression of HDAC6. <i>In vitro</i>, virus-challenged porcine alveolar macrophages (PAMs) overexpressed <i>HDAC6</i>, which suppressed viral gene expression and PRRSV production. <i>In vivo</i>, resistance to PRRSV in TG pigs was evaluated by direct or cohabitation mediated infection with a highly pathogenic PRRSV (HP-PRRSV) strain. Compared with non-TG (NTG) siblings, TG pigs showed a significantly lower viral load in the lungs and an extended survival time after infection with HP-PRRSV via intramuscular injection. In the cohabitation study, NTG pigs housed with challenged NTG pigs exhibited significantly worse clinical symptoms than the other three in-contact groups. These results collectively suggest that HDAC6 overexpression enhances resistance to PRRSV infection both <i>in vitro</i> and <i>in vivo</i>. Our findings suggest the potential involvement of HDAC6 in the response to PRRSV, which will facilitate the development of novel therapies for PRRSV.</p></div

Transgenic pigs developed a lower level of viremia and had a lower virus load in the lungs.

<p>(A) Survival curves for the pigs in the two challenged groups after infection with PRRSV JXA1. The pigs in challenged TG group survived for a longer period than the pigs in the challenged NTG group (log rank test; P = 0.328). Group: challenged TG group, n = 6; challenged NTG group, n = 6. (B) Rectal temperature curves for the pigs from the two challenged groups after infection with PRRSV JXA1. (C) Body weight curves for pigs in the two challenged groups after infection with PRRSV JXA1. (D) Analysis of the viral loads in serum samples obtained from challenged TG and challenged NTG pigs at the indicated times. (E) Viral load in the lungs. The samples were collected from the lungs of dying challenged TG and challenged NTG pigs. The data in panels B, C, D and E are presented as the mean±SD. The data in panels B, C, D and E were analyzed using Student’s t-test. *, P<0.05.</p

HDAC6 Overexpression enhanced resistance to infection during cohabitation with PRRSV-infected pigs.

<p>(A) The survival curves of the pigs housed with challenged pigs (log rank test; P = 0.153). n = 5 for each group. (B) Rectal temperature curves for the 4 groups housed with challenged pigs. (C) Body weight curves for the 4 groups housed with challenged pigs. The data were analyzed using ANOVA, which revealed that the body weights of the pigs in the NTG/NTG group were significantly lower than the body weights of the other groups after 5 dpi (P<0.05). (D) Analysis of the viral load in the serum samples obtained from pigs in the 4 housed groups at 4 dpi. (E) Antibody positive tests (S/P>0.4) indicating the level of the immune response <i>in vivo</i>. (F) Survival curves for the two groups of pigs that were housed in two separate rooms (log rank test; P = 0.712). (G) Body weight curves for pigs in the two groups that were housed in two separate rooms. The body weights of the pigs housed with NTG pigs were significantly higher than those of pigs housed with TG pigs at 6, 9 and 10 dpi. (H) Rectal temperature curves for the pigs from the two groups that were housed in two separate rooms. The rectal temperatures of the pigs housed with NTG pigs were significantly higher than were those of pigs housed with TG pigs at 2 and 3 dpi. (I) Survival curves for the housed pigs with different genotypes (log rank test; P = 0.046). (J) Rectal temperature curves for the housed pigs with different genotypes. (K) Body weight curves for the housed pigs with different genotypes. The data in panels B, C and D are presented as the mean±SD. The data in panels B, C and D were analyzed using ANOVA. The data in panels G, H, J, and K are presented as the mean±SE. The statistical significance of these data was analyzed using a t-test. *, P<0.05; **, P<0.01.</p

Determination of HDAC6 expression in F1 generation transgenic pigs.

<p>(A) qRT-PCR analysis of exogenous <i>HDAC6</i> expression in the lungs and skin of F1 TG (n = 9) and sibling NTG pigs (lung, n = 10; skin, n = 6) using the Q-GFP-F/R primer pair. The data are presented as the mean ± SD. The relative expression of <i>GFP</i> was calculated using the 2^-Δct method. (B) qRT-PCR analysis of <i>HDAC6</i> expression in lungs of F1 TG (n = 9) and sibling NTG pigs (n = 10) using the Q-HDAC6-F/R primer pair. (C) qRT-PCR analysis of HDAC6 expression in the skin of F1 TG (n = 9) and sibling NTG pigs (n = 6) using the Q-HDAC6-F/R primer pair. The data are presented as the mean±SD. The mRNA relative expression of total HDAC6 was calculated using the 2^-ΔΔct method (RNA from NTG pigs was used as the reference sample). GAPDH was used as an internal qRT-PCR control. Statistical significance was analyzed using a t-test. ***, P<0.001 **; P<0.01. (D) Western blot analysis of F1 pigs. The samples were collected from lung biopsies of six TG pigs and three sibling NTG pigs. The protein samples were probed with an anti-GFP antibody. GAPDH was used as an internal control for western blot analysis.</p

<i>HDAC6</i> Overexpression inhibits viral gene expression and PRRSV production in PAMs.

<p>(A) qRT-PCR analysis of <i>HDAC6</i> expression in PAMs isolated from pigs using the Q-HDAC6-F/R primer pair. <i>HDAC6</i> expression is presented as a ratio relative to the level of <i>GAPDH</i>. The data are presented as the mean±SD from three independent experiments. (B) Western blot analysis of PAMs isolated from F1 pigs. The protein samples were probed with anti-Actub and GAPDH antibodies. (C) qRT-PCR analysis of viral <i>ORF7</i> RNA levels in TG and NTG PAMs that were inoculated with the PRRSV strain CH-1a (MOI = 0.5) for 24, 48 and 72 h. The data represent the results of three independent experiments (mean±SD). (D) qRT-PCR analysis of viral <i>ORF7</i> RNA levels in TG and NTG PAMs that were inoculated with the PRRSV strain JXA1 (MOI = 0.25) for 24 and 48 h. The data are presented relative to the expression of <i>GAPDH</i> mRNA and represent the results of three independent experiments (mean±SD). RNA from NTG PAMs at 24 hpi was used as the reference sample. Statistical significance was analyzed using a t-test. *, P<0.05; **, P<0.01; ***; P<0.001. (E) The supernatant containing PRRSV RNA was analyzed based on absolute quantitative RT-PCR values at the indicated time points. PAMs were infected with PRRSV CH-1a (MOI = 0.05), and the supernatant was collected and used for RNA extraction and absolute qPCR analysis of the virions at 24, 48 and 72 hpi. The data are representative of the results of three independent experiments (mean±SD). Statistical significance was analyzed using Student’s t-test. **, P<0.01; ***, P<0.001.</p

Production and identification of F0 TG pigs.

<p>(A) Schematic diagram of the transgenic vector (pCMV-pHDAC6-Puro). The pair of black arrows indicates the primers used to identify the transgenic insert in TG pigs and in fibroblast colonies. The pair of green arrows indicates the primers used to identify exogenous <i>HDAC6</i> mRNA in pigs. The pair of red arrows indicates the primers used to determine total <i>HDAC6</i> mRNA levels in F1 pigs. (B) PCR assay to detect founder transgenic pigs. The PCR product (771 bp) was the GFP tag of the inserted transgene, which was amplified using the HDAC6-F/R primer pair. M, 100 bp DNA ladder; Lanes 1–9, founders No. 1–9; w, water; P, plasmid control; N, wild-type pig genomic DNA, used as the negative control. (C) qRT-PCR analysis of exogenous <i>HDAC6</i> expression in different tissues (blood, liver, kidney, skin, lung, intestine and spleen) of the F0 transgenic pig using the Q-GFP-F/R primer pair. The blood sample data are presented as the mean±SD from 3 individuals (No. 1, 2 and 3). The data from other tissues are presented as the mean±SD from 3 repeated experiments. RNA from the intestines was used as the reference sample. WT, wild-type control. (D) Western blot analysis of F0 transgenic pigs. The samples were collected from tissues of TG (No.1) and WT pigs. The protein samples were probed with an anti-GFP antibody.</p

Mutation of the key interface residues in nectin-1 undermines the interaction with PRV gD.

<p>(A) SPR tests of the binding between nectin-1 mutants and PRV gD337. The kinetic profiles are recorded and shown. (B) Decreased cell fusion with the mutated nectin-1 receptors. CHO-K1 cells expressing PRV gD/gB/gH/gL and T7 luciferase were mixed and incubated with those expressing T7 polymerase in combination with wild type or mutant HU-nectin-1. The histogram shows the efficiencies of cell fusion with the indicated nectin-1 mutants in comparison to that with the wild type receptor. The results are expressed as means ± SD from three independent experiments.</p

Mutation of the key interface residues in nectin-1 undermines the interaction with PRV gD.

<p>(A) SPR tests of the binding between nectin-1 mutants and PRV gD337. The kinetic profiles are recorded and shown. (B) Decreased cell fusion with the mutated nectin-1 receptors. CHO-K1 cells expressing PRV gD/gB/gH/gL and T7 luciferase were mixed and incubated with those expressing T7 polymerase in combination with wild type or mutant HU-nectin-1. The histogram shows the efficiencies of cell fusion with the indicated nectin-1 mutants in comparison to that with the wild type receptor. The results are expressed as means ± SD from three independent experiments.</p

Structure of the PRV-gD/SW-nectin-1 complex.

<p>(A, B) Cartoon representation of the overall structure. The gD molecule is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006314#ppat.1006314.g003" target="_blank">Fig 3A</a>, and the membrane-distal IgV domain of SW-nectin-1 is shown in cyan. Those elements referred to in the text, including the secondary structure elements of SW-nectin-1 IgV and the interface elements in PRV gD, are labeled. The free PRV gD structure (in gray) was also aligned to the complex structure in (B) to highlight the reorientation of the gD N-terminal loop upon receptor binding. (A) The complex structure of PRV gD bound to SW-nectin-1. (B) The same complex structure that is shown after horizontal rotation of about 180 degrees. (C) Comparison of the PRV-gD/SW-nectin-1 (PRV gD in green and SW-nectin-1 is cyan) complex structure with previously reported HSV-1-gD/HU-nectin-1 (HSV-1 gD in yellow and HU-nectin-1 in orange) and HSV-2-gD/HU-nectin-1 (HSV-2 gD in magenta and HU-nectin-1 in gray) complex structures. The CC' loop of variant conformations in nectin-1, the 3.5 Å shift between the bound PRV and HSV gDs, and the unique α2' helix in PRV gD bulged towards the CC' loop of nectin-1 are highlighted and labeled. For clarity, the view of the structure in panel (C) is clockwise rotated along the vertical axis for about 90 degrees relative to that in panel (B).</p