Figure 1.
Phylogenetic relationships of all the fish, chicken, mouse and human Mx protein sequences in GenBank
(Danio rerio MxD and MxF sequences are not included due to just partial sequences available). Maximum-likelihood phylogenetic tree generated from a MAFT alignment and MEGA 5.1 program. Haliotis discus discus Mx was employed as the outgroup. The bar indicates the distance. CiMx1, CiMx2 and CiMx3 were marked with triangle (▴), diamond (♦) and circle (•), respectively. The protein IDs are as follows: Ctenopharyngodon idella Mx1 ADU33870, Mx2 AAQ95584, Mx3 ADZ44601; Carassius auratus Mx1 AAP68828, Mx2 AAP68827; Danio rerio MxA NP_891987, MxB Q800G8, MxC NP_001007285, MxE NP_878287, MxG CAD67761; Dicentrarchus labrax Mx AAR99718; Epinephelus fuscoguttatus Mx ADE80885; Epinephelus coioides Mx1 ABD95979, Mx2 ABD95982; Gobiocypris rarus Mx ABL61237; Gallus gallus Mx CAA80686; Homo sapiens Mx1 NP_001171517, Mx2 NP_002454; Hippoglossus hippoglossus Mx AAF66055; Ictalurus punctatus Mx1 Q7T2P0, Mx2 AAY33864; Lates calcarifer Mx AAW22002; Larimichthys crocea Mx ABJ56003; Mus musculus Mx1 NP_034976, Mx2 NP_038634; Oplegnathus fasciatus Mx1 ACF75866, Mx2 ACF75867, Mx3 ACF75868; Oncorhynchus mykiss Mx1 AAA87839, Mx2 AAC60214, Mx3 AAC60215; Paralichthys olivaceus Mx BAC76769; Scophthalmus maximus Mx1 AAT57877, Mx2 AAT57878; Sparus aurata Mx1 ACK99554, Mx2 ACK99553, Mx3 ACN22085; Salmo salar Mx1 AAB40994, Mx2 AAB40995, Mx3 AAB40996; Solea senegalensis Mx AAV49303; Siniperca chuatsi Mx AAQ91382; Takifugu rubripes Mx AAO37934; Haliotis discus discus Mx ABI53802.
Table 1.
Sequence identities of the deduced amino acid sequences of Mx genes among fishes and human with serial members.
Figure 2.
Alignments and characterizations of deduced amino acid sequences of grass carp Mx isoforms.
Identical amino acids are in black background, and similar amino acids are in dark gray background. Tripartite GTP-binding motif consensus elements (GXXXSGKS/T, DXXG and T/NKXD), dynamin family signature (LPRG(S/K)GIVTR) are in blue boxes. The leucine residues of the LZ are shown in green. The potential N-linked glycosylation sites (NXT/S) are in pink. The bipartite nuclear localization signals ((K/R)(K/R)X10–12(K/R)3/5) are double lined. The nuclear export signals (L/I/V/F/M)X3(L/I/V/F/M)X2(L/I/V/F/M)X(L/I/V/F/M) are underlined. The positions of dynamin domain, central interactive domain, GTPase effector domain, isoelectric point and localization in the corresponding isoforms are listed at the bottom.
Figure 3.
The spatial structures of three grass carp Mx isoforms predicted by SWISS-MODEL program.
Blue, α-helices; pink, β-sheets; black, random coil. The hollow arrows mark the various structure among the three isoforms. In amino acid position 509–540, Glu517-Lys523 (overlapping with CID) and Leu534-Asp540 form two β-sheets in CiMx1; Ser509-Asn516 (overlapping with CID) forms α-helix in CiMx2; Ala512-Asp519 (overlapping with CID) and Thr525-Thr538 (overlapping with GED) form two α-helices in CiMx3.
Figure 4.
RT-PCR-based expression analysis of different Mx genes in various tissues of healthy grass carp.
cDNAs from three animals for corresponding tissues were pooled for detection analysis. Reverse transcription and amplification by PCR with the specific primers were carried out for analyzing CiMx1, CiMx2, CiMx3 expression, and 18S rRNA was used as an internal reference. The 15 tested tissues are indicated above each lane. The top panel demonstrates 18S rRNA expression, and bottom three panels show the expression of different Mx genes. Gene names are indicated to the right of the panel.
Figure 5.
The mRNA expression profiles of three grass carp Mx genes and IFN-I gene post-GCRV injection in spleen, head kidney and gill tissues.
18S rRNA was employed as an internal control. A: spleen; B: head kidney; C: gill. Asterisks (*) mark the significant difference between experimental and control groups (P<0.05). Error bars indicate standard error.
Figure 6.
qRT-PCR-based expression analyses of different Mx genes and IFN-I gene in CIK cell culture.
The cells were infected with GCRV and collected at different time-points (0, 2, 8, 24 and 48 h) after infection, then used in RNA extraction and qRT-PCR. EF1α was employed as an internal reference. Asterisks (*) mark the significant difference between experimental and control groups (p<0.05). Error bars indicate standard deviation.
Figure 7.
mRNA expression patterns of three grass carp Mx isoforms and IFN-I gene post poly(I:C) stimulation in CIK cells.
EF1α was utilized as an internal control gene. The gene expression levels were measured at 2, 8 and 24 h post-stimulation. A: Mx1 transcription; B: Mx2 transcription; C: Mx3 transcription; D: IFN-I transcription. Asterisks (*) mark the significant difference between experimental and control groups (P<0.05). Error bars indicate standard error.
Figure 8.
Analyses of the levels of three grass carp Mx, IFN-I and viral VP4 expression post-GCRV infection in transgenic cell lines.
EF1α was used as an internal control gene. The gene expression levels were examined at 0, 24 and 48 h post-challenge. A: Mx1 expression levels; B: Mx2 expression levels; C: Mx3 expression levels; D: IFN-I expression levels; E: VP4 expression levels. Asterisks (*) mark significant differences between experimental and control groups (P<0.05). Error bars indicate standard error.
Figure 9.
Antiviral activities of three grass carp Mx genes against GCRV in transgenic cells.
Stable transgenic cells were cultured in 96-well plate for 24 h at 28°C. Monolayer cells were infected with GCRV in duplicate at the indicated densities. Cells were fixed with 4% paraformaldehyde and stained with 3% crystal violet at 60 h post-GCRV challenge (A). The culture supernatants from transgenic cells infected with GCRV were collected at 0 h, 12 h and 48 h post-infection, and the viral titers were determined for each culture by plaque assay in triplicate (B). Asterisks (*) mark significant differences between experimental and control groups (P<0.05). Error bars indicate standard error.
Table 2.
Primer sequences and their designated applications in this study.