Fig 1.
cDNA structure and expression of GP33.
(A) The GP33 cDNA structure determined by RACE analyses is depicted with a TATA box and a poly A signal (pA). The numbers in the GPCMV genome positions are based on Kanai et al. [33]. The ORF predicted initially as GP33 (hatched box; GP33s) and the ORF determined by RACE (closed boxes; GP33) are shown. An open arrowhead indicates the location of the DRY motif. “ins TGAT” is the mutation containing a stop codon and a 1-bp insertion to generate the GP33-defective virus, Δ33. (B) Multiple sequence alignment of GP33 homologs, HCMV UL33, MCMV M33, and RCMV R33, was performed by ClustalW analysis (DDBJ, ver. 2.1) with the default parameters except for GAP OPEN 100, and the obtained bootstrapped tree is depicted by TreeViewX software. (C) Localization of GP33-EGFP fusion protein in COS-7 cells transiently transfected with pEGFP-GP33. (D) Lysates of cells transfected with pcDNA3 empty vector (lane 1) or pcDNA-GP33F (lane 2) were separated on SDS-10% polyacrylamide gel. Proteins in the gel were transferred to polyvinylidene difluoride membrane (Millipore), and reacted with monoclonal anti-FLAG tag antibody followed by peroxidase-conjugated anti-mouse IgG. An arrowhead indicates the expected full-length GP33 band.
Fig 2.
Signal pathways activated by GP33.
(A-C) COS-7 cells were transfected with 50 ng of a reporter plasmid, pCRE-luc (A), pNFAT-luc (B) or pNFκB-luc (C), 0.5 ng of pRL-EF1α, and the indicated amount (ng) of an empty vector (vec) or an effector plasmid. pcDNA-GP33s (GP33s), -GP33 (GP33), -UL33 (UL33), pF4A-CMV-GαqQ209L (Gαq), and a plasmid expressing FLAG-MyD88 (MyD88) were used as the effector and control plasmids. Means and standard error of means (SEM) of fold induction obtained in triplicated wells using the relative luciferase activity of the cells transfected with an empty vector (vec) as a standard are shown. **: p<0.01. (D) TNF-α mRNA levels in uninfected GPL cells, or those infected with Δ33 or r33 at an MOI of 1. RNA samples were prepared from GPL cells at 1 day p.i. The amounts of TNF-α cDNA relative to those of β-actin were measured using real-time RT-PCR assay. Means and SEM of the fold increases obtained in triplicated wells using the relative TNF-α level in the uninfected cells as a standard are shown. *: p<0.05.
Fig 3.
(A) GPL cells in 48-well plates were infected with GPCMV r33 and Δ33 at an MOI of 0.05, and culture supernatants were recovered and frozen at the indicated hours after infection. The viral titers (IUs) were determined by counting GFP-positive cell foci at 2 days after infection. Means and SEMs of the virus stocks prepared in triplicated wells are plotted for r33 (open circles) and Δ33 (closed circles). (B) Representative images of GFP-positive cells after infection of GPE-7 cells and macrophages (Mϕ) with r33 or Δ33 at an MOI of 10 (the virus titers were determined in GPL cells) at 24 hrs and 36 hrs p.i., respectively.
Fig 4.
Viral loads in organs after infection of normal animals.
Female 4-week-old guinea pigs (Hartley) were infected i.p. with r33 (open circles) or Δ33 (closed circles) at a low dose (n = 8 each, 1x106 IUs/animal) (A) and at a high dose (n = 4 each, 2x107 IUs/animal) (B). GPCMV genome copies per 106 cells of the indicated organs obtained at 1 week after the infection are plotted. Means and SEMs are shown.
Fig 5.
Viral loads in organs at 6 days after infection of dams at 4 weeks’ GA.
Pregnant guinea pigs (Hartley) at 4 weeks’ GA were infected s.c. with GPCMV r33 or Δ33 (n = 4 each, 2x107 IUs/animal) and sacrificed at 6 days p.i. to collect specimens. (A) Relative body weights of dams infected with r33 (open circles) or Δ33 (closed circles) (n = 4 each) are shown using the body weight of each animal at the time of GPCMV infection as a 100% control. Comparison of the weights of placentas (B) and fetuses (C) from dams infected with r33 (open circles) or Δ33 (closed circles). Each circle indicates one placenta or fetus. Means and SEMs are shown. (D-H) GPCMV genome copies per 106 cells of the indicated organs from the animals infected with r33 (open circles) or Δ33 (closed circles). Means and SEMs are shown. (I) GPCMV genome copies per 106 cells of the placentas obtained from the dams described above.
Fig 6.
Appearance of the organs obtained from infected dams at 6 days p.i.
Images of the lungs (A) and livers (B) obtained from the dams described in the legend for Fig 5 are shown in the order of abnormalities in appearance from most severe to mildest.
Fig 7.
Pathological findings in r33- and Δ33-infected dams at 6 days p.i.
Formalin-fixed specimens prepared from the dams described in the legend for Fig 5 were histologically analyzed. (A, B) Severe inflammatory cell infiltration with hemorrhage was observed more frequently in the lungs of the r33-infected dams (A) than in those of Δ33-infected dams (B). (C, D) Severe inflammation was observed in the portal area in the liver of r33 virus-infected guinea pigs, and GPCMV-antigen-positive cells were found by immunohistochemical analysis (inset) (C). Minor inflammatory infiltration (arrow) was found in the liver of Δ33-infected guinea pigs (D). (E, H) HE staining shows intranuclear inclusion bodies in the lymph nodes of one of the r33-infected guinea pigs (E, arrows). Immunohistochemical analysis of cells demonstrated GPCMV-positive cells in the lymph nodes (H). (F, G, I, J) Intranuclear inclusion bodies (F, arrows) and many GPCMV-positive cells were found in the spleens of r33-infected guinea pigs (I) by HE staining and by immunohistochemical analysis, respectively. In contrast, no abnormalities or viral antigens were detected in the spleens of Δ33-infected guinea pigs (G, J).
Fig 8.
Viral loads in organs at 3 weeks after infection of dams at 4 weeks’ GA.
(A, B) Pregnant guinea pigs (Hartley) at 4 weeks’ GA were infected s.c. with GPCMV r33 or Δ33 (n = 4 each; 2x107 IUs/animal) and sacrificed at 3 weeks p.i. to collect specimens. Comparison of the weights of placentas (A) and fetuses (B) from dams infected with r33 (open circles) or Δ33 (closed circles). Each circle indicates one placenta or fetus. Means and SEMs of the weights are shown. (C-F) GPCMV genome copies per 106 cells of the livers (C), lungs (D), spleens (E), and salivary glands (F) obtained from the dams infected with r33 (open circles) or Δ33 (closed circles). Means and SEMs of genome copies are shown. (G-I) GPCMV genome copies per 106 cells of the placentas (G), fetal livers (H) and fetal lungs (I) obtained from fetuses of the dams described above. Means and SEMs of genome copies are shown.
Fig 9.
Pathological findings in r33- and Δ33-infected dams at 3 weeks p.i.
(A) Images of the lungs obtained from the dams infected with r33 (upper row) or Δ33 (lower row), which are described in the legend for Fig 8, are shown in the order of abnormalities in appearance from most severe to mildest. (B) Low and high magnifications of the HE staining of sections prepared from the lungs of the dams infected with r33 or Δ33. Asterisks and arrows indicate infiltration of inflammatory cells and alveolar hemorrhage, respectively. (C) Average number of lesions observed in two sections prepared from each of the dams infected with r33 (open circles) or Δ33 (closed circles). Means and SEMs are shown. (D) Relationship between visible lesion scores on the surface of the lungs determined blindly as described in the Method section and the average number of lesions per cut section. Each circle indicates a lung from each dam.
Fig 10.
BAC-derived GPCMV strains showed attenuated phenotypes.
Pregnant guinea pigs (Hartley) at 4 weeks’ GA were infected s.c. with GPCMVΔ9K WT or with GPCMV SG (n = 4 each; 3x106 IUs/animal) and sacrificed at 6 days p.i. to collect specimens. (A) Images of the lungs obtained from the dams infected with GPCMVΔ9K WT (upper row) or GPCMV SG (lower row) are shown in the order of abnormalities in appearance from most severe to mildest. (B) An image of the liver with necrotic lesion that was obtained from one of the dams infected with GPCMV SG. (C-F) GPCMV genome copies per 106 cells of the spleens (C), livers (D), lungs (E), and salivary glands (F) obtained from the dams infected with GPCMVΔ9K WT (open circles) or GPCMV SG (squares). Means and SEMs are shown.