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. 2005 Apr;79(7):4298-307.
doi: 10.1128/JVI.79.7.4298-4307.2005.

"V体育官网入口" Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation

Misako Yajima  1 , Teru Kanda, Kenzo Takada
Affiliations

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation

VSports注册入口 - Misako Yajima et al. J Virol. 2005 Apr.

Abstract

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation VSports手机版. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes. .

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Figures

FIG. 1.
FIG. 1.
Experimental strategies for the construction of EBER knockout EBV in Akata cells. The EBER genes (black boxes), neomycin resistance gene (neor; open boxes), and hygromycin resistance gene (hygr; gray boxes), and loxP sites (white arrowheads) are indicated. Akata cells having episomes of NeorEBV were subjected to the EBER targeting by using the Hygr gene as a marker gene. The Hygr gene was then removed from the genome of the homologous recombinant by the excision reaction of cre recombinase.
FIG. 2.
FIG. 2.
(A) Map of the genome of Akata strain EBV surrounding the EBER locus before and after the EBER targeting. The EBER genes of Neor EBV were replaced with the hygromycin resistance gene by homologous recombination. Subsequently, the Hygr gene was removed by the transient expression of cre recombinase in Akata cells. The positions of restriction enzyme recognition sequences are indicated by B (BamHI), Bs (BstPI), Sa (SacI), A (AccI), RI (EcoRI), and RV (EcoRV). A gray line indicates a probe used for Southern blotting. White arrowheads indicate loxP sites. The EBER genes (black boxes) and hygromycin resistance gene (hygr; open box) are also indicated. (B) Southern blotting of Akata cell clones harboring various recombinant EBVs. The results from a NeorEBV-infected cell clone (lane 1), a targeted cell clone (recombinant; lane 2), an EBER(−)HygrEBV-infected cell clone (lane 3), the same cell clone with transient cre expression (lane 4), and EBER(−)EBV-infected cell clones (lanes 5 through 8) are shown. (C) Northern blotting of EBERs. The results from a EBV-negative Akata cell clone (Akata−; lane 1), a NeorEBV-infected cell clone (lane 2), and EBER(−)EBV-infected cell clones (lanes 3 through 6) are shown. (D) EBV gene expression in EBER(−) EBV-infected cell clones. Immunoblotting was used to examine the expression of EBNA proteins and LMP-1 protein (top), whereas RT-PCR analysis was used to examine BamHI Q promoter (Qp) usage and the expressions of other latent genes (bottom). The results from an LCL (lane 1), an EBV-negative Akata cell clone (Akata−; lane 2), a NeorEBV-infected cell clone (lane 3), and EBER(−)EBV-infected cell clones (lanes 4 through 7) are shown.
FIG. 3.
FIG. 3.
(A) Comparison of the amounts of the produced recombinant viruses. Akata cell clones harboring either NeorEBV or EBER(−)EBV were treated with anti-IgG, and the culture supernatants were harvested. Viral DNAs were extracted from the pelleted virions, digested with EcoRI, and analyzed by agarose gel electrophoresis. (B) EBV-negative Akata cells infected with either NeorEBV (left) or EBER(−)EBV (right) were processed for immunofluorescence analysis to detect the expression of EBNA proteins at 48 h postinfection (bottom). The corresponding differential interference contrast (DIC) images are shown (top). The percentages of cells that are positive for EBNA staining are also indicated.
FIG. 4.
FIG. 4.
(A) Map of the genome of Akata strain EBV surrounding the EBER locus before and after the EBER knock-in. The EBER genes were reconstituted in the genome of EBER(−)EBV via homologous recombination by using the hygromycin resistance gene as a marker gene. Subsequently, the Hygr gene was removed by the transient expression of cre recombinase in Akata cells. The positions of restriction enzyme recognition sequences are indicated by B (BamHI), Bs (BstPI), Sa (SacI), RI (EcoRI), and RV (EcoRV). A gray line indicates a probe used for Southern blot analysis. White arrowheads indicate loxP sites. The EBER genes (black boxes) and the hygromycin resistance gene (hygr; open box) are also indicated. (B) Southern blot analysis of Akata cell clones harboring various recombinant EBVs.The results from an EBER(−)EBV-infected cell clone (lane 1), a targeted cell clone (recombinant; lane 2), an EBER(+)HygrEBV-infected cell clone (lane 3), the same cell clone with transient cre expression (lane 4), and EBER(+)EBV-infected cell clones (lanes 5 through 8) are shown. (C) Northern blot analysis of EBERs. The results from an EBV-negative Akata cell clone (Akata−; lane 1), a NeorEBV-infected cell clone (lane 2), a EBER(−)EBV-infected cell clone (lane 3), and EBER(+)EBV-infected cell clones (lanes 4 through 7) are shown.
FIG. 5.
FIG. 5.
Immunoblot analysis of EBNA proteins and LMP-1 protein in the established LCLs. The results from two independent NeorEBV-infected LCLs (lanes 1 and 2), two independent EBER(−)EBV-infected LCLs (lanes 3 and 4), and four independent EBER(+)EBV-infected LCLs (lanes 5 through 8) are shown. Note that all of the LCLs express similar amounts of EBNA-1, EBNA-2, EBNA-3s, and LMP-1.
FIG. 6.
FIG. 6.
Comparison of the growth rates of the established LCLs at low cell densities. Two independent sets of LCLs (panels A and B), each derived from cord blood lymphocytes of different donors, were subjected to the assay. The LCLs established by infection with NeorEBV, EBER(−)EBV, or EBER(+)EBV were plated to the wells of 24-well plates at a density of either 5 × 104 (A) or 2 × 105 (B) cells per ml, and the numbers of cells were counted daily (except for day 6). The results for NeorEBV-infected LCLs (open circles), EBER(−)EBV-infected LCLs (solid circles), and EBER(+)EBV-infected LCLs (open triangles) are shown. The growth curves represent the averaged results (means ± standard deviations) obtained with four independent LCLs (A) or two independent LCLs (B).
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References

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