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Mutual inhibition between Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus lytic replication initiators in dually-infected primary effusion lymphoma.

Jiang Y, Xu D, Zhao Y, Zhang L - PLoS ONE (2008)

Bottom Line: Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are members of the human gamma herpesvirus family: each is associated with various human cancers.The majority of AIDS-associated primary effusion lymphoma (PEL) are co-infected with both KSHV and EBV.The leucine heptapeptide repeat (LR) region in K-RTA and leucine zipper region in EBV-Z are involved in the physical interactions of the two molecules.

View Article: PubMed Central - PubMed

Affiliation: Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska, USA.

ABSTRACT

Background: Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are members of the human gamma herpesvirus family: each is associated with various human cancers. The majority of AIDS-associated primary effusion lymphoma (PEL) are co-infected with both KSHV and EBV. Dually-infected PELs selectively switch from latency to lytic replication of either KSHV or EBV in response to chemical stimuli. KSHV replication and transcription activator (K-RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication, while EBV BZLF1 gene product (EBV-Z) is a critical initiator for induction of EBV lytic replication.

Methodology/principal findings: We show K-RTA and EBV-Z are co-localized and physically interact with each other in dually-infected PELs. K-RTA inhibits the EBV lytic replication by ifying EBV-Z-mediated EBV lytic gene activation. EBV-Z inhibits KSHV lytic gene expression by blocking K-RTA-mediated transactivations. The physical interaction between K-RTA and EBV-Z are required for the mutual inhibition of the two molecules. The leucine heptapeptide repeat (LR) region in K-RTA and leucine zipper region in EBV-Z are involved in the physical interactions of the two molecules. Finally, initiation of KSHV lytic gene expression is correlated with the reduction of EBV lytic gene expression in the same PEL cells.

Conclusions/significance: In this report, how the two viruses interact with each other in dually infected PELs is addressed. Our data may provide a possible mechanism for maintaining viral latency and for selective lytic replication in dually infected PELs, i.e., through mutual inhibition of two critical lytic replication initiators. Our data about putative interactions between EBV and KSHV would be applicable to the majority of AIDS-associated PELs and may be relevant to the pathogenesis of PELs.

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K-RTA inhibits EBV-Z-mediated EBV lytic gene expression.A. K-RTA inhibits EBV-Z-mediated EBV lytic gene expression. EBV-Z expression plasmid (0, 0.1, and 0.2 µg) plus K-RTA (0.2 µg) were transfected into BRLF1KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis 24 hours later. The same membrane was stripped and reprobed with other antibodies. The identity of proteins is as shown. B. Dose-dependent inhibition of EBV-Z-mediated lytic gene expression by K-RTA. Fix amount of EBV-Z expression plasmid (0.1 µg) plus various amounts of K-RTA (0, 0.05, 0.1, 0.2, 0.4 µg) were transfected into BRLF1-KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis. Same cell lysates were used. C. K-RTA inhibits the synergistic activation of EA-D. EBV-Z expression plasmid (0.025 µg), E-RTA (0.1 µg), and K-RTA (0.2 µg) were transfected with different combinations into BRLF1-KO cells as shown on the top. The same cell lysates were used for western blot analysis. The identity of proteins is as shown.
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pone-0001569-g002: K-RTA inhibits EBV-Z-mediated EBV lytic gene expression.A. K-RTA inhibits EBV-Z-mediated EBV lytic gene expression. EBV-Z expression plasmid (0, 0.1, and 0.2 µg) plus K-RTA (0.2 µg) were transfected into BRLF1KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis 24 hours later. The same membrane was stripped and reprobed with other antibodies. The identity of proteins is as shown. B. Dose-dependent inhibition of EBV-Z-mediated lytic gene expression by K-RTA. Fix amount of EBV-Z expression plasmid (0.1 µg) plus various amounts of K-RTA (0, 0.05, 0.1, 0.2, 0.4 µg) were transfected into BRLF1-KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis. Same cell lysates were used. C. K-RTA inhibits the synergistic activation of EA-D. EBV-Z expression plasmid (0.025 µg), E-RTA (0.1 µg), and K-RTA (0.2 µg) were transfected with different combinations into BRLF1-KO cells as shown on the top. The same cell lysates were used for western blot analysis. The identity of proteins is as shown.

Mentions: A serial of cell lines, 293-EBV, BRLF1-KO, and BZLF1-KO, was used for the experiments. 293-EBV harbors wild type EBV genome. BRLF1-KO and BZLF1-KO contain the EBV genome missing BRLF1 (E-RTA) or BZLF1 (EBV-Z) gene respectively [38]. BRLF1-KO was used primarily because expression of EA-D was not very sensitive to EBV-Z expression (data not shown). As shown in the Fig. 2, EBV-Z is able to induce the lytic replication of EBV as indicated by the induction of EA-D protein. However in the presence of K-RTA, the expression of EA-D is inhibited (Fig. 2A). The inhibition is dose-dependent phenomena (Fig. 2B). Interestingly, EBV-Z and E-RTA can synergistically induce EBV lytic replication [61]–[64]. By reducing the expression of EBV-Z with less plasmid in transfection, we could observe the reported synergy and K-RTA was a potent inhibitor of the synergistic activation (Fig. 2C). Essentially the same results can be obtained from 293-EBV and BZLF1-KO cell lines (data not shown). The use of more than one cell lines is to ensure the results are not cell-line dependent. These data suggested that K-RTA inhibited EBV-Z-mediated lytic gene expression.


Mutual inhibition between Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus lytic replication initiators in dually-infected primary effusion lymphoma.

Jiang Y, Xu D, Zhao Y, Zhang L - PLoS ONE (2008)

K-RTA inhibits EBV-Z-mediated EBV lytic gene expression.A. K-RTA inhibits EBV-Z-mediated EBV lytic gene expression. EBV-Z expression plasmid (0, 0.1, and 0.2 µg) plus K-RTA (0.2 µg) were transfected into BRLF1KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis 24 hours later. The same membrane was stripped and reprobed with other antibodies. The identity of proteins is as shown. B. Dose-dependent inhibition of EBV-Z-mediated lytic gene expression by K-RTA. Fix amount of EBV-Z expression plasmid (0.1 µg) plus various amounts of K-RTA (0, 0.05, 0.1, 0.2, 0.4 µg) were transfected into BRLF1-KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis. Same cell lysates were used. C. K-RTA inhibits the synergistic activation of EA-D. EBV-Z expression plasmid (0.025 µg), E-RTA (0.1 µg), and K-RTA (0.2 µg) were transfected with different combinations into BRLF1-KO cells as shown on the top. The same cell lysates were used for western blot analysis. The identity of proteins is as shown.
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Related In: Results  -  Collection

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pone-0001569-g002: K-RTA inhibits EBV-Z-mediated EBV lytic gene expression.A. K-RTA inhibits EBV-Z-mediated EBV lytic gene expression. EBV-Z expression plasmid (0, 0.1, and 0.2 µg) plus K-RTA (0.2 µg) were transfected into BRLF1KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis 24 hours later. The same membrane was stripped and reprobed with other antibodies. The identity of proteins is as shown. B. Dose-dependent inhibition of EBV-Z-mediated lytic gene expression by K-RTA. Fix amount of EBV-Z expression plasmid (0.1 µg) plus various amounts of K-RTA (0, 0.05, 0.1, 0.2, 0.4 µg) were transfected into BRLF1-KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis. Same cell lysates were used. C. K-RTA inhibits the synergistic activation of EA-D. EBV-Z expression plasmid (0.025 µg), E-RTA (0.1 µg), and K-RTA (0.2 µg) were transfected with different combinations into BRLF1-KO cells as shown on the top. The same cell lysates were used for western blot analysis. The identity of proteins is as shown.
Mentions: A serial of cell lines, 293-EBV, BRLF1-KO, and BZLF1-KO, was used for the experiments. 293-EBV harbors wild type EBV genome. BRLF1-KO and BZLF1-KO contain the EBV genome missing BRLF1 (E-RTA) or BZLF1 (EBV-Z) gene respectively [38]. BRLF1-KO was used primarily because expression of EA-D was not very sensitive to EBV-Z expression (data not shown). As shown in the Fig. 2, EBV-Z is able to induce the lytic replication of EBV as indicated by the induction of EA-D protein. However in the presence of K-RTA, the expression of EA-D is inhibited (Fig. 2A). The inhibition is dose-dependent phenomena (Fig. 2B). Interestingly, EBV-Z and E-RTA can synergistically induce EBV lytic replication [61]–[64]. By reducing the expression of EBV-Z with less plasmid in transfection, we could observe the reported synergy and K-RTA was a potent inhibitor of the synergistic activation (Fig. 2C). Essentially the same results can be obtained from 293-EBV and BZLF1-KO cell lines (data not shown). The use of more than one cell lines is to ensure the results are not cell-line dependent. These data suggested that K-RTA inhibited EBV-Z-mediated lytic gene expression.

Bottom Line: Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are members of the human gamma herpesvirus family: each is associated with various human cancers.The majority of AIDS-associated primary effusion lymphoma (PEL) are co-infected with both KSHV and EBV.The leucine heptapeptide repeat (LR) region in K-RTA and leucine zipper region in EBV-Z are involved in the physical interactions of the two molecules.

View Article: PubMed Central - PubMed

Affiliation: Nebraska Center for Virology, University of Nebraska, Lincoln, Nebraska, USA.

ABSTRACT

Background: Both Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) are members of the human gamma herpesvirus family: each is associated with various human cancers. The majority of AIDS-associated primary effusion lymphoma (PEL) are co-infected with both KSHV and EBV. Dually-infected PELs selectively switch from latency to lytic replication of either KSHV or EBV in response to chemical stimuli. KSHV replication and transcription activator (K-RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication, while EBV BZLF1 gene product (EBV-Z) is a critical initiator for induction of EBV lytic replication.

Methodology/principal findings: We show K-RTA and EBV-Z are co-localized and physically interact with each other in dually-infected PELs. K-RTA inhibits the EBV lytic replication by ifying EBV-Z-mediated EBV lytic gene activation. EBV-Z inhibits KSHV lytic gene expression by blocking K-RTA-mediated transactivations. The physical interaction between K-RTA and EBV-Z are required for the mutual inhibition of the two molecules. The leucine heptapeptide repeat (LR) region in K-RTA and leucine zipper region in EBV-Z are involved in the physical interactions of the two molecules. Finally, initiation of KSHV lytic gene expression is correlated with the reduction of EBV lytic gene expression in the same PEL cells.

Conclusions/significance: In this report, how the two viruses interact with each other in dually infected PELs is addressed. Our data may provide a possible mechanism for maintaining viral latency and for selective lytic replication in dually infected PELs, i.e., through mutual inhibition of two critical lytic replication initiators. Our data about putative interactions between EBV and KSHV would be applicable to the majority of AIDS-associated PELs and may be relevant to the pathogenesis of PELs.

Show MeSH
Related in: MedlinePlus