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Epstein-Barr virus IL-10 gene expression by a recombinant murine gammaherpesvirus in vivo enhances acute pathogenicity but does not affect latency or reactivation.

Lindquester GJ, Greer KA, Stewart JP, Sample JT - Herpesviridae (2014)

Bottom Line: Many viral genes affect cytokine function within infected hosts, with interleukin 10 (IL-10) as a commonly targeted mediator.Recombinant murine gammaherpesvirus expressing EBV vIL-10 rose to significantly higher titers in lungs and promoted an increase in spleen cell number in infected mice in comparison to MHV strains lacking the vIL-10 gene.Given that EBV and MHV wild-type strains contain other genes that induce cIL-10 expression in latency (e.g. LMP-1 and M2, respectively), vIL-10 may have evolved to serve the specific role in acute infection of enlarging the permissive host cell population, perhaps to facilitate initial survival and dissemination of viral-infected cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Rhodes College, Memphis, TN 38112, USA.

ABSTRACT

Background: Many viral genes affect cytokine function within infected hosts, with interleukin 10 (IL-10) as a commonly targeted mediator. Epstein-Barr virus (EBV) encodes an IL-10 homologue (vIL-10) expressed during productive (lytic) infection and induces expression of cellular IL-10 (cIL-10) during latency. This study explored the role of vIL-10 in a murine gammaherpesvirus (MHV) model of viral infection.

Methods: The EBV vIL-10 gene was inserted into MHV-76, a strain which lacks the ability to induce cIL-10, by recombination in transfected mouse cells. Mice were infected intranasally with the recombinant, vIL-10-containing MHV-76 or control virus strains and assayed at various days post infection for lung virus titer, spleen cell number, percentage of latently infected spleen cells and ability to reactivate virus from spleen cells.

Results: Recombinant murine gammaherpesvirus expressing EBV vIL-10 rose to significantly higher titers in lungs and promoted an increase in spleen cell number in infected mice in comparison to MHV strains lacking the vIL-10 gene. However, vIL-10 expression did not alter the quantity of latent virus in the spleen or its ability to reactivate.

Conclusions: In this mouse model of gammaherpesvirus infection, EBV vIL-10 appears to influence acute-phase pathogenicity. Given that EBV and MHV wild-type strains contain other genes that induce cIL-10 expression in latency (e.g. LMP-1 and M2, respectively), vIL-10 may have evolved to serve the specific role in acute infection of enlarging the permissive host cell population, perhaps to facilitate initial survival and dissemination of viral-infected cells.

No MeSH data available.


Related in: MedlinePlus

Schematic of approach to generating recombinant viruses. Expression cassettes were created in the pGL3-Basic vector (Promega) by insertion of the EBV vIL-10 gene along with the MHV76 gp150 promoter (Pgp150). Targeting cassettes were created by insertion of the expression cassette between the MHV76 terminal repeat segment (TR) and unique sequences of the left hand end (LHE, prototype orientation) in the pBSLHE-TR vector (see Methods). The targeting cassette was generated for this study with transcription from Pgp150 in the leftward direction, as determined by the asymmetric regeneration of the BamHI site during insertion (X = no restriction site). The targeting cassette and MHV76 DNA were co-transfected into NIH-3T3 cells for recombination. Once isolated, recombinant viral DNA was co-transfected along with the TR-LHE segment from pBSLHE-TR into NIH-3T3 cells to generate revertant viruses.
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Figure 1: Schematic of approach to generating recombinant viruses. Expression cassettes were created in the pGL3-Basic vector (Promega) by insertion of the EBV vIL-10 gene along with the MHV76 gp150 promoter (Pgp150). Targeting cassettes were created by insertion of the expression cassette between the MHV76 terminal repeat segment (TR) and unique sequences of the left hand end (LHE, prototype orientation) in the pBSLHE-TR vector (see Methods). The targeting cassette was generated for this study with transcription from Pgp150 in the leftward direction, as determined by the asymmetric regeneration of the BamHI site during insertion (X = no restriction site). The targeting cassette and MHV76 DNA were co-transfected into NIH-3T3 cells for recombination. Once isolated, recombinant viral DNA was co-transfected along with the TR-LHE segment from pBSLHE-TR into NIH-3T3 cells to generate revertant viruses.

Mentions: Targeting cassette: Plasmid pBS76LHE (courtesy of James Stewart) contains an approximately 3-kbp fragment from the left hand end (LHE) unique sequence of the MHV-76 genome[53] and was modified to generate pBS76LHE-TR as follows. A portion of a terminal repeat fragment and its immediately adjacent unique sequence was amplified from MHV-68 DNA by PCR under conditions favorable for GC rich sequences (Roche). Primers were 5′-AGGCAGGCACCAACAG-3′ and 5′-CAGCATCAGCCCCGGATCTC-3′. This fragment, designated TR, represents terminal repeat sequences immediately to the left (in the prototype orientation) of the LHE fragment in MHV-76. TR was inserted next to the LHE fragment in pBS76LHE to generate pBSLHE-TR; a BamHI site separates TR from LHE. Furthermore, PmeI restriction enzyme sites were inserted on either side of the TR-LHE sequence to be able to liberate the TR-LHE targeting cassette. Next, the vIL10 expression cassette was liberated from its plasmid as a fragment with a BglII restriction site on the upstream end and a BamHI restriction site on the downstream end. This fragment was inserted into the BamHI site of pBS76LHE-TR and restriction enzyme analysis revealed clones containing the expression cassette in either orientation bounded by the TR and LHE components. Figure 1 shows a general schematic of construction of the targeting cassette.


Epstein-Barr virus IL-10 gene expression by a recombinant murine gammaherpesvirus in vivo enhances acute pathogenicity but does not affect latency or reactivation.

Lindquester GJ, Greer KA, Stewart JP, Sample JT - Herpesviridae (2014)

Schematic of approach to generating recombinant viruses. Expression cassettes were created in the pGL3-Basic vector (Promega) by insertion of the EBV vIL-10 gene along with the MHV76 gp150 promoter (Pgp150). Targeting cassettes were created by insertion of the expression cassette between the MHV76 terminal repeat segment (TR) and unique sequences of the left hand end (LHE, prototype orientation) in the pBSLHE-TR vector (see Methods). The targeting cassette was generated for this study with transcription from Pgp150 in the leftward direction, as determined by the asymmetric regeneration of the BamHI site during insertion (X = no restriction site). The targeting cassette and MHV76 DNA were co-transfected into NIH-3T3 cells for recombination. Once isolated, recombinant viral DNA was co-transfected along with the TR-LHE segment from pBSLHE-TR into NIH-3T3 cells to generate revertant viruses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4199788&req=5

Figure 1: Schematic of approach to generating recombinant viruses. Expression cassettes were created in the pGL3-Basic vector (Promega) by insertion of the EBV vIL-10 gene along with the MHV76 gp150 promoter (Pgp150). Targeting cassettes were created by insertion of the expression cassette between the MHV76 terminal repeat segment (TR) and unique sequences of the left hand end (LHE, prototype orientation) in the pBSLHE-TR vector (see Methods). The targeting cassette was generated for this study with transcription from Pgp150 in the leftward direction, as determined by the asymmetric regeneration of the BamHI site during insertion (X = no restriction site). The targeting cassette and MHV76 DNA were co-transfected into NIH-3T3 cells for recombination. Once isolated, recombinant viral DNA was co-transfected along with the TR-LHE segment from pBSLHE-TR into NIH-3T3 cells to generate revertant viruses.
Mentions: Targeting cassette: Plasmid pBS76LHE (courtesy of James Stewart) contains an approximately 3-kbp fragment from the left hand end (LHE) unique sequence of the MHV-76 genome[53] and was modified to generate pBS76LHE-TR as follows. A portion of a terminal repeat fragment and its immediately adjacent unique sequence was amplified from MHV-68 DNA by PCR under conditions favorable for GC rich sequences (Roche). Primers were 5′-AGGCAGGCACCAACAG-3′ and 5′-CAGCATCAGCCCCGGATCTC-3′. This fragment, designated TR, represents terminal repeat sequences immediately to the left (in the prototype orientation) of the LHE fragment in MHV-76. TR was inserted next to the LHE fragment in pBS76LHE to generate pBSLHE-TR; a BamHI site separates TR from LHE. Furthermore, PmeI restriction enzyme sites were inserted on either side of the TR-LHE sequence to be able to liberate the TR-LHE targeting cassette. Next, the vIL10 expression cassette was liberated from its plasmid as a fragment with a BglII restriction site on the upstream end and a BamHI restriction site on the downstream end. This fragment was inserted into the BamHI site of pBS76LHE-TR and restriction enzyme analysis revealed clones containing the expression cassette in either orientation bounded by the TR and LHE components. Figure 1 shows a general schematic of construction of the targeting cassette.

Bottom Line: Many viral genes affect cytokine function within infected hosts, with interleukin 10 (IL-10) as a commonly targeted mediator.Recombinant murine gammaherpesvirus expressing EBV vIL-10 rose to significantly higher titers in lungs and promoted an increase in spleen cell number in infected mice in comparison to MHV strains lacking the vIL-10 gene.Given that EBV and MHV wild-type strains contain other genes that induce cIL-10 expression in latency (e.g. LMP-1 and M2, respectively), vIL-10 may have evolved to serve the specific role in acute infection of enlarging the permissive host cell population, perhaps to facilitate initial survival and dissemination of viral-infected cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Rhodes College, Memphis, TN 38112, USA.

ABSTRACT

Background: Many viral genes affect cytokine function within infected hosts, with interleukin 10 (IL-10) as a commonly targeted mediator. Epstein-Barr virus (EBV) encodes an IL-10 homologue (vIL-10) expressed during productive (lytic) infection and induces expression of cellular IL-10 (cIL-10) during latency. This study explored the role of vIL-10 in a murine gammaherpesvirus (MHV) model of viral infection.

Methods: The EBV vIL-10 gene was inserted into MHV-76, a strain which lacks the ability to induce cIL-10, by recombination in transfected mouse cells. Mice were infected intranasally with the recombinant, vIL-10-containing MHV-76 or control virus strains and assayed at various days post infection for lung virus titer, spleen cell number, percentage of latently infected spleen cells and ability to reactivate virus from spleen cells.

Results: Recombinant murine gammaherpesvirus expressing EBV vIL-10 rose to significantly higher titers in lungs and promoted an increase in spleen cell number in infected mice in comparison to MHV strains lacking the vIL-10 gene. However, vIL-10 expression did not alter the quantity of latent virus in the spleen or its ability to reactivate.

Conclusions: In this mouse model of gammaherpesvirus infection, EBV vIL-10 appears to influence acute-phase pathogenicity. Given that EBV and MHV wild-type strains contain other genes that induce cIL-10 expression in latency (e.g. LMP-1 and M2, respectively), vIL-10 may have evolved to serve the specific role in acute infection of enlarging the permissive host cell population, perhaps to facilitate initial survival and dissemination of viral-infected cells.

No MeSH data available.


Related in: MedlinePlus