Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells

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Published inJournal of Virology Vol. 74; no. 9; pp. 4028 - 4038
Main Authors Czauderna, F, Fischer, N, Boller, K, Kurth, R, Tönjes, R R
Format Journal Article
LanguageEnglish
Published United States American Society for Microbiology 01.05.2000
Subjects
Animals
Binding Sites
Cell Line
Cell Line, Transformed
Cloning, Molecular
Endogenous Retroviruses - genetics
Endogenous Retroviruses - physiology
Endogenous Retroviruses - ultrastructure
Gene Library
HeLa Cells
Humans
Open Reading Frames
pigs
porcine endogenous retrovirus
Retrovirus
RNA Splicing
Swine
Terminal Repeat Sequences
Transcription, Genetic
Transfection
Virus Replication
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Abstract Article Usage Stats Services JVI Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue Spotlights in the Current Issue JVI About JVI Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy JVI RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0022-538X Online ISSN: 1098-5514 Copyright © 2014 by the American Society for Microbiology.   For an alternate route to JVI .asm.org, visit: JVI       
AbstractList The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p. t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3. 1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.
The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.
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ABSTRACT The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming immunological and physiological barriers, the existence of numerous porcine microorganisms poses the risk of initiating a xenozoonosis. Recently, different classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We therefore examined whether completely intact proviruses exist that produce infectious and replication-competent virions. Several proviral PERV sequences were cloned and characterized. One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A second clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-A mutation in the first start codon (Met to Ile) of the env gene, preventing this provirus from replicating. However, a genetic recombinant, PERV-B(33)/ATG, carrying a restored env start codon, became infectious and could be serially passaged on 293 cells similar to virus clone PERV-B(43). PERV protein expression was detected 24 to 48 h posttransfection (p.t.) using cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic env transcript of 3.1 kb. PERV-B(33) and PERV-B(43) differ in the number of copies of a 39-bp segment in the U3 region of the long terminal repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-free animals.
Author Nicole Fischer
Klaus Boller
Ralf R. Tönjes
Frank Czauderna
Reinhard Kurth
AuthorAffiliation Paul-Ehrlich-Institut, D-63225 Langen, Germany
AuthorAffiliation_xml – name: Paul-Ehrlich-Institut, D-63225 Langen, Germany
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  fullname: Kurth, R
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  fullname: Tönjes, R R
BackLink https://www.ncbi.nlm.nih.gov/pubmed/10756014$$D View this record in MEDLINE/PubMed
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Present address: Atugen Biotechnology AG, D-13125 Berlin, Germany.
Corresponding author. Mailing address: Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany. Phone: 49 6103 775304. Fax: 49 6103 771255. E-mail: toera@pei.de.
Present address: Robert-Koch-Institut, D-13353 Berlin, Germany.
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The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems overcoming...
ABSTRACT The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. In addition to the problems...
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SubjectTerms Animals
Binding Sites
Cell Line
Cell Line, Transformed
Cloning, Molecular
Endogenous Retroviruses - genetics
Endogenous Retroviruses - physiology
Endogenous Retroviruses - ultrastructure
Gene Library
HeLa Cells
Humans
Open Reading Frames
pigs
porcine endogenous retrovirus
Retrovirus
RNA Splicing
Swine
Terminal Repeat Sequences
Transcription, Genetic
Transfection
Virus Replication
Title Establishment and Characterization of Molecular Clones of Porcine Endogenous Retroviruses Replicating on Human Cells
URI http://jvi.asm.org/content/74/9/4028.abstract
https://www.ncbi.nlm.nih.gov/pubmed/10756014
https://search.proquest.com/docview/17514633
https://search.proquest.com/docview/71031069
https://pubmed.ncbi.nlm.nih.gov/PMC111916
Volume 74
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