Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself
D Michel, P Schaarschmidt, K Wunderlich, M Heuschmid, L Simoncini, D Muhlberger, A Zimmermann, I Pavic and T Mertens Abteilung Virologie der Universitat Ulm, Germany. detlef.michel@medizin.uni-ulm.de In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5&#...
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| Veröffentlicht in: | Journal of general virology 1998-09, Vol.79 (9), p.2105-2112 |
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| creator | Michel, D Schaarschmidt, P Wunderlich, K Heuschmid, M Simoncini, L Muhlberger, D Zimmermann, A Pavic, I Mertens, T |
| description | D Michel, P Schaarschmidt, K Wunderlich, M Heuschmid, L Simoncini, D Muhlberger, A Zimmermann, I Pavic and T Mertens
Abteilung Virologie der Universitat Ulm, Germany. detlef.michel@medizin.uni-ulm.de
In order to identify functional regions of the human cytomegalovirus
protein pUL97 (i) different 5' fragments of the UL97 open reading frame
(ORF) were fused to the coding region of the green fluorescent protein and
(ii) recombinant vaccinia viruses (rVV) were generated carrying two
full-length and 11 mutated UL97 ORFs. The results indicated the presence of
an N-terminal region within pUL97 which changed the intracellular
distribution of the fusion proteins. pUL97 was localized in the nucleus,
but not in the nucleoli, and was detected in the nuclear matrix fraction.
Expression of all pUL97 mutants could be confirmed by Western blot
analysis. pUL97-associated ganciclovir (GCV) phosphorylation in
rVV-infected cells, determined quantitatively by HPLC analysis, was
abolished completely using individual UL97 deletion mutants.
Phosphorylation of full-length and some of the mutated pUL97 was detected
in cells infected with the rVVs. The UL97 constructs carrying point
mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V,
and the 4 aa deletion 590AACR593, also resulted in decreased but not
abolished phosphorylation of GCV in the rVV system, whereas the
phosphorylation of pUL97 itself was not influenced. The rVV system is a
suitable method for quantitatively testing the functional relevance of
pUL97 mutations. |
| doi_str_mv | 10.1099/0022-1317-79-9-2105 |
| format | Article |
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Abteilung Virologie der Universitat Ulm, Germany. detlef.michel@medizin.uni-ulm.de
In order to identify functional regions of the human cytomegalovirus
protein pUL97 (i) different 5' fragments of the UL97 open reading frame
(ORF) were fused to the coding region of the green fluorescent protein and
(ii) recombinant vaccinia viruses (rVV) were generated carrying two
full-length and 11 mutated UL97 ORFs. The results indicated the presence of
an N-terminal region within pUL97 which changed the intracellular
distribution of the fusion proteins. pUL97 was localized in the nucleus,
but not in the nucleoli, and was detected in the nuclear matrix fraction.
Expression of all pUL97 mutants could be confirmed by Western blot
analysis. pUL97-associated ganciclovir (GCV) phosphorylation in
rVV-infected cells, determined quantitatively by HPLC analysis, was
abolished completely using individual UL97 deletion mutants.
Phosphorylation of full-length and some of the mutated pUL97 was detected
in cells infected with the rVVs. The UL97 constructs carrying point
mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V,
and the 4 aa deletion 590AACR593, also resulted in decreased but not
abolished phosphorylation of GCV in the rVV system, whereas the
phosphorylation of pUL97 itself was not influenced. The rVV system is a
suitable method for quantitatively testing the functional relevance of
pUL97 mutations.</description><identifier>ISSN: 0022-1317</identifier><identifier>EISSN: 1465-2099</identifier><identifier>DOI: 10.1099/0022-1317-79-9-2105</identifier><identifier>PMID: 9747718</identifier><language>eng</language><publisher>England: Soc General Microbiol</publisher><subject>Amino Acid Sequence ; Antiviral Agents - metabolism ; Antiviral Agents - pharmacokinetics ; Base Sequence ; Binding Sites ; Biological Transport, Active ; Cell Line ; Cell Nucleus - metabolism ; Cytomegalovirus - genetics ; Cytomegalovirus - metabolism ; DNA Primers - genetics ; Ganciclovir - metabolism ; Ganciclovir - pharmacokinetics ; Genes, Viral ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins - chemistry ; Luminescent Proteins - genetics ; Luminescent Proteins - metabolism ; Molecular Sequence Data ; Nuclear Matrix - metabolism ; Open Reading Frames ; Phosphorylation ; Phosphotransferases (Alcohol Group Acceptor) - chemistry ; Phosphotransferases (Alcohol Group Acceptor) - genetics ; Phosphotransferases (Alcohol Group Acceptor) - metabolism ; Point Mutation ; Polymerase Chain Reaction ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Sequence Deletion ; Vaccinia virus - genetics</subject><ispartof>Journal of general virology, 1998-09, Vol.79 (9), p.2105-2112</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c409t-e46a5dc2cb9701c4621cae8a92e460385c63ade6ec05ef4a0a2ba876e29fdeb83</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,3732,3733,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9747718$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Michel, D</creatorcontrib><creatorcontrib>Schaarschmidt, P</creatorcontrib><creatorcontrib>Wunderlich, K</creatorcontrib><creatorcontrib>Heuschmid, M</creatorcontrib><creatorcontrib>Simoncini, L</creatorcontrib><creatorcontrib>Muhlberger, D</creatorcontrib><creatorcontrib>Zimmermann, A</creatorcontrib><creatorcontrib>Pavic, I</creatorcontrib><creatorcontrib>Mertens, T</creatorcontrib><title>Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself</title><title>Journal of general virology</title><addtitle>J Gen Virol</addtitle><description>D Michel, P Schaarschmidt, K Wunderlich, M Heuschmid, L Simoncini, D Muhlberger, A Zimmermann, I Pavic and T Mertens
Abteilung Virologie der Universitat Ulm, Germany. detlef.michel@medizin.uni-ulm.de
In order to identify functional regions of the human cytomegalovirus
protein pUL97 (i) different 5' fragments of the UL97 open reading frame
(ORF) were fused to the coding region of the green fluorescent protein and
(ii) recombinant vaccinia viruses (rVV) were generated carrying two
full-length and 11 mutated UL97 ORFs. The results indicated the presence of
an N-terminal region within pUL97 which changed the intracellular
distribution of the fusion proteins. pUL97 was localized in the nucleus,
but not in the nucleoli, and was detected in the nuclear matrix fraction.
Expression of all pUL97 mutants could be confirmed by Western blot
analysis. pUL97-associated ganciclovir (GCV) phosphorylation in
rVV-infected cells, determined quantitatively by HPLC analysis, was
abolished completely using individual UL97 deletion mutants.
Phosphorylation of full-length and some of the mutated pUL97 was detected
in cells infected with the rVVs. The UL97 constructs carrying point
mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V,
and the 4 aa deletion 590AACR593, also resulted in decreased but not
abolished phosphorylation of GCV in the rVV system, whereas the
phosphorylation of pUL97 itself was not influenced. The rVV system is a
suitable method for quantitatively testing the functional relevance of
pUL97 mutations.</description><subject>Amino Acid Sequence</subject><subject>Antiviral Agents - metabolism</subject><subject>Antiviral Agents - pharmacokinetics</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Biological Transport, Active</subject><subject>Cell Line</subject><subject>Cell Nucleus - metabolism</subject><subject>Cytomegalovirus - genetics</subject><subject>Cytomegalovirus - metabolism</subject><subject>DNA Primers - genetics</subject><subject>Ganciclovir - metabolism</subject><subject>Ganciclovir - pharmacokinetics</subject><subject>Genes, Viral</subject><subject>Green Fluorescent Proteins</subject><subject>Humans</subject><subject>Luminescent Proteins - chemistry</subject><subject>Luminescent Proteins - genetics</subject><subject>Luminescent Proteins - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Nuclear Matrix - metabolism</subject><subject>Open Reading Frames</subject><subject>Phosphorylation</subject><subject>Phosphotransferases (Alcohol Group Acceptor) - chemistry</subject><subject>Phosphotransferases (Alcohol Group Acceptor) - genetics</subject><subject>Phosphotransferases (Alcohol Group Acceptor) - metabolism</subject><subject>Point Mutation</subject><subject>Polymerase Chain Reaction</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Sequence Deletion</subject><subject>Vaccinia virus - genetics</subject><issn>0022-1317</issn><issn>1465-2099</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc-O1SAUxonRjHdGn8CYsDJuqkBpKUszmX_JTdw4a0LpaYuhcIX2mjuP4RNLpzfj0gXhhO87P3LOh9AHSr5QIuVXQhgraElFIWQhC0ZJ9QrtKK-rgmX9Ndq9ON6iy5R-EkI5r8QFupCCC0GbHfpzu3gz2-C1wxGGXCQcejyPgMdl0h6b0xwmGLQLRxuXhA8xzGA9PjzupcDWH4M7QpcL7BfjQEfsgtHOPumVirXv8GEMKZ94cttb5g_aG2uemZtlo80JXP8Ovem1S_D-fF-hx9ubH9f3xf773cP1t31hOJFzAbzWVWeYaaUg1PCaUaOh0ZJlhZRNZepSd1CDIRX0XBPNWt2IGpjsO2ib8gp92rh5pF8LpFlNNhlwTnsIS1KilIyLuvyvkdYVJawk2VhuRhNDShF6dYh20vGkKFFrZGoNRK2BKCGVVGtkuevjGb-0E3QvPeeMsv5500c7jL9tBDWAn2z-o7VB5Q3-Q_0FaCijcw</recordid><startdate>19980901</startdate><enddate>19980901</enddate><creator>Michel, D</creator><creator>Schaarschmidt, P</creator><creator>Wunderlich, K</creator><creator>Heuschmid, M</creator><creator>Simoncini, L</creator><creator>Muhlberger, D</creator><creator>Zimmermann, A</creator><creator>Pavic, I</creator><creator>Mertens, T</creator><general>Soc General Microbiol</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7U9</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>19980901</creationdate><title>Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself</title><author>Michel, D ; Schaarschmidt, P ; Wunderlich, K ; Heuschmid, M ; Simoncini, L ; Muhlberger, D ; Zimmermann, A ; Pavic, I ; Mertens, T</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c409t-e46a5dc2cb9701c4621cae8a92e460385c63ade6ec05ef4a0a2ba876e29fdeb83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Amino Acid Sequence</topic><topic>Antiviral Agents - metabolism</topic><topic>Antiviral Agents - pharmacokinetics</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Biological Transport, Active</topic><topic>Cell Line</topic><topic>Cell Nucleus - metabolism</topic><topic>Cytomegalovirus - genetics</topic><topic>Cytomegalovirus - metabolism</topic><topic>DNA Primers - genetics</topic><topic>Ganciclovir - metabolism</topic><topic>Ganciclovir - pharmacokinetics</topic><topic>Genes, Viral</topic><topic>Green Fluorescent Proteins</topic><topic>Humans</topic><topic>Luminescent Proteins - chemistry</topic><topic>Luminescent Proteins - genetics</topic><topic>Luminescent Proteins - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Nuclear Matrix - metabolism</topic><topic>Open Reading Frames</topic><topic>Phosphorylation</topic><topic>Phosphotransferases (Alcohol Group Acceptor) - chemistry</topic><topic>Phosphotransferases (Alcohol Group Acceptor) - genetics</topic><topic>Phosphotransferases (Alcohol Group Acceptor) - metabolism</topic><topic>Point Mutation</topic><topic>Polymerase Chain Reaction</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Sequence Deletion</topic><topic>Vaccinia virus - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Michel, D</creatorcontrib><creatorcontrib>Schaarschmidt, P</creatorcontrib><creatorcontrib>Wunderlich, K</creatorcontrib><creatorcontrib>Heuschmid, M</creatorcontrib><creatorcontrib>Simoncini, L</creatorcontrib><creatorcontrib>Muhlberger, D</creatorcontrib><creatorcontrib>Zimmermann, A</creatorcontrib><creatorcontrib>Pavic, I</creatorcontrib><creatorcontrib>Mertens, T</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of general virology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Michel, D</au><au>Schaarschmidt, P</au><au>Wunderlich, K</au><au>Heuschmid, M</au><au>Simoncini, L</au><au>Muhlberger, D</au><au>Zimmermann, A</au><au>Pavic, I</au><au>Mertens, T</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself</atitle><jtitle>Journal of general virology</jtitle><addtitle>J Gen Virol</addtitle><date>1998-09-01</date><risdate>1998</risdate><volume>79</volume><issue>9</issue><spage>2105</spage><epage>2112</epage><pages>2105-2112</pages><issn>0022-1317</issn><eissn>1465-2099</eissn><abstract>D Michel, P Schaarschmidt, K Wunderlich, M Heuschmid, L Simoncini, D Muhlberger, A Zimmermann, I Pavic and T Mertens
Abteilung Virologie der Universitat Ulm, Germany. detlef.michel@medizin.uni-ulm.de
In order to identify functional regions of the human cytomegalovirus
protein pUL97 (i) different 5' fragments of the UL97 open reading frame
(ORF) were fused to the coding region of the green fluorescent protein and
(ii) recombinant vaccinia viruses (rVV) were generated carrying two
full-length and 11 mutated UL97 ORFs. The results indicated the presence of
an N-terminal region within pUL97 which changed the intracellular
distribution of the fusion proteins. pUL97 was localized in the nucleus,
but not in the nucleoli, and was detected in the nuclear matrix fraction.
Expression of all pUL97 mutants could be confirmed by Western blot
analysis. pUL97-associated ganciclovir (GCV) phosphorylation in
rVV-infected cells, determined quantitatively by HPLC analysis, was
abolished completely using individual UL97 deletion mutants.
Phosphorylation of full-length and some of the mutated pUL97 was detected
in cells infected with the rVVs. The UL97 constructs carrying point
mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V,
and the 4 aa deletion 590AACR593, also resulted in decreased but not
abolished phosphorylation of GCV in the rVV system, whereas the
phosphorylation of pUL97 itself was not influenced. The rVV system is a
suitable method for quantitatively testing the functional relevance of
pUL97 mutations.</abstract><cop>England</cop><pub>Soc General Microbiol</pub><pmid>9747718</pmid><doi>10.1099/0022-1317-79-9-2105</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0022-1317 |
| ispartof | Journal of general virology, 1998-09, Vol.79 (9), p.2105-2112 |
| issn | 0022-1317 1465-2099 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_73924763 |
| source | MEDLINE; Microbiology Society; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Amino Acid Sequence Antiviral Agents - metabolism Antiviral Agents - pharmacokinetics Base Sequence Binding Sites Biological Transport, Active Cell Line Cell Nucleus - metabolism Cytomegalovirus - genetics Cytomegalovirus - metabolism DNA Primers - genetics Ganciclovir - metabolism Ganciclovir - pharmacokinetics Genes, Viral Green Fluorescent Proteins Humans Luminescent Proteins - chemistry Luminescent Proteins - genetics Luminescent Proteins - metabolism Molecular Sequence Data Nuclear Matrix - metabolism Open Reading Frames Phosphorylation Phosphotransferases (Alcohol Group Acceptor) - chemistry Phosphotransferases (Alcohol Group Acceptor) - genetics Phosphotransferases (Alcohol Group Acceptor) - metabolism Point Mutation Polymerase Chain Reaction Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Sequence Deletion Vaccinia virus - genetics |
| title | Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself |
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