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Evidence that human cytomegalovirus DNA cleavage involves a cellular DNA polymerase
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Abstracts
C M V Latency and Replication - Posters G0-03 Evidence that human cytomegalovirus DNA cleavage involves a cellular DNA polymerase MICHAEL McVOY Medical College of Virginia campus of Virginia Commonwealth University, USA Duplication of human cytomegalovirus terminal a sequences occurs during replication and may be integral to cleavage and packaging of viral DNA. We hypothesized that DNA synthesis may be required for a sequence duplication and therefore involved in cleavage. The effects of DNA polymerase inhibitors on cleavage were assessed by blocking cleavage in infected cells with the inhibitor BDCRB while allowing accumulation of concatemers. Resumption of cleavage was then measured by detection of 230 kb genomes 24 h after BDCRB removal. Drugs that selectively inhibited the viral DNA polymerase (PFA, HPMPA, and PMEA) did not block cleavage when added after BDCRB was removed, whereas drugs that also inhibited host DNA synthesis (aphidicolin and PMEG) fully blocked cleavage, as did a high dose of PFA sufficient to block host synthesis. Likewise, cleavage was fully blocked by a dose of DHPG predicted to inhibit host polymerases in infected cells. These results suggest that the viral DNA polymerase is not involved in cleavage but that an aphidicolinsensitive DNA polymerase such as host DNA polymerases ~,i~, or e may be required for cleavage.
G0-04 Distinct, longitudinal localization patterns of RhCMV in the spleen following experimental inoculation of rhesus macaques PETER BARRY University of California, Davis, USA Juvenile rhesus macaques (Macacca mulatta) were inoculated with rhesus cytomegalovirus (RhCMV) to identify virologic and host parameters that might influence the development of persistent infections. All animals exhibited virologic, immunologic, hematologic, and molecular evidence of RhCMV infection, although all remained clinically healthy. RhCMV DNA was detected at 2 weeks post infection (wPI) in the plasma of five animals and in multiple tissues b y 4 w P I . Viral DNA was sporadically observed in plasma after 2 wPI, and fewer tissues had detectable levels of viral DNA at later time points. Antiviral IgM antibodies were observed 1 wPI with peak titers occurring at 2 wPI. Neutralizing antibody titers were first observed 2-4 wPI, coincident with the appearance of antiviral IgG antibodies directed against a limited number of viral proteins. Immunohistochemical analysis demonstrated that the spleen was the predominant site for viral gene expression. The location of virus-expressing cells shifted within the spleen during the course of infection. At 4 wPI, the majority of cells expressing the immediate-early 1 (IE1) protein were associated with the follicles. Fewer virus-positive cells were observed in the inter-follicular spaces. At 25 wPl, most IEl-expressing cells were confined to the red pulp. Endothelial cells and tissue macrophages appeared to support RhCMV expression, although the vast majority of cells expressing viral antigens were an indeterminate cell type. The relative patterns of cells expressing IE1 suggested that the cell type supporting viral replication and/or the locations of these cells within a tissue are important for the establishment and maintenance of persistence.
Abstracts
C M V Latency and Replication - Posters G0-03 Evidence that human cytomegalovirus DNA cleavage involves a cellular DNA polymerase MICHAEL McVOY Medical College of Virginia campus of Virginia Commonwealth University, USA Duplication of human cytomegalovirus terminal a sequences occurs during replication and may be integral to cleavage and packaging of viral DNA. We hypothesized that DNA synthesis may be required for a sequence duplication and therefore involved in cleavage. The effects of DNA polymerase inhibitors on cleavage were assessed by blocking cleavage in infected cells with the inhibitor BDCRB while allowing accumulation of concatemers. Resumption of cleavage was then measured by detection of 230 kb genomes 24 h after BDCRB removal. Drugs that selectively inhibited the viral DNA polymerase (PFA, HPMPA, and PMEA) did not block cleavage when added after BDCRB was removed, whereas drugs that also inhibited host DNA synthesis (aphidicolin and PMEG) fully blocked cleavage, as did a high dose of PFA sufficient to block host synthesis. Likewise, cleavage was fully blocked by a dose of DHPG predicted to inhibit host polymerases in infected cells. These results suggest that the viral DNA polymerase is not involved in cleavage but that an aphidicolinsensitive DNA polymerase such as host DNA polymerases ~,i~, or e may be required for cleavage.
G0-04 Distinct, longitudinal localization patterns of RhCMV in the spleen following experimental inoculation of rhesus macaques PETER BARRY University of California, Davis, USA Juvenile rhesus macaques (Macacca mulatta) were inoculated with rhesus cytomegalovirus (RhCMV) to identify virologic and host parameters that might influence the development of persistent infections. All animals exhibited virologic, immunologic, hematologic, and molecular evidence of RhCMV infection, although all remained clinically healthy. RhCMV DNA was detected at 2 weeks post infection (wPI) in the plasma of five animals and in multiple tissues b y 4 w P I . Viral DNA was sporadically observed in plasma after 2 wPI, and fewer tissues had detectable levels of viral DNA at later time points. Antiviral IgM antibodies were observed 1 wPI with peak titers occurring at 2 wPI. Neutralizing antibody titers were first observed 2-4 wPI, coincident with the appearance of antiviral IgG antibodies directed against a limited number of viral proteins. Immunohistochemical analysis demonstrated that the spleen was the predominant site for viral gene expression. The location of virus-expressing cells shifted within the spleen during the course of infection. At 4 wPI, the majority of cells expressing the immediate-early 1 (IE1) protein were associated with the follicles. Fewer virus-positive cells were observed in the inter-follicular spaces. At 25 wPl, most IEl-expressing cells were confined to the red pulp. Endothelial cells and tissue macrophages appeared to support RhCMV expression, although the vast majority of cells expressing viral antigens were an indeterminate cell type. The relative patterns of cells expressing IE1 suggested that the cell type supporting viral replication and/or the locations of these cells within a tissue are important for the establishment and maintenance of persistence.