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Reevaluation of the coding potential and proteomic analysis of the BAC-derived rhesus cytomegalovirus strain 68-1.

ABSTRACT: Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown, and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several related Old World monkey (OWM) CMVs, we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs could be detected. Taken together, these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes, and OWMs than previously assumed.

SUBMITTER: Malouli D

PROVIDER: S-EPMC3416160 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Reevaluation of the coding potential and proteomic analysis of the BAC-derived rhesus cytomegalovirus strain 68-1.

Malouli Daniel D Nakayasu Ernesto S ES Viswanathan Kasinath K Camp David G DG Chang W L William WL Barry Peter A PA Smith Richard D RD Früh Klaus K

Journal of virology 20120620 17

Cytomegaloviruses are highly host restricted, resulting in cospeciation with their hosts. As a natural pathogen of rhesus macaques (RM), rhesus cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68 ...[more]

PMID: 22718821

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Project description:UnlabelledThe UL128 complex of human cytomegalovirus (CMV) is a major determinant of viral entry into epithelial and endothelial cells and a target for vaccine development. The UL/b' region of rhesus CMV contains several open reading frames, including orthologs of the UL128 complex. We recently showed that the coding content of the rhesus CMV (RhCMV) UL/b' region predicts acute endothelial tropism and long-term shedding in vivo in the rhesus macaque model of CMV infection. The laboratory-passaged RhCMV 180.92 strain has a truncated UL/b' region but an intact UL128 complex. To investigate whether the presence of the UL128 complex alone was sufficient to confer endothelial and epithelial tropism in vivo, we investigated tissue dissemination and viral excretion following experimental RhCMV 180.92 inoculation of RhCMV-seronegative rhesus macaques. We show the presence of at least two virus variants in the RhCMV 180.92 infectious virus stock. A rare variant noted for a nontruncated wild-type-virus-like UL/b' region, rapidly emerged during in vivo replication and showed high-level replication in blood and tissues and excretion in urine and saliva, features similar to those previously reported in naturally occurring wild-type RhCMV infection. In contrast, the predominant truncated version of RhCMV 180.92 showed significantly lower plasma DNAemia and limited tissue dissemination and viral shedding. These data demonstrate that the truncated RhCMV 180.92 variant is attenuated in vivo and suggest that additional UL/b' genes, besides the UL128 complex, are required for optimal in vivo CMV replication and dissemination.ImportanceAn effective vaccine against human CMV infection will need to target genes that are essential for virus propagation and transmission. The human CMV UL128 complex represents one such candidate antigen since it is essential for endothelial and epithelial cell tropism, and is a target for neutralizing antibodies in CMV-infected individuals. In this study, we used the rhesus macaque animal model of CMV infection to investigate the in vivo function of the UL128 complex. Using experimental infection of rhesus macaques with a rhesus CMV virus variant that contained an intact UL128 complex but was missing several other genes, we show that the presence of the UL128 complex alone is not sufficient for widespread tissue dissemination and virus excretion. These data highlight the importance of in vivo studies in evaluating human CMV gene function and suggest that additional UL/b' genes are required for optimal CMV dissemination and transmission.

Dataset Information

Reevaluation of the coding potential and proteomic analysis of the BAC-derived rhesus cytomegalovirus strain 68-1.

Publications

Reevaluation of the coding potential and proteomic analysis of the BAC-derived rhesus cytomegalovirus strain 68-1.

Similar Datasets

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