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The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

ABSTRACT: Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234-474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.

SUBMITTER: Hornig J

PROVIDER: S-EPMC5483178 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

Hornig Julia J Choi K Yeon KY McGregor Alistair A

Virology 20170210

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to ...[more]

PMID: 28189970

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Project description:Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene knockout results were similar to HCMV, except in the case of the gO homolog, which was non-essential in epithelial tropic virus but essential in lab adapted GPCMV. Overall, the findings demonstrate the similarity between HCMV and GPCMV glycoproteins and strengthen the relevance of this model for development of CMV intervention strategies.

Project description:Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.

Dataset Information

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

Publications

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

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