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Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus.

ABSTRACT:

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The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle.

Importance

Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.

SUBMITTER: Chang PJ

PROVIDER: S-EPMC4136336 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus.

Chang Pey-Jium PJ Hung Chien-Hui CH Wang Shie-Shan SS Tsai Ping-Hsin PH Shih Ying-Ju YJ Chen Li-Yu LY Huang Hsiao-Yun HY Wei Ling-Huei LH Yen Ju-Bei JB Lin Chun-Liang CL Chen Lee-Wen LW

Journal of virology 20140625 17

<h4>Unlabelled</h4>The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, con ...[more]

PMID: 24965448

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-oncogenic herpesvirus, and both lytic and latent infections play important roles in its pathogenesis and tumorigenic properties. Multiple cellular pathways and diverse mediators are hijacked by viral proteins and are used to support KSHV lytic replication. In previous studies, we revealed that KSHV ORF45 promoted KSHV transcription and translation by inducing sustained p90 ribosomal S6 kinase (RSK) activation and the phosphorylation of its substrates c-Fos and eIF4B. However, the cellular mediators required for lytic replication remain largely unknown. Here, we reveal that ORF45 activates eIF2α phosphorylation and ATF4 translation and then upregulates the expression of lysosome-associated membrane protein 3 (LAMP3) in an ATF4-dependent manner during KSHV lytic replication. Consequently, LAMP3 promotes Akt and ERK activation and then facilitates lytic gene expression and virion production. Furthermore, ATF4 enhances lytic replication through LAMP3, and LAMP3 acts in an ATF4-independent manner. Our findings suggest that the ATF4-LAMP3 axis is upregulated by ORF45 through ER stress activation during the KSHV lytic life cycle and, in turn, facilitates optimal lytic replication. IMPORTANCE The lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) reprograms cellular transcription and translation to generate viral proteins and virion particles. Here, we show that the mediator of ER stress ATF4 and the expression of the downstream gene LAMP3 are upregulated by ORF45 during lytic replication. Consequently, increased LAMP3 expression activates Akt and ERK and promotes lytic replication. Although several UPR transcription factors are able to promote KSHV lytic replication, the proviral effect of ATF4 on lytic replication is attenuated by LAMP3 silencing, whereas the effect of LAMP3 does not directly require ATF4 expression, indicating that LAMP3 primarily exerts effects on KSHV lytic replication downstream of ATF4 and ER stress. Taken together, our findings suggest that the ORF45-upregulated ATF4-LAMP3 axis plays an essential role in KSHV lytic replication.

Project description:UnlabelledThe human oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) expresses a set of ∼20 viral microRNAs (miRNAs). miR-K10a stands out among these miRNAs because its entire stem-loop precursor overlaps the coding sequence for the Kaposin (Kap) A/C proteins. The ectopic expression of KapA has been reported to lead to transformation of rodent fibroblasts. However, these experiments inadvertently also introduced miR-K10a, which raises the question whether the transforming activity of the locus could in fact be due to miR-K10a expression. To answer this question, we have uncoupled miR-K10a and KapA expression. Our experiments revealed that miR-K10a alone transformed cells with an efficiency similar to that when it was coexpressed with KapA. Maintenance of the transformed phenotype was conditional upon continued miR-K10a but not KapA protein expression, consistent with its dependence on miRNA-mediated changes in gene expression. Importantly, miR-K10a taps into an evolutionarily conserved network of miR-142-3p targets, several of which are expressed in 3T3 cells and are also known inhibitors of cellular transformation. In summary, our studies of miR-K10a serve as an example of an unsuspected function of an mRNA whose precursor is embedded within a coding transcript. In addition, our identification of conserved miR-K10a targets that limit transformation will point the way to a better understanding of the role of this miRNA in KSHV-associated tumors.ImportanceKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus. The viral Kaposin locus has known oncogenic potential, which has previously been attributed to the encoded KapA protein. Here we show that the virally encoded miR-K10a miRNA, whose precursor overlaps the KapA-coding region, may account for the oncogenic properties of this locus. Our data suggest that miR-K10a mimics the cellular miRNA miR-142-3p and thereby represses several known inhibitors of oncogenic transformation. Our work demonstrates that functional properties attributed to a coding region may in fact be carried out by an embedded noncoding element and sheds light on the functions of viral miR-K10a.

Dataset Information

Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus.

Unlabelled

Importance

Publications

Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus.

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