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Mutagenesis of the herpesvirus saimiri terminal repeat region reveals important elements for virus production.

ABSTRACT: Deletion of the terminal repeats (TR) from herpesvirus saimiri (HVS) renders it unable to produce infectious virus or generate plaques. However, a TR-deleted HVS bacterial artificial chromosome can form replication compartments. Complementation of this mutant shows that one copy of the TR, plus the right junction of the genome with the TR, is sufficient for efficient plaque formation and generation of infectious virus. Within the TR unit, the region around the cleavage site of the genome appears both necessary and sufficient for virus production. Analysis of episomes from productive cells indicates a propensity to amplify TR numbers during the lytic cycle.

SUBMITTER: White RE

PROVIDER: S-EPMC1900079 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Mutagenesis of the herpesvirus saimiri terminal repeat region reveals important elements for virus production.

White Robert E RE Carline Lindsay L Allday Martin J MJ

Journal of virology 20070411 12

Deletion of the terminal repeats (TR) from herpesvirus saimiri (HVS) renders it unable to produce infectious virus or generate plaques. However, a TR-deleted HVS bacterial artificial chromosome can form replication compartments. Complementation of this mutant shows that one copy of the TR, plus the right junction of the genome with the TR, is sufficient for efficient plaque formation and generation of infectious virus. Within the TR unit, the region around the cleavage site of the genome appears ...[more]

PMID: 17428860

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Project description:The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown. The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line cancer- predisposing mutations in this region abolish transcription activation, whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions NH2-terminal to the BRCTs retained wild-type activity, whereas insertions in the BRCTs were, with few exceptions, deleterious; and (c) site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the COOH terminus. Deletion analysis revealed that the integrity of the most COOH-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay.

Project description:UnlabelledIn latently infected marmoset T cells, Herpesvirus saimiri (HVS) expresses six microRNAs (known as miR-HSURs [H. saimiri U-rich RNAs]). The viral miR-HSURs are processed from chimeric primary transcripts, each containing a noncoding U-rich RNA (HSUR) and a pre-miRNA hairpin. To uncover the functions of miR-HSURs, we identified mRNA targets in infected cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). HITS-CLIP revealed hundreds of robust Argonaute (Ago) binding sites mediated by miR-HSURs that map to the host genome but few in the HVS genome. Gene ontology analysis showed that several pathways regulating the cell cycle are enriched among cellular targets of miR-HSURs. Interestingly, miR-HSUR4-3p represses expression of the p300 transcriptional coactivator by binding the open reading frame of its mRNA. miR-HSUR5-3p directly regulates BiP, an endoplasmic reticulum (ER)-localized chaperone facilitating maturation of major histocompatibility complex class I (MHC-I) and the antiviral response. miR-HSUR5-3p also robustly downregulates WEE1, a key negative regulator of cell cycle progression, leading to reduced phosphorylation of its substrate, cyclin-dependent kinase (Cdk1). Consistently, inhibition of miR-HSUR5-3p in HVS-infected cells decreases their proliferation. Together, our results shed light on the roles of viral miRNAs in cellular transformation and viral latency.ImportanceViruses express miRNAs during various stages of infection, suggesting that viral miRNAs play critical roles in the viral life cycle. Compared to protein-coding genes, the functions of viral miRNAs are not well understood. This is because it has been challenging to identify their mRNA targets. Here, we focused on the functions of the recently discovered HVS miRNAs, called miR-HSURs. HVS is an oncogenic gammaherpesvirus that causes acute T-cell lymphomas and leukemias in New World primates and transforms human T cells. A better understanding of HVS biology will help advance our knowledge of virus-induced oncogenesis. Because numerous cellular miRNAs play crucial roles in cancer, viral miRNAs from the highly oncogenic HVS might also be important for transformation. Here, we found that the miR-HSURs preferentially modulate expression of host cell cycle regulators, as well as antiviral response factors. Our work provides further insight into the functions of herpesviral miRNAs in virus-induced oncogenesis and latency.

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Mutagenesis of the herpesvirus saimiri terminal repeat region reveals important elements for virus production.

Publications

Mutagenesis of the herpesvirus saimiri terminal repeat region reveals important elements for virus production.

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