Dataset Information

ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin ?1 for non-classical nuclear import.

ABSTRACT: The latency-associated nuclear antigens (LANA) of KSHV and macaque RFHVMn, members of the RV1 rhadinovirus lineage, are closely related with conservation of complex nuclear localization signals (NLS) containing bipartite KR-rich motifs and RG-rich domains, which interact distinctly with importins ? and ß1 for nuclear import via classical and non-classical pathways, respectively. RV1 LANAs are expressed in the nucleus of latently-infected cells where they inhibit replication and establish a dominant RV1 latency. Here we show that LANA homologs of macaque RRV and MneRV2 from the more distantly-related RV2 lineage, lack the KR-rich NLS, and instead have a large RG-rich NLS with multiple RG dipeptides and a conserved RGG motif. The RG-NLS interacts uniquely with importin ?1, which mediates nuclear import and accumulation of RV2 LANA in the nucleolus. The alternative nuclear import and localization of RV2 LANA homologs may contribute to the dominant RV2 lytic replication phenotype.

SUBMITTER: Howard K

PROVIDER: S-EPMC5929975 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin β1 for non-classical nuclear import.

Howard Kellie K Cherezova Lidia L DeMaster Laura K LK Rose Timothy M TM

Virology 20170829

The latency-associated nuclear antigens (LANA) of KSHV and macaque RFHVMn, members of the RV1 rhadinovirus lineage, are closely related with conservation of complex nuclear localization signals (NLS) containing bipartite KR-rich motifs and RG-rich domains, which interact distinctly with importins α and ß1 for nuclear import via classical and non-classical pathways, respectively. RV1 LANAs are expressed in the nucleus of latently-infected cells where they inhibit replication and establish a domin ...[more]

PMID: 28850829

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Project description:ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin ?1 in a RanGTP-sensitive manner and serves as an NLS in the importin ?1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin ? family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin ?/?-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency.

Project description:Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response. TraR and DksA increase or decrease transcription initiation depending on the kinetic features of the promoter by binding directly to RNA polymerase without binding to DNA. Unlike DksA, whose full activity requires ppGpp as a cofactor, TraR is fully active by itself and unaffected by ppGpp. TraR belongs to a family of divergent proteins encoded by proteobacterial bacteriophages and other mobile elements. Here, we experimentally addressed whether other members of the TraR family function like the F element-encoded TraR. Purified TraR and all 5 homologs that were examined bound to RNA polymerase, functioned at lower concentrations than DksA, and complemented a dksA-null strain for growth on minimal medium. One of the homologs, λ Orf73, encoded by bacteriophage lambda, was examined in greater detail. λ Orf73 slowed host growth and increased phage burst size. Mutational analysis suggested that λ Orf73 and TraR have a similar mechanism for inhibiting rRNA and r-protein promoters. We suggest that TraR and its homologs regulate host transcription to divert cellular resources to phage propagation or conjugation without induction of ppGpp and a stringent response. IMPORTANCE TraR is a distant homolog of the transcription factor DksA and the founding member of a large family of small proteins encoded by proteobacterial phages and conjugative plasmids. Reprogramming transcription during the stringent response requires the interaction of DksA not only with RNA polymerase but also with the stress-induced regulatory nucleotide ppGpp. We show here that five phage TraR homologs by themselves, without ppGpp, regulate transcription of host promoters, mimicking the effects of DksA and ppGpp together. During a stringent response, ppGpp independently binds directly to, and inhibits the activities of, many proteins in addition to RNA polymerase, including translation factors, enzymes needed for ribonucleotide biosynthesis, and other metabolic enzymes. Here, we suggest a physiological role for TraR-like proteins: bacteriophages utilize TraR homologs to reprogram host transcription in the absence of ppGpp induction and thus without inhibiting host enzymes needed for phage development.

Dataset Information

ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin ?1 for non-classical nuclear import.

Publications

ORF73 LANA homologs of RRV and MneRV2 contain an extended RGG/RG-rich nuclear and nucleolar localization signal that interacts directly with importin β1 for non-classical nuclear import.

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