Construction and Manipulation of a New Kaposi's Sarcoma-Associated Herpesvirus Bacterial Artificial Chromosome Clone

Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is a 1,162-amino-acid protein that mediates the maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division and the segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal-repeat (TR) DNA and to mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate the segregation of episomes to daughter nuclei. The N-terminal region of LANA binds histones H2A and H2B to attach to mitotic chromosomes, while the C-terminal region binds TR DNA and also associates with chromosomes. Both the N-and C-terminal regions of LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequences results in highly deficient episome maintenance. Here we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N-and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and to bind TR DNA, as assessed by electrophoretic mobility shift assays (EMSA). Deletion of the internal regions did not reduce the half-life of LANA. Notably, deletions within either the repeat elements or the unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on the ability of LANA to retain green fluorescent protein (GFP) expression from TR-containing episomes deficient in DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient in episome persistence, and the deficiencies ranged from minor to severe. Mutants deficient in DNA replication that contained deletions within the unique internal sequence had the most-severe deficits. These data suggest that internal LANA regions exert critical roles in LANA-mediated DNA replication, segregation, and episome persistence, likely through interactions with key host cell factors.

Section: Discussionmentioning

confidence: 92%

Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence

Vázquez

Carey

Kaye

2013

“…Telomerase-immortalized human umbilical vein endothelial cells (HUVECs) with KSHV infection (TIVE-KSHV cells) (77), KS tumor cells carrying the recombinant KSHV bacterial artificial chromosome 16 (BAC16) (iSLK-BAC16 cells) (76), and human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS). KSHV-positive PEL cells (BCBL1 cells) (50) were grown in RPMI 1640 medium plus 10% FBS.…”

Section: Methodsmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N ⁶ -Adenosine Methylation To Promote Lytic Replication

Chen

Nilsen

2017

125

155

N 6 -adenosine methylation (m 6 A) is the most common posttranscriptional RNA modification in mammalian cells. We found that most transcripts encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) genome undergo m 6 A modification. The levels of m 6 A-modified mRNAs increased substantially upon stimulation for lytic replication. The blockage of m 6 A inhibited splicing of the pre-mRNA encoding the replication transcription activator (RTA), a key KSHV lytic switch protein, and halted viral lytic replication. We identified several m 6 A sites in RTA pre-mRNA crucial for splicing through interactions with YTH domain containing 1 (YTHDC1), an m 6 A nuclear reader protein, in conjunction with serine/arginine-rich splicing factor 3 (SRSF3) and SRSF10. Interestingly, RTA induced m 6 A and enhanced its own premRNA splicing. Our results not only demonstrate an essential role of m 6 A in regulating RTA pre-mRNA splicing but also suggest that KSHV has evolved a mechanism to manipulate the host m 6 A machinery to its advantage in promoting lytic replication.IMPORTANCE KSHV productive lytic replication plays a pivotal role in the initiation and progression of Kaposi's sarcoma tumors. Previous studies suggested that the KSHV switch from latency to lytic replication is primarily controlled at the chromatin level through histone and DNA modifications. The present work reports for the first time that KSHV genome-encoded mRNAs undergo m 6 A modification, which represents a new mechanism at the posttranscriptional level in the control of viral replication.KEYWORDS KSHV, N 6 -adenosine methylation, RNA splicing, lytic replication G ene expression is controlled not only at the chromatin level through histone and DNA modifications but also at the posttranscriptional level through RNA modifications. N 6 -adenosine methylation (m 6 A) is the most abundant RNA modification found in ϳ25% of RNA species in mammalian cells (1, 2). Despite its discovery decades ago (3-7), the biochemical pathways responsible for m 6 A and the biological functions of this process were not fully defined until very recently (8-10). Three methyltransferases, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associated protein (WTAP), act as m 6 A writers and catalyze RNA m 6 A at specific sites with the consensus sequence [(G/A)GAC, where the underlined adenosine is the methylation site] (11, 12). Two demethylases, fat mass-and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), both of which act as m 6 A erasers, reverse this process (13-17). Most m 6 A sites are located near the transcription start sites, exonic regions flanking splicing sites, stop codons, and the 3= untranslated region (3= UTR) (1,

“…Our data demonstrated that the same region in KSHV ORF45 is involved in interaction with ORF33 and is critical for accumulation of ORF33 protein in cells. To determine the roles of this conserved region of ORF45 in KSHV lytic replication, we deleted the C-terminal 19 aa by introducing stop codons following leucine-389 of ORF45 in BAC16, an infectious bacterial artificial chromosome clone of the entire KSHV genome (39). Similarly, we repaired the mutations and created a revertant version of BAC16 (Fig.…”

Section: Identification Of Viral and Cellular Proteins Associated Withmentioning

confidence: 99%

A Survey of the Interactome of Kaposi's Sarcoma-Associated Herpesvirus ORF45 Revealed Its Binding to Viral ORF33 and Cellular USP7, Resulting in Stabilization of ORF33 That Is Required for Production of Progeny Viruses

Gillen

Liang

et al. 2015

Self Cite

The ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ϳ38 kDa and ϳ130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33-or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication. K aposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, the most common malignancy in HIV/AIDS patients. It is also associated with two lymphoproliferative disorders: primary effusion lymphoma and multicentric Castleman's disease (1-3). Like other herpesviruses, KSHV exhibits two alternative life cycles, a quiescent latent stage and a productiv...