EBV noncoding RNA EBER2 interacts with host RNA-binding proteins to regulate viral gene expression

Abstract

Epstein-Barr virus (EBV) produces a highly abundant noncoding RNA called EBV-encoded RNA 2 (EBER2) that interacts indirectly with the host transcription factor paired box protein 5 (PAX5) to regulate viral latent membrane protein 1/2 (LMP1/2) gene expression as well as EBV lytic replication. To identify intermediary proteins, we isolated EBER2-PAX5-containing complexes and analyzed the protein components by mass spectrometry. The top candidates include three host proteins splicing factor proline and glutamine rich (SFPQ), non-POU domaincontaining octamer-binding protein (NONO), and RNA binding motif protein 14 (RBM14), all reported to be components of nuclear bodies called paraspeckles. In vivo RNA-protein crosslinking indicates that SFPQ and RBM14 contact EBER2 directly. Binding studies using recombinant proteins demonstrate that SFPQ and NONO associate with PAX5, potentially bridging its interaction with EBER2. Similar to EBER2 or PAX5 depletion, knockdown of any of the three host RNA-binding proteins results in the up-regulation of viral LMP2A mRNA levels, supporting a physiologically relevant interaction of these newly identified factors with EBER2 and PAX5. Identification of these EBER2-interacting proteins enables the search for cellular noncoding RNAs that regulate host gene expression in a manner similar to EBER2.noncoding RNA | Epstein-Barr virus | paraspeckle | RNA binding protein E pstein-Barr virus (EBV)-encoded RNA 2 (EBER2) is one of two highly abundant nuclear noncoding RNAs (ncRNAs) expressed during both latent and lytic infection of human B cells by the gamma herpesvirus EBV (1). Previous genome-wide location analysis of EBER2 using capture hybridization analysis of RNA targets (CHART) (2) revealed that EBER2 binds to the socalled terminal repeat (TR) regions of the double-stranded EBV genome (3). After DNA circularization to form the latent episome, these repeats are located in the first intron of the viral transcripts encoding latent membrane protein (LMP) 2A and 2B that are generated by alternative promoter use (4). Until recently, only the RNA chaperone La was known to interact with EBER2 through the stretch of uridylates at its 3′ end (5). Prompted by chromatin colocalization of EBER2 and the host transcription factor paired box protein 5 (PAX5) at the TR regions (6), we showed that PAX5 associates with EBER2 as well (3). However, negative results in electrophoretic mobility shift assays (EMSAs) using recombinantly expressed EBER2 and PAX5 suggested that the interaction between the ncRNA and host transcription factor might be bridged by an intermediate protein(s).Within EBER2 are two accessible regions available for hybridization with complementary nucleic acids based on ribonuclease H sensitivity (3). One of these sites (Fig. 1A, Top) engages in RNA-RNA interactions with nascent transcripts from the TR regions, thus facilitating the recruitment and accumulation of PAX5 at this locus (3). Loss of either EBER2 or PAX5 binding to the TR regions results in up-regulation of the nearby LMP1, 2A,...

Structural Basis for the Inhibition of Host Gene Expression by Porcine Epidemic Diarrhea Virus nsp1

“…After invasion of host cells, some virus-encoded proteins are used to regulate the host's environment, thus helping to increase virus replication (50)(51)(52)(53).Previous studies have revealed that the nsp1 proteins of coronaviruses can regulate the host protein synthesis and help the virus escape the host's immune interference (25,27,43,45,54). The highly variable sequence homology of the coronavirus nsp1 proteins may lead to the diverse mechanisms for inhibiting the host gene expression.…”

Stratified UWHAM and Its Stochastic Approximation for Multicanonical Simulations Which Are Far from Equilibrium

“…Two maximum likelihood-based methods, the dynamic histogram analysis method (DHAM) and the general transition-based reweighting analysis method (TRAM), 59–62 were proposed recently to provide free energy estimates for multi-state simulations when the simulations at some λ -states are only locally equilibrated. As we propose for Stratified-UWHAM, both DHAM and TRAM require building MSMs first for further analyses.…”

“…The maximum likelihood function of TRAM is a product of the maximum likelihood functions of binless WHAM (population counts) and DHAM (transition counts). 62 Unlike Stratified-UWHAM, TRAM stratifies every λ -state into configuration states (macrostates) of MSMs. The local free energy of each configuration state at each λ -state is calculated during each iteration of TRAM analysis: the free energy differences between the same configuration states at different λ -states are calculated in a binless manner; the free energy differences between configuration states at each λ -state are calculated based on the transition counts and the detailed balance condition.…”

“…In Ref. [62] TRAM was applied to obtain the thermodynamic and kinetic information of a protein-ligand binding complex successfully while the MBAR/UWHAM or WHAM analysis was found to be unfeasible or less efficient.…”

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naive B cells, and facilitates recruitment of transcription factors to the viral genome

“…Notably, this region includes the terminal repeats, and the virus – linear in the virion – needs to recircularize before LMP2 can be transcribed, and perhaps before this region is properly regulated. In addition the terminal repeats contain a binding site for PAX5, which is directed to the viral genome by EBER2 [74]. Two of the factors reported to bind to the EBER2/PAX5 complex (NONO and SPFQ) have also been reported to bind to EBNA-LP in a tandem affinity mass spectrometry experiment [75], although these two proteins both have a high background signal in such experiments according to the CRAPome repository [76].…”