ICP27 Phosphorylation Site Mutants Are Defective in Herpes Simplex Virus 1 Replication and Gene Expression

The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same ␣-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus. IMPORTANCEThe UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy. E very mammalian or avian herpesvirus encodes a member of the ICP27 protein family which functions as a posttranscriptional activator by facilitating the nuclear export of intronless mRNAs ...

Section: Importancementioning

confidence: 56%

pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication

Thomas

Sonntag

Müller

et al. 2015

“…Therefore, the flexibility of this region may be required for ICP27 to undergo successive interactions with proteins and RNA. Interestingly, the phosphorylation site mutants, which were found to be even more disordered than wild-type ICP27, were severely impaired in their ability to interact with proteins that interact with wild-type ICP27 (6,33). Despite the effects on protein interactions, the phosphorylation site mutants were able to bind gC sequences with the same affinity and specificity as those of wild-type ICP27 (6).…”

Section: Discussionmentioning

confidence: 93%

Three Arginine Residues within the RGG Box Are Crucial for ICP27 Binding to Herpes Simplex Virus 1 GC-Rich Sequences and for Efficient Viral RNA Export

Corbin-Lickfett¹,

Souki²,

Cocco

et al. 2010

Self Cite

ICP27 is a multifunctional protein that is required for herpes simplex virus 1 mRNA export. ICP27 interacts with the mRNA export receptor TAP/NXF1 and binds RNA through an RGG box motif. Unlike other RGG box proteins, ICP27 does not bind G-quartet structures but instead binds GC-rich sequences that are flexible in structure. To determine the contribution of arginines within the RGG box, we performed in vitro binding assays with N-terminal proteins encoding amino acids 1 to 160 of wild-type ICP27 or arginine-to-lysine substitution mutants. The R138,148,150K triple mutant bound weakly to sequences that were bound by the wild-type protein and single and double mutants. Furthermore, during infection with the R138,148,150K mutant, poly(A) ؉ RNA and newly transcribed RNA accumulated in the nucleus, indicating that viral RNA export was impaired. To determine if structural changes had occurred, nuclear magnetic resonance (NMR) analysis was performed on N-terminal proteins consisting of amino acids 1 to 160 from wild-type ICP27 and the R138,148,150K mutant. This region of ICP27 was found to be highly flexible, and there were no apparent differences in the spectra seen with wild-type ICP27 and the R138,148,150K mutant. Furthermore, NMR analysis with the wild-type protein bound to GC-rich sequences did not show any discernible folding. We conclude that arginines at positions 138, 148, and 150 within the RGG box of ICP27 are required for binding to GC-rich sequences and that the N-terminal portion of ICP27 is highly flexible in structure, which may account for its preference for binding flexible sequences.The herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is required for productive viral infection. ICP27 interacts with a number of cellular proteins, and it binds RNA (35). One of the functions that ICP27 performs is to escort viral mRNAs from the nucleus to the cytoplasm for translation (2,3,5,10,13,21,34). ICP27 binds viral RNAs (5, 34) and interacts directly with the cellular mRNA export receptor TAP/NXF1 (2, 21), which is required for the export of HSV-1 mRNAs (20,21). ICP27 also interacts with the export adaptor proteins Aly/REF (2,3,23) and UAP56 (L. A. Johnson, H. Swesey, and R. M. Sandri-Goldin, unpublished results), which form part of the TREX complex that binds to the 5Ј end of mRNA through an interaction with CBP80 (26, 32, 41). Aly/REF does not appear to bind viral RNA directly (3), and it is not essential for HSV-1 RNA export based upon small interfering RNA (siRNA) knockdown studies (20), but it contributes to the efficiency of viral RNA export (3, 23). ICP27 also interacts with the SR splicing proteins SRp20 and 9G8 (11,36), which have been shown to shuttle between the nucleus and the cytoplasm (1). SRp20 and 9G8 have also been shown to facilitate the export of some cellular RNAs (16,17,27) by binding RNA and interacting with TAP/ NXF1 (14,16,18). The knockdown of SRp20 or 9G8 adversely affects HSV-1 replication and specifically results in a nuclear accumulation of new...

“…Phosphorylation of YY1 by CKII␣ prevents cleavage by caspase-7 during apoptosis (66). CKII phosphorylation during virus infection plays an important role in regulation of ICP27 and EB2 binding activities to cellular cofactors and promotes virus replication of HSV-1 and EBV (63,67,68). ORF57 and its homologues are self-interacting proteins via their C-terminal domains (41,47,50).…”

Section: Discussionmentioning

confidence: 99%

Stability of Structured Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Is Regulated by Protein Phosphorylation and Homodimerization

Majerčiak

Pripuzova

Chan

et al. 2015

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches, we determined that ORF57 contains two structurally and functionally distinct domains: a disordered nonstructural N-terminal domain (amino acids [aa] 1 to 152) and a structured ␣-helix-rich C-terminal domain (aa 153 to 455). The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II (CKII) at S21, T32, and S43, and other cellular kinases at S95 and S97 residues in proximity of the caspase-7 cleavage site, 30-DETD-33, inhibits caspase-7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57, and the critical region for this function was mapped carefully to ␣-helices 7 to 9. Introduction of point mutations into ␣-helix 7 at ORF57 aa 280 to 299, a region highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C terminus prevents ORF57 from degrading and allows two structure-free N termini of the dimerized ORF57 to work coordinately for host factor interactions, leading to productive KSHV lytic infection and pathogenesis. IMPORTANCEKSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. The KSHV ORF57 protein is essential for KSHV lytic replication, as it regulates the expression of viral lytic genes at the posttranscriptional level. This report provides evidence that the structural conformation of the ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase-7. The C-terminal domain of ORF57, which is rich in ␣-helices, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of the ORF57 structure not only enables us to better understand ORF57 stability and functions but also provides an important tool for us to modulate ORF57's activity with the aim to inhibit KSHV lytic replication. K aposi's sarcoma-associated herpesvirus (KSHV) ORF57 (also known as Mta) is expressed early in the KSHV lytic cycle and is required for the efficient expression of a subset of viral genes, including KSHV PAN, ORF59, K8, viral interleukin-6 (vIL-6), ORF47, and others (1-7). A KSHV genome lacking ORF57 expression is associated with a defective lytic cycle incapable of producing infectious virions (8, 9).KSHV ORF57 functions as a posttranscriptional regulator of viral gene expression by affecting RNA stability (PAN, ORF59, and ORF47), splicing (ORF50 and K8), polyadenylation (ORF59), and translation (vIL-6) (1, 2, 4-7, 10) but ...