Functional Analysis of Epstein-Barr Virus SM Protein: Identification of Amino Acids Essential for Structure, Transactivation, Splicing Inhibition, and Virion Production

Abstract

The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxyterminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication.

Epstein-Barr Virus (EBV) SM Protein Induces and Recruits Cellular Sp110b To Stabilize mRNAs and Enhance EBV Lytic Gene Expression

“…The SM-deleted EBV which was generated in 293 cells was passaged into AGS cells, and EBV infection was confirmed by observation of green fluorescent protein expression and detection of EBV antigen by immunofluorescence microscopy (data not shown). The SM-deleted virus is defective for EBV lytic replication unless complemented with SM (19,39). Upon induction of lytic viral replication by transfection with BZLF1 and SM but not by BZLF1 alone, Sp110b mRNA levels were increased in both cell types ( Fig.…”

“…SM transactivates RL⌬i less strongly than it does the CAT reporter (39). It should also be noted that with both reporters, Sp110 or Sp110b in the absence of SM had little effect on reporter gene activity, indicating that Sp110 and Sp110b were not acting at the transcriptional level to increase the amounts of target gene mRNAs.…”

“…AGS cells, a gastric epithelial cell line (ATCC CRL-1739), and AGS cells carrying an SM-knockout recombinant EBV (SMKO-AGS) were grown in F-12 nutrient mixture (Intvitrogen) supplemented with 10% fetal calf serum and glutamine. SMKO-AGS cells were generated by infecting AGS cells with supernatant from SMKO-293 cells which were induced to permit lytic EBV replication by transfection with Z and SM expression plasmids as previously described (19,39). , P3HR1, an EBV-positive Burkitt's lymphoma-derived cell line (36), and BJAB, an EBV-negative B lymphoma cell line (33), were grown in RPMI (Invitrogen) supplemented with 10% fetal calf serum and glutamine.…”

“…SM increases the accumulation of several viral intronless lytic mRNA transcripts and specific cellular transcripts in both the nucleus and cytoplasm (38,40,42). SM also inhibits splicing and inhibits expression of the majority of cellular genes (38,39). Thus, SM affects multiple cellular pathways involved in RNA processing and transport.…”

General and Target-Specific RNA Binding Properties of Epstein-Barr Virus SM Posttranscriptional Regulatory Protein

“…Furthermore, the presence of these sequences conferred responsiveness to SM. These data are consistent with a mechanism of action similar to that of hnRNPs, which exert sequence-specific effects on gene expression despite having multiple degenerate consensus binding sites common to a large number of RNAs.Epstein-Barr virus (EBV) SM is an RNA binding protein that is essential for lytic EBV replication (1,5,6,8,14,15,17,(33)(34)(35)42). The SM gene is homologous to immediate-early or early genes expressed by several other human and animal herpesviruses, including those encoding herpes simplex virus type 1 (HSV-1) ICP27, human cytomegalovirus (HCMV) UL69, varicella-zoster virus open reading frame 4 (ORF4) protein, and Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein/Mta (2,24,27,30,36,47).…”