Tanel Punga

The adenovirus-2 E1B-55K protein interacts with a mSin3A/histone deacetylase 1 complex.

The adenovirus E1B‐55K protein is a multifunctional phosphoprotein that regulates nuclear to cytoplasmic export of host cell and viral mRNAs during lytic viral growth. E1B‐55K also blocks apoptosis by binding and functionally inactivating the human tumor suppressor protein p53. Here, we show that E1B‐55K interacts with histone deacetylase 1 (HDAC1) and the transcriptional corepressor protein mSin3A, both in the adenovirus‐transformed 293 cell line and during a lytic adenovirus infection. Furthermore, we show that the central amino acids 156–261 in E1B‐55K are necessary for efficient HDAC1 interaction. Importantly, the E1B‐55K/mSin3A/HDAC1 complex is also enzymatically active, catalyzing deacetylation of a histone substrate peptide. Collectively, our results suggest that E1B‐55K interaction with mSin3A/HDAC1 containing complexes may be significant for one or several of the multiple activities ascribed to this protein.

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus‐infected cells (SR‐Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus‐induced partial de‐phosphorylation. Here, we show that SR proteins purified from late vaccinia‐virus‐infected cells (SR‐VV) are also hypo‐phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR‐Ad proteins under conditions that restore the phospho‐epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re‐phosphorylation of SR‐VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.

Circulating miRNAs in myasthenia gravis: miR-150-5p as a new potential biomarker

Myasthenia gravis (MG) is a chronic autoimmune disorder where autoantibodies target the nicotinic acetylcholine receptors (AChR+) in about 85% of cases, in which the thymus is considered to play a pathogenic role. As there are no reliable biomarkers to monitor disease status in MG, we analyzed circulating miRNAs in sera of MG patients to find disease‐specific miRNAs.

Disease specific signature of circulating miR-150-5p and miR-21-5p in myasthenia gravis patients

PURPOSE Reliable biological markers for patients with the autoimmune neuromuscular disorder myasthenia gravis (MG) are lacking. We determined whether levels of the circulating immuno-microRNAs miR-150-5p and miR-21-5p were elevated in sera from clinically heterogeneous MG patients, with and without immunosuppression, as compared to healthy controls and patients with other autoimmune disorders. METHODS Sera from 71 MG patients and 55 healthy controls (HC) were analyzed for the expression levels of miR-150-5p and miR-21-5p with qRT-PCR. Sera were also assayed from 23 patients with other autoimmune disorders (AID; psoriasis, Addisons and Crohns diseases). RESULTS 34 MG patients had no immunosuppressive drug treatment (MG-0) and 37 patients were under stable immunosuppressive drug treatment since ≥ 6 months (MG+IMM). Serum levels of miR-150-5p and miR-21-5p were higher in the MG-0 patients compared to HC (p<0.0001). Further, miR-150-5p levels were 41% lower and miR-21-5p levels were 25% lower in the MG+IMM compared to MG-0 (p=0.0051 and 0.0419). In the AID patients, mean miR-150-5p and miR-21-5p were comparable with healthy controls (p=0.66). CONCLUSIONS The immuno-microRNAs miR-150-5p and miR-21-5p show a disease specific signature, which suggests these microRNAs as possible biological autoimmune markers of MG.

Adenovirus 2 E1B-55K protein relieves p53-mediated transcriptional repression of the survivin and MAP4 promoters

It is well established that adenovirus E1B‐55K protein functions as an inhibitor of the tumor suppressor protein p53 by binding and inactivating p53 as a transcriptional activator protein. Here we show that the adenovirus 2 E1B‐55K protein also blocks p53 as a transcriptional repressor protein of the survivin and the MAP4 promoters. The repression is dependent on the ability of E1B‐55K to bind to p53 and is enhanced by coexpression of the adenovirus E4orf6 protein. Overexpression of the transcriptional corepressor protein Sin3A partially relieves the inhibitory effect of E1B‐55K, suggesting that E1B‐55K blocks p53 functions by interfering with the Sin3 complex.

Disease specific enrichment of circulating let-7 family microRNA in MuSK + myasthenia gravis

Myasthenia gravis (MG) patients with antibodies against the muscle specific tyrosine kinase (MuSK+) have predominantly involvement of cranio-bulbar muscles and do not display thymus pathology, as do acetylcholine receptor antibody seropositive (AChR+) MG patients. In search of novel biomarkers for MuSK+ MG, we evaluated circulating serum microRNAs. Four analyzed microRNAs were specifically elevated in MuSK+ MG patient serum samples: let-7a-5p, let-7f-5p, miR-151a-3p and miR-423-5p. The circulating microRNA profile in MuSK+ MG differs from the profile previously observed in the serum of AChR+ MG, thus indicating the etiological difference between these two entities. We propose that the identified microRNAs could serve as potential serum biomarkers for MuSK+ MG.

Two Cellular Protein Kinases, DNA-PK and PKA, Phosphorylate the Adenoviral L4-33K Protein and Have Opposite Effects on L1 Alternative RNA Splicing

Accumulation of the complex set of alternatively processed mRNA from the adenovirus major late transcription unit (MLTU) is subjected to a temporal regulation involving both changes in poly (A) site choice and alternative 3′ splice site usage. We have previously shown that the adenovirus L4-33K protein functions as an alternative splicing factor involved in activating the shift from L1-52,55K to L1-IIIa mRNA. Here we show that L4-33K specifically associates with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) in uninfected and adenovirus-infected nuclear extracts. Further, we show that L4-33K is highly phosphorylated by DNA-PK in vitro in a double stranded DNA-independent manner. Importantly, DNA-PK deficient cells show an enhanced production of the L1-IIIa mRNA suggesting an inhibitory role of DNA-PK on the temporal switch in L1 alternative RNA splicing. Moreover, we show that L4-33K also is phosphorylated by protein kinase A (PKA), and that PKA has an enhancer effect on L4-33K-stimulated L1-IIIa splicing. Hence, we demonstrate that these kinases have opposite effects on L4-33K function; DNA-PK as an inhibitor and PKA as an activator of L1-IIIa mRNA splicing. Taken together, this is the first report identifying protein kinases that phosphorylate L4-33K and to suggest novel regulatory roles for DNA-PK and PKA in adenovirus alternative RNA splicing.

Adenovirus Precursor pVII Protein Stability Is Regulated By Its Propeptide Sequence

Adenovirus encodes for the pVII protein, which interacts and modulates virus DNA structure in the infected cells. The pVII protein is synthesized as the precursor protein and undergoes proteolytic processing by viral proteinase Avp, leading to release of a propeptide sequence and accumulation of the mature VII protein. Here we elucidate the molecular functions of the propeptide sequence present in the precursor pVII protein. The results show that the propeptide is the destabilizing element targeting the precursor pVII protein for proteasomal degradation. Our data further indicate that the propeptide sequence and the lysine residues K26 and K27 regulate the precursor pVII protein stability in a co-dependent manner. We also provide evidence that the Cullin-3 E3 ubiquitin ligase complex alters the precursor pVII protein stability by association with the propeptide sequence. In addition, we show that inactivation of the Cullin-3 protein activity reduces adenovirus E1A gene expression during early phase of virus infection. Collectively, our results indicate a novel function of the adenovirus propeptide sequence and involvement of Cullin-3 in adenovirus gene expression.

Circulating microRNAs as potential biomarkers in myasthenia gravis patients

MicroRNAs (miRNAs) are small noncoding RNA molecules that bind to specific mRNA targets and regulate a wide range of important biological processes within cells. Circulating miRNAs are released into the extracellular space and can be measured in most biofluids, including blood serum and plasma. Recently, circulating miRNAs have emerged as easily accessible markers in various body fluids with different profiles and quantities specific for different human disorders, including autoimmune diseases. In myasthenia gravis (MG), diagnostic tests such as titers of serum autoantibodies specific for either the acetylcholine receptor (AChR+) or muscle‐specific tyrosine kinase (MuSK+) do not necessarily reflect disease progression, and there is a great need for reliable objective biomarkers to monitor the disease course and therapeutic response. Recent studies in AChR+ MG revealed elevated levels of the immuno‐miRNAs miR‐150‐5p and miR‐21‐5p. Of particular importance, levels of miR‐150‐5p were lower in immunosuppressed patients and in patients with clinical improvement following thymectomy. In MuSK+ MG, another profile of circulating miRNAs was found, including upregulation of the let‐7 family of miRNAs. Here, we summarize the potential role of circulating miRNAs as biomarkers in general and in MG, and highlight important considerations for the analysis of circulating miRNA.

Opposite expression of CYP51A1 and its natural antisense transcript AluCYP51A1 in adenovirus type 37 infected retinal pigmented epithelial cells

Cytochrome P450 family member CYP51A1 is a key enzyme in cholesterol biosynthesis whose deregulation is implicated in numerous diseases, including retinal degeneration. Here we describe that HAdV‐37 infection leads to downregulation of CYP51A1 expression and overexpression of its antisense non‐coding Alu element (AluCYP51A1) in retinal pigment epithelium (RPE) cells. This change in gene expression is associated with a reversed accumulation of a positive histone mark at the CYP51A1 and AluCYP51A1 promoters. Further, transient AluCYP51A1 RNA overexpression correlates with reduced CYP51A1 mRNA accumulation. Collectively, our data suggest that AluCYP51A1 might control CYP51A1 gene expression in HAdV‐37‐infected RPE cells.