Limits...
Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

View Article: PubMed Central - PubMed

ABSTRACT

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.

No MeSH data available.


Related in: MedlinePlus

Virus vs. human selenoprotein antisense interactions demonstrated at the DNA level for the Ebola nucleoprotein and HIV nef regions shown in Fig. 1. Target-specific in vitro DNA hybridization of the cognate virus-selenoprotein pairs is shown by gel shift assay using ~40mer synthetic single stranded ssDNA oligos. The matched pairs of oligos from the respective viral and host mRNAs, corresponding to the sequence regions involved in the antisense interactions shown in Fig. 1, were as follows: EBOV nucleoprotein (Enp) vs. TR3, and HIV-1 nef/LTR region (Hnf) vs. TR1. Lanes have either a single oligo that runs as ssDNA (the lowest bands) or an incubated pair of oligos, as follows: 1. Enp; 2. 1:1 Enp+TR3; 3. 2:1 Enp+TR3; 4. TR3; 5. Hnf; 6. 1:1 Hnf+TR1; 7. 2:1 Hnf+TR1; 8. TR1; 9. 1:1 Enp+TR3 plus sheered herring sperm DNA; 10. 1:1 Hnf+TR1 plus herring DNA; 11. 1:1 Enp+TR1; 12. 1:1 Hnf+TR3; 13. 1:1 Hnf+Enp; 14. DNA size markers. The bright bands at the size of ~40mer double stranded dsDNA correspond to the expected hybridizations (Enp+TR3 or Hnf+TR1) at 1:1 (lanes 2,6,9,10) and 2:1 ratios (lanes 3,7). The faint bands above the 50 size marker (e.g. lanes 2,3,6,7) are possibly from trimer formation (e.g. TR3+Enp+TR3, etc.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4997913&req=5

Figure 2: Virus vs. human selenoprotein antisense interactions demonstrated at the DNA level for the Ebola nucleoprotein and HIV nef regions shown in Fig. 1. Target-specific in vitro DNA hybridization of the cognate virus-selenoprotein pairs is shown by gel shift assay using ~40mer synthetic single stranded ssDNA oligos. The matched pairs of oligos from the respective viral and host mRNAs, corresponding to the sequence regions involved in the antisense interactions shown in Fig. 1, were as follows: EBOV nucleoprotein (Enp) vs. TR3, and HIV-1 nef/LTR region (Hnf) vs. TR1. Lanes have either a single oligo that runs as ssDNA (the lowest bands) or an incubated pair of oligos, as follows: 1. Enp; 2. 1:1 Enp+TR3; 3. 2:1 Enp+TR3; 4. TR3; 5. Hnf; 6. 1:1 Hnf+TR1; 7. 2:1 Hnf+TR1; 8. TR1; 9. 1:1 Enp+TR3 plus sheered herring sperm DNA; 10. 1:1 Hnf+TR1 plus herring DNA; 11. 1:1 Enp+TR1; 12. 1:1 Hnf+TR3; 13. 1:1 Hnf+Enp; 14. DNA size markers. The bright bands at the size of ~40mer double stranded dsDNA correspond to the expected hybridizations (Enp+TR3 or Hnf+TR1) at 1:1 (lanes 2,6,9,10) and 2:1 ratios (lanes 3,7). The faint bands above the 50 size marker (e.g. lanes 2,3,6,7) are possibly from trimer formation (e.g. TR3+Enp+TR3, etc.).

Mentions: Target-specific in vitro DNA hybridization of the cognate virus-selenoprotein pairs was demonstrated by electrophoretic mobility shift assays, using ~40mer synthetic single stranded ssDNA oligomers (Integrated DNA Technologies, Inc., Coralville, IA). Oligos (~1 μg each in 10 μl PBS), either singly or in cognate or mismatched pairs, were incubated at 37°C for 15 hours, except those in lanes 9 and 10 (Fig. 2), which were heated to 90°C for 10 minutes in the presence of 10 μg of sheered herring sperm DNA (Promega D1811, Madison, WI), followed by cooling to room temperature over 1 hour. The matched pairs of oligos from the respective viral and host mRNAs, corresponding to the sequence regions involved in the antisense interactions shown in Fig. 1, were as follows: EBOV nucleoprotein (Enp) vs. TR3, and HIV-1 nef/LTR region (Hnf) vs. TR1. Mismatched (non-cognate) mRNA pairs, e.g. Enp vs. TR1, were used as negative controls (see legend to Fig. 2 for details). Separation was on a 4% agarose gel with ethidium bromide visualization. GeneRuler 1 kb Plus DNA ladder (Thermo Scientific, Waltham, MA) was used as a guide.


Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry
Virus vs. human selenoprotein antisense interactions demonstrated at the DNA level for the Ebola nucleoprotein and HIV nef regions shown in Fig. 1. Target-specific in vitro DNA hybridization of the cognate virus-selenoprotein pairs is shown by gel shift assay using ~40mer synthetic single stranded ssDNA oligos. The matched pairs of oligos from the respective viral and host mRNAs, corresponding to the sequence regions involved in the antisense interactions shown in Fig. 1, were as follows: EBOV nucleoprotein (Enp) vs. TR3, and HIV-1 nef/LTR region (Hnf) vs. TR1. Lanes have either a single oligo that runs as ssDNA (the lowest bands) or an incubated pair of oligos, as follows: 1. Enp; 2. 1:1 Enp+TR3; 3. 2:1 Enp+TR3; 4. TR3; 5. Hnf; 6. 1:1 Hnf+TR1; 7. 2:1 Hnf+TR1; 8. TR1; 9. 1:1 Enp+TR3 plus sheered herring sperm DNA; 10. 1:1 Hnf+TR1 plus herring DNA; 11. 1:1 Enp+TR1; 12. 1:1 Hnf+TR3; 13. 1:1 Hnf+Enp; 14. DNA size markers. The bright bands at the size of ~40mer double stranded dsDNA correspond to the expected hybridizations (Enp+TR3 or Hnf+TR1) at 1:1 (lanes 2,6,9,10) and 2:1 ratios (lanes 3,7). The faint bands above the 50 size marker (e.g. lanes 2,3,6,7) are possibly from trimer formation (e.g. TR3+Enp+TR3, etc.).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997913&req=5

Figure 2: Virus vs. human selenoprotein antisense interactions demonstrated at the DNA level for the Ebola nucleoprotein and HIV nef regions shown in Fig. 1. Target-specific in vitro DNA hybridization of the cognate virus-selenoprotein pairs is shown by gel shift assay using ~40mer synthetic single stranded ssDNA oligos. The matched pairs of oligos from the respective viral and host mRNAs, corresponding to the sequence regions involved in the antisense interactions shown in Fig. 1, were as follows: EBOV nucleoprotein (Enp) vs. TR3, and HIV-1 nef/LTR region (Hnf) vs. TR1. Lanes have either a single oligo that runs as ssDNA (the lowest bands) or an incubated pair of oligos, as follows: 1. Enp; 2. 1:1 Enp+TR3; 3. 2:1 Enp+TR3; 4. TR3; 5. Hnf; 6. 1:1 Hnf+TR1; 7. 2:1 Hnf+TR1; 8. TR1; 9. 1:1 Enp+TR3 plus sheered herring sperm DNA; 10. 1:1 Hnf+TR1 plus herring DNA; 11. 1:1 Enp+TR1; 12. 1:1 Hnf+TR3; 13. 1:1 Hnf+Enp; 14. DNA size markers. The bright bands at the size of ~40mer double stranded dsDNA correspond to the expected hybridizations (Enp+TR3 or Hnf+TR1) at 1:1 (lanes 2,6,9,10) and 2:1 ratios (lanes 3,7). The faint bands above the 50 size marker (e.g. lanes 2,3,6,7) are possibly from trimer formation (e.g. TR3+Enp+TR3, etc.).
Mentions: Target-specific in vitro DNA hybridization of the cognate virus-selenoprotein pairs was demonstrated by electrophoretic mobility shift assays, using ~40mer synthetic single stranded ssDNA oligomers (Integrated DNA Technologies, Inc., Coralville, IA). Oligos (~1 μg each in 10 μl PBS), either singly or in cognate or mismatched pairs, were incubated at 37°C for 15 hours, except those in lanes 9 and 10 (Fig. 2), which were heated to 90°C for 10 minutes in the presence of 10 μg of sheered herring sperm DNA (Promega D1811, Madison, WI), followed by cooling to room temperature over 1 hour. The matched pairs of oligos from the respective viral and host mRNAs, corresponding to the sequence regions involved in the antisense interactions shown in Fig. 1, were as follows: EBOV nucleoprotein (Enp) vs. TR3, and HIV-1 nef/LTR region (Hnf) vs. TR1. Mismatched (non-cognate) mRNA pairs, e.g. Enp vs. TR1, were used as negative controls (see legend to Fig. 2 for details). Separation was on a 4% agarose gel with ethidium bromide visualization. GeneRuler 1 kb Plus DNA ladder (Thermo Scientific, Waltham, MA) was used as a guide.

View Article: PubMed Central - PubMed

ABSTRACT

Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation.

No MeSH data available.


Related in: MedlinePlus