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The combined effect of environmental and host factors on the emergence of viral RNA recombinants.

Jaag HM, Nagy PD - PLoS Pathog. (2010)

Bottom Line: Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication.Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication.Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, University of Kentucky, Plant Science Building, Lexington, Kentucky, United States of America.

ABSTRACT
Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.

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Increased stability of the TBSV repRNA in met22Δ yeast.(A) The DI-72 repRNA was expressed in both BY4741 parental strain and met22Δ strain from the GAL1 promoter (in the absence of p92), followed by suppression of repRNA synthesis by glucose. Total RNA samples were analyzed at various time points (as shown) by Northern blotting using a DI-72(+) repRNA-specific probe. The ribozyme cleaved repRNA was quantified and the half-life of the repRNA is shown in minutes. (B) Similar profile of recRNAs and degRNAs in double deletion and in xrn1Δ yeasts. Northern blot analysis was performed on total RNA samples from the shown yeast strains replicating DI-72 repRNA. See further details in Fig. 1.
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ppat-1001156-g003: Increased stability of the TBSV repRNA in met22Δ yeast.(A) The DI-72 repRNA was expressed in both BY4741 parental strain and met22Δ strain from the GAL1 promoter (in the absence of p92), followed by suppression of repRNA synthesis by glucose. Total RNA samples were analyzed at various time points (as shown) by Northern blotting using a DI-72(+) repRNA-specific probe. The ribozyme cleaved repRNA was quantified and the half-life of the repRNA is shown in minutes. (B) Similar profile of recRNAs and degRNAs in double deletion and in xrn1Δ yeasts. Northern blot analysis was performed on total RNA samples from the shown yeast strains replicating DI-72 repRNA. See further details in Fig. 1.

Mentions: To test this model, we estimated the half-life of TBSV RNAs in met22Δ yeast. Indeed, the stability of TBSV repRNA increased by ∼3-fold in met22Δ when compared with the wt yeast (Fig. 3A, lanes 6–10 versus 1–5). The increased half-life for TBSV repRNA is in agreement with the possible inhibition of Xrn1p activity. In addition, the double-deletion (met22Δ xrn1Δ) yeast supported increased level of recRNA accumulation (by 26-fold, Fig. 3B, lanes 1–3) when compared with BY4741 (see Fig. 1, lane 6), similar to the high recombination rate in single-deletion met22Δ yeast or in xrn1Δ yeast (Fig. 3B). The profile of TBSV degRNAs accumulating in these yeasts suggest that the double-deletion strain is more similar to xrn1Δ than to met22Δ yeasts since met22Δ xrn1Δ yeast strain accumulates mostly the longer degRNA1 product (Fig. 3B). It is proposed that the degRNA1 product is due to a cleavage by an endoribonuclease [34]. On the contrary, met22Δ yeast accumulates mostly the shorter degRNA2 product, suggesting that a limited 5′-to-3′ degradation of degRNA1 by the incompletely inhibited Xrn1p nuclease still takes place in met22Δ yeast to give rise to degRNA2. Also, over-expression of Met22p in xrn1Δ yeast did not result in decreased level of recRNA accumulation (not shown), unlike when Met22p was expressed in the met22Δ yeast strain (Fig. 2B, lanes 15–16). Altogether, these data support the model that MET22 and XRN1 are both inhibitors of TBSV recombination and they act in the same pathway.


The combined effect of environmental and host factors on the emergence of viral RNA recombinants.

Jaag HM, Nagy PD - PLoS Pathog. (2010)

Increased stability of the TBSV repRNA in met22Δ yeast.(A) The DI-72 repRNA was expressed in both BY4741 parental strain and met22Δ strain from the GAL1 promoter (in the absence of p92), followed by suppression of repRNA synthesis by glucose. Total RNA samples were analyzed at various time points (as shown) by Northern blotting using a DI-72(+) repRNA-specific probe. The ribozyme cleaved repRNA was quantified and the half-life of the repRNA is shown in minutes. (B) Similar profile of recRNAs and degRNAs in double deletion and in xrn1Δ yeasts. Northern blot analysis was performed on total RNA samples from the shown yeast strains replicating DI-72 repRNA. See further details in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958810&req=5

ppat-1001156-g003: Increased stability of the TBSV repRNA in met22Δ yeast.(A) The DI-72 repRNA was expressed in both BY4741 parental strain and met22Δ strain from the GAL1 promoter (in the absence of p92), followed by suppression of repRNA synthesis by glucose. Total RNA samples were analyzed at various time points (as shown) by Northern blotting using a DI-72(+) repRNA-specific probe. The ribozyme cleaved repRNA was quantified and the half-life of the repRNA is shown in minutes. (B) Similar profile of recRNAs and degRNAs in double deletion and in xrn1Δ yeasts. Northern blot analysis was performed on total RNA samples from the shown yeast strains replicating DI-72 repRNA. See further details in Fig. 1.
Mentions: To test this model, we estimated the half-life of TBSV RNAs in met22Δ yeast. Indeed, the stability of TBSV repRNA increased by ∼3-fold in met22Δ when compared with the wt yeast (Fig. 3A, lanes 6–10 versus 1–5). The increased half-life for TBSV repRNA is in agreement with the possible inhibition of Xrn1p activity. In addition, the double-deletion (met22Δ xrn1Δ) yeast supported increased level of recRNA accumulation (by 26-fold, Fig. 3B, lanes 1–3) when compared with BY4741 (see Fig. 1, lane 6), similar to the high recombination rate in single-deletion met22Δ yeast or in xrn1Δ yeast (Fig. 3B). The profile of TBSV degRNAs accumulating in these yeasts suggest that the double-deletion strain is more similar to xrn1Δ than to met22Δ yeasts since met22Δ xrn1Δ yeast strain accumulates mostly the longer degRNA1 product (Fig. 3B). It is proposed that the degRNA1 product is due to a cleavage by an endoribonuclease [34]. On the contrary, met22Δ yeast accumulates mostly the shorter degRNA2 product, suggesting that a limited 5′-to-3′ degradation of degRNA1 by the incompletely inhibited Xrn1p nuclease still takes place in met22Δ yeast to give rise to degRNA2. Also, over-expression of Met22p in xrn1Δ yeast did not result in decreased level of recRNA accumulation (not shown), unlike when Met22p was expressed in the met22Δ yeast strain (Fig. 2B, lanes 15–16). Altogether, these data support the model that MET22 and XRN1 are both inhibitors of TBSV recombination and they act in the same pathway.

Bottom Line: Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication.Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication.Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, University of Kentucky, Plant Science Building, Lexington, Kentucky, United States of America.

ABSTRACT
Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.

Show MeSH
Related in: MedlinePlus