41 research outputs found

    Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane

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    <div><p>RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of <i>PAH1</i> (<u>p</u>hosphatidic <u>a</u>cid phospho<u>h</u>ydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in <i>pah1Δ</i> yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in <i>pah1</i>Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the <i>Arabidopsis</i> Pah2p in <i>Nicotiana benthamiana</i> decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.</p></div

    Inhibition of tombusvirus and RCNMV RNA accumulation in plants by over-expression of AtPah2p in <i>N. benthamiana</i>.

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    <p>(A) Expression of the yeast <i>PAH1</i> homolog AtPah2p (lanes 5–8) was done in <i>N. benthamiana</i> leaves by agroinfiltration. Two days later, the same leaves were infiltrated with <i>Agrobacterium</i> carrying a plasmid to launch CNV replication from the 35S promoter. The control samples were obtained from leaves expressing no proteins (lanes 1–4). Total RNA was extracted from leaves 5 days after agroinfiltration that launched CNV replication. The accumulation of CNV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting (Top panel). The ribosomal RNA (rRNA) was used as a loading control and shown in agarose gel stained with ethidium-bromide (Second panel). (B) Over-expression of AtPah2p in <i>N. benthamiana</i> protects the plant from rapid necrosis caused by systemic CNV infection. The pictures were taken 12 days after agroinfiltration. (C) Inhibition of the peroxisomal TBSV replication by over-expression of AtPah2p in <i>N. benthamiana</i>. The agro-infiltrated leaves were inoculated with TBSV two days later, followed by sampling of the same leaves after 3 day of incubation. The accumulation of TBSV gRNA and subgenomic (sg)RNAs in <i>N. benthamiana</i> leaves was measured by Northern blotting. See additional details in panel A. (D) The lack of inhibition of the mitochondrial CIRV tombusvirus by over-expression of AtPah2p in <i>N. benthamiana</i>. See additional details in panel A. (E) Inhibition of the distantly related RCNMV (which uses ER membranes for replication) by over-expression of AtPah2p in <i>N. benthamiana</i>. The agro-infiltrated leaves were inoculated with RCNMV two days later, followed by sampling of the same leaves after 3 day of incubation. The accumulation of RCNMV RNA1 in <i>N. benthamiana</i> leaves was measured by Northern blotting. See additional details in panel A.</p

    Deletion of the single yeast lipin gene (<i>PAH1</i>) enhances TBSV repRNA accumulation.

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    <p>(A) The role of Pah1p phosphatidate phosphatase and Dgk1p diacylglycerol kinase in lipid synthesis in yeast. To convert phosphatidic acid (PA) to diacylglycerol (DAG), Pah1p is dephosphorylated (activated) by the ER-localized Nem1p/Spo7p complex. (B) Top panel: Replication of the TBSV repRNA in wt and <i>pah1Δ</i> yeast was measured by Northern blotting 24 h after initiation of TBSV replication. Yeast co-expressed the TBSV (lanes 1–6) and the CNV (lanes 7–12) p33 and p92 replication proteins. The accumulation level of repRNA was normalized based on the ribosomal (r)RNA. Each sample is obtained from different yeast colonies. Middle and bottom panels: The accumulation levels of FLAG-p92 and 6×His-p33 were tested by Western blotting. Each experiment was repeated. (C–D) Expression of wt Pah1p and a phosphorylation deficient, constitutively active Pah1p, called Pah1-7A, which contains alanine substitutions for all seven phosphorylation sites, reduces TBSV replication in <i>pah1Δ</i> and wt yeasts. Northern blotting was done as in panel B. (E) Stimulatory effect of deletion of <i>NEM1</i> and <i>SPO7</i>, which form the dephosphorylation complex in the ER membrane, on TBSV repRNA accumulation is shown by Northern blotting. Note that Nem1p and Spo7p are required to dephosphorylate Pah1p, leading to the activation and relocalization of Pah1p from the cytosol to the ER membrane.</p

    Enhanced activity of the affinity-purified tombusvirus replicase assembled in CFE from <i>pah1Δ nem1Δ</i> yeast.

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    <p>(A) Scheme of the tombusvirus replicase assay. CFEs were prepared from wt and <i>pah1Δ nem1Δ</i> yeasts, followed by the addition of purified recombinant tombusvirus p33 and p92<sup>pol</sup> replication proteins and DI-72 (+)repRNA in the presence of rATP and rGTP. The <i>in vitro</i> assays were programmed with RI/III (−)RNA in the presence of rATP/rCTP/rGTP and <sup>32</sup>P-rUTP. (B) Top panel: Representative denaturing gel of <sup>32</sup>P-labeled RNA products synthesized by the purified tombusvirus replicase <i>in vitro</i>. Each experiment was repeated. Bottom panel: Western blot analysis of p33 in the shown replicase samples.</p

    Only a small portion of the tombusvirus p33 replication protein co-localize with peroxisomes in <i>pah1Δ nem1Δ</i> yeast.

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    <p>(A) YFP-tagged p33 and CFP-Pex13 (a peroxisomal membrane marker) were co-expressed in WT (RS453) or (B) <i>pah1Δ nem1Δ</i> yeasts. The confocal images were taken 24 hours after the induction of YFP-p33 expression at 23°C. See further details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003944#ppat-1003944-g005" target="_blank">Fig. 5</a>.</p

    Increased TBSV repRNA replication and enhanced p33 stability in yeast lacking Pah1p.

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    <p>(A–C) Time points experiments to show the accumulation levels of the TBSV repRNA, 6×His-p33 (CNV) and 6×His-p92 (CNV) in wt and <i>pah1Δ nem1Δ</i> yeasts. Asterisk marks a detergent-resistant p33 dimer band. Note that this mutant yeast behaves similarly to <i>pah1Δ</i> yeast during tombusvirus replication. See details in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003944#ppat-1003944-g001" target="_blank">Fig. 1B</a>. (D) The scheme of the <i>in vitro</i> TBSV replication assay based on the isolated membrane fraction carrying the tombusvirus replicase and the bound RNA template. (E–F) Top panels: Increased <i>in vitro</i> replication of TBSV repRNA in the isolated membrane fraction from <i>pah1Δ nem1Δ</i> yeast when compared with that from wt yeast. Note that the levels of p92 replication protein were normalized as shown in the second panel. The third, fourth and fifth panels show the accumulation levels of 6×His-p33, and the cellular Sec61p (an ER marker) and Ssa1p Hsp70. Samples in panel E and F were taken at 5 and 24 h time points, respectively. (G) Increased stability of the p33 replication protein in yeast lacking Pah1p. The accumulation level of 6×His-p33 in wt and <i>pah1Δ nem1Δ</i> yeasts was measured by Western blotting at the shown time points. The production of 6×His-p33 (CNV) lasted for 3 h from the inducible <i>GAL1</i> promoter, followed by turning off transcription and stopping translation by changing the galactose media to a new media containing glucose and cyclohexamide (taken as “0 time point”). The p33 level at the 0 time point was taken as 100%.</p

    Generation of infectious recombinant Adeno-associated virus in <i>Saccharomyces cerevisiae</i>

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    <div><p>The yeast <i>Saccharomyces cerevisiae</i> has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast <i>S</i>. <i>cerevisiae</i> would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.</p></div

    Encapsidation of AAV DNA.

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    <p>(A) Analysis of GFP DNA co-purified with AAV capsids. AAV capsids were purified by AVB affinity chromatography from yeast carrying plasmids DB021, DB027, DB029 and DB040 (No AAP); DB155, DB149, DB029 and DB040 (All AAV); pESC-HIS, DB022, DB023, DB040 (No VPs); DB155, DB149, DB028 and DB040 (No Rep78). GFP DNA was quantified by ddPCR while capsids were quantified by ELISA. Asterisks (*) indicate capsid content bellow the limit of detection. (B) Detection by western blot of VP proteins in affinity purified capsids from yeast carrying plasmids DB155, DB149, DB029 and DB040 (All AAV) or from yeast carrying plasmids pESC-HIS, pESC-LEU, DB081 and DB040 (Rep78). Note that plasmid DB081 contains a VP2 expression cassette, however VP2 alone does not form capsids and is not purified by AVB affinity chromatography. (C) Detection by southern blot of GFP DNA co-purified with AAV capsids. Samples were treated with benzonase (b) or not treated before DNA extraction and run as single stranded DNA on alkaline gels. A band of the expected size for the ITR-GFP-ITR rAAV DNA product was observed (white arrow) as well as additional material of smaller size (black arrow) which is likely the result of partial encapsidation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173010#pone.0173010.ref008" target="_blank">8</a>]. Refer to panel B for sample descriptions.</p

    Replication of AAV DNA in yeast.

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    <p>(A) Southern blot with a GFP specific probe of yeast samples carrying plasmids DB046, DB026, DB028 and DB040 (No Rep) or DB046, DB027, DB029 and DB040 (Rep78, Rep52). The top band (black arrow) corresponds to plasmid DB040-pAAV-GFP-2mic-URA3 (pAAV-GFP/URA3). The bottom band (white arrow) likely corresponds to the rescued ITR-GFP-ITR DNA. The middle band (*) could be a dimer of the ITR-GFP-ITR DNA. (B) Western blot with 303.9 anti-Rep antibody of samples from yeast carrying plasmids DB046, DB026, DB028 and DB040 (No Rep); DB046, DB027, DB029 and DB040 (Rep78, Rep52); DB046, DB027, DB135 and DB040 (Rep78-op, Rep52); DB046, DB138, DB029 and DB040 (Rep78, Rep52-op); DB046, DB138, DB135 and DB040 (Rep78-op, Rep52-op). Additional bands not consistent with the expected size for Rep78 and Rep52 were also observed (*). (C) Southern blot with a GFP specific probe of yeast samples described above. See band descriptions in panel A and sample descriptions in panel B. (D) quantification by ddPCR of GFP and URA3 DNA species. ddPCR was performed with primers and TaqMan probes specific for GFP, URA3 and 18S ribosomal DNA sequences. Black bars indicate the average (2 to 3 samples) GFP copy number relative to 18S rDNA copy number. White bars indicate the average URA3 copy number relative to 18S rDNA. The ratio of GFP to URA3 copies is indicated below the diagram. See sample descriptions in panel B.</p

    Detection of AAV proteins expressed in yeast.

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    <p>Yeast samples were processed for SDS-PAGE and western blot. (A) Detection of VP capsid proteins with B1 antibody. Samples from yeast carrying plasmids pESC-HIS, DB022, DB023 and DB040 (left lane, No VPs) or DB046, DB027, DB029 and DB040 (right lane, VPs). (B) Detection of HA-tagged AAP with anti-HA antibody. Samples from yeast carrying plasmids DB232, DB138, DB029 and DB040 (left lane, AAP-HA); DB046, DB228, DB029 and DB040 (middle lane, AAP no HA) or DB233, DB138, DB029 and DB040 (right lane, AAP-op-HA). (C) Detection of Rep proteins with 303.1 anti-Rep antibody. Samples from yeast carrying plasmids DB046, DB026, DB028 and DB040 (left lane, No Rep) or DB046, DB027, DB029 and DB040 (right lane, Rep78/Rep52).</p
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