The yeast mitochondrial porin represses Snf1/AMP kinase signaling to attenuate viral replication.

Introduction

Recent sequencing studies have revealed a vast array of RNA viruses that infect many fungal species ([SATO ANDSUZUKI2023](#ref-SATO ANDSUZUKI2023)). Fungal mycoviruses are well known to persist as endemic infections transmitted through cell division or fusion, with no known extracellular route. Although sometimes believed to be asymptomatic, mycoviruses can profoundly affect their fungal hosts and are better understood of as spanning the symbiotic spectrum as opposed to being inconsequential travelers ([WU ANDLI2025](#ref-WU ANDLI2025)). For example, mycovirus infections of the Rapeseed phytopathogenLeptosphaeria biglobosapromote survival of the fungus through seasonal periods of high temperature that intervene crop cycles ([ZHOUet al. 2026](#ref-ZHOUet al. 2026)). In other cases, mycovirus infection apparently disadvantage the host as seen in phytopathogens where ‘hypoviruses’ limit the fungus’s ability to mount robust plant infections (Kotta-Loizou2021). Recent findings show that mycoviruses in human fungal pathogens elicit an opposite effect, causing hyper-virulence in cell culture and mouse infection models ([LAUet al. 2018](#ref-LAUet al. 2018);[APPLENCLANCEYet al. 2020](#ref-APPLENCLANCEYet al. 2020);[PARKet al. 2020](#ref-PARKet al. 2020);[ROCHAet al. 2025](#ref-ROCHAet al. 2025)). Despite the emergence of mycoviruses as modulators of fungal pathogenesis, insights into how fungi control replication of these endosymbionts remain limited.

TheS. cerevisiaeL-A virus is the most comprehensively studied mycovirus, belonging to the broadly dispersedTotiviridaefamily of endogenous double stranded RNA (dsRNA) viruses. The 4.6 Kb L-A genome is encapsidated within a viral particle and extruded L-A transcripts encode for the capsid proteins (Gag) that form it. The L-A transcript also encodes a Gag-pol fusion protein that contains the canonical RNA dependent RNA polymerase (Pol) found in all RNA viruses. Each particle contains one or two Gag-pol proteins, which facilitates L-A genome replication and transcription. In many strains, L-A enables replication of satellite dsRNA segments called “M” that parasitize L-A viral particles. M satellites encode secreted toxins and a cell-autonomously acting toxin immunity factor. These “Killer” toxins thus cause lethality in neighboring sensitive yeast ([WICKNERet al. 2013](#ref-WICKNERet al. 2013)).

L-A replication is maintained at a low level through theSKI2, 3, and8genes, which encode subunits of a conserved translational surveillance complex that facilitates 3’-5’ exonucleolytic RNA decay and opposes the translation of transcripts that lack polyA tails like those encoded by L-A ([TOHet al. 1978](#ref-TOHet al. 1978);[BROWNet al. 2000](#ref-BROWNet al. 2000);[SEARFOSS ANDWICKNER2000](#ref-SEARFOSS ANDWICKNER2000)). Separate pathways of L-A attenuation act through Xrn1, a 5’-3’ exoribonuclease that degrades RNAs lacking 5’ methyl caps, and Nuc1, a versatile nuclease localized to the mitochondria ([BALLet al. 1984](#ref-BALLet al. 1984);[ROWLEYet al. 2016](#ref-ROWLEYet al. 2016);[GAOet al. 2019](#ref-GAOet al. 2019);[CHAUet al. 2023](#ref-CHAUet al. 2023)). Although L-A is asymptomatic in wild-type cells, in cells lackingNUC1, XRN1, and/orSKIgenes, high levels of L-A and/or of Killer toxin cause lethality at extreme temperatures or in meiotic spore progeny ([RIDLEYet al. 1984](#ref-RIDLEYet al. 1984);[EDWARDSet al. 2014](#ref-EDWARDSet al. 2014);[GAOet al. 2019](#ref-GAOet al. 2019);[CHAUet al. 2023](#ref-CHAUet al. 2023)).

Por1 is a well-known mitochondrial protein, but less well-characterized in the context of yeast antiviral responses. Earlier studies showed that L-A viral particles accumulate to high levels inpor1Δ mutant cells grown on the non-fermentable carbon glycerol for several days ([DIHANICHet al. 1987](#ref-DIHANICHet al. 1987);[DIHANICHet al. 1989](#ref-DIHANICHet al. 1989)).POR1encodes the yeast voltage dependent anion channel (VDAC), a highly abundant mitochondrial membrane protein. VDAC beta-barrel proteins span the outer mitochondrial membrane and control small molecule flux.POR1is essential for respiratory growth at high temperatures and plays important roles in mitochondrial osmotic stability, phospholipid metabolism, autophagy, mitochondrial protein import, and movement of phospholipids from one membrane bilayer leaflet to the other, a process known as lipid scrambling ([BLACHLY-DYSONet al. 1997](#ref-BLACHLY-DYSONet al. 1997);[SANCHEZet al. 2001](#ref-SANCHEZet al. 2001);[MIYATAet al. 2018](#ref-MIYATAet al. 2018);[SAKAUEet al. 2019](#ref-SAKAUEet al. 2019);[BROESKAMPet al. 2021](#ref-BROESKAMPet al. 2021);[JAHNet al. 2023](#ref-JAHNet al. 2023);[TAKEDAet al. 2025](#ref-TAKEDAet al. 2025)). In some contexts, VDACs assemble into higher order oligomers thought to facilitate movement of larger molecules, and recent findings show that Por1 lipid scramblase activity requires its oligomerized form ([JAHNet al. 2023](#ref-JAHNet al. 2023);[TAKEDAet al. 2025](#ref-TAKEDAet al. 2025)). If and how any of these functions relate to L-A repression is unknown.

Here we show thatPOR1prevents L-A from accumulating to high levels in stationary phase cells following sustained culture in standard growth media. Using genetic and biochemical experiments, we elucidate thatPOR1represses L-A by preventing hyper-activation of Snf1, the yeast homolog of AMP-activated Kinase (AMPK). By investigating Snf1 signaling, we show that thisPOR1-SNF1regulatory mechanism represses L-A replication in stationary phase cells by limiting amino acids. Our findings identify a novelPOR1-SNF1regulatory mechanism in budding yeast that may be similarly functional in other eukaryotes.

Materials and Methods

Yeast strains, media, and plasmids

StandardS. cerevisiaegenetic and strain manipulation techniques were used for strain construction. All strains are derivatives of BY4742 constructed through crossing and dissection. Generation of L-A cured strains was achieved using a previously described genetic backcrossing strategy ([GAOet al. 2019](#ref-GAOet al. 2019);[CHAUet al. 2023](#ref-CHAUet al. 2023)). Refer to Table S2 for all yeast strains used. For all experiments, strains were first grown in log phase for at least 10 doublings by serially splitting back of cultures. Time 0 for all stationary phase experiments corresponded to these log phase cells at an OD600of 1. All experiments were carried at 30 °C in synthetic complete media (SC; 0.348% yeast nitrogen base, 1% ammonium sulphate, and 2% glucose) with the appropriate amino acid powder mix (Sunrise Science) unless otherwise specified.

Measurement of yeast growth and physiological activity

Saturated cultures were diluted to an OD600of 0.1 in 200 µL in a 96-well plate. The plate was sealed a Breathe-Easy membrane (MilliporeSigma) and growth curve data was generated at 30 °C using an S&P growth curve robot (S&P Robotics Inc). Plates were shaken, and the optical density readings were taken every 15 min for 24 h. The data were plotted using R studio ggplot2. Glucose and ethanol concentration in media were measured using assay kits from MyBioSource (MBS8243232 and MBS8309715) according to the manufacturer’s instructions. The luminescent signals were detected using Varioskan LUX Multimode Microplate Reader (ThermoFisher).

Protein extraction and western blotting

Cells were harvested at indicated time points and permeabilized with 0.1N NaOH at room temperature for 5 min. The cells were then pelleted and resuspended in SDS/PAGE buffer. Cells were disrupted by bead-beating for 3 minutes before heating at 100°C for 10 minutes. The samples were centrifuged to isolate the soluble fraction for western blotting. Protein concentrations were determined with an RC/DC assay (BioRad 5000121). For Snf1-pT210 western blotting, cells were boiled at 100°C for 3 minutes before extraction.

Equal amounts of protein were electrophoresed on 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated in primary antibody at 4°C overnight and probed with 1:3,000 horseradish peroxidase (HRP)-conjugated horse anti-mouse (7075; Cell Signaling Technology) or goat anti-rabbit (7074; Cell Signaling Technology) secondary antibody. The proteins were detected with Luminata Forte Western HRP Substrate (EMD Millipore) and imaged with the Bio-Rad ChemiDoc XRS+ system. Images were processed with the Image Lab software package (Bio-Rad). The primary antibodies and their dilutions were 1:1,000 anti-FLAG M2 (F1804; Sigma-Aldrich), 1:1,000 anti-VDAC1/Porin (ab110326; Abcam), 1:5,000 anti-Pgk1 (ab113687; Abcam), 1:2,000 anti–L-A Gag (obtained from Reed Wickner), and 1:1000 anti-Phospho-AMPKα (Thr172) (2535; Cell Signaling Technology).

To quantify the relative fold change of protein levels, band intensities of protein of interest were measured in ImageJ and normalized to the band intensities of the corresponding loading control, Pgk1 protein. The average relative fold change and standard deviation was plotted using R studio ggplot2.

RT-qPCR

RNA was prepared from 10 ODs of cells harvested from 7-day cultures and used for RT-qPCR as previously described with some modifications. Briefly, harvested 7-day cell pellets were resuspended in Trizol (15596026; Invitrogen) and subjected to bead-beating (Mini Bead beater, Biospec Products) for 1 minute followed by 2-minute incubations on ice for 8 cycles. Samples were then incubated for 30 minutes at 65°C in acidic phenol (P4682; Sigma-Aldrich), SDS, and buffer AE (10mM Tris-HCl, 0.5mM EDTA pH 9.0) solution. The phase-separated supernatant was washed with chloroform and precipitated overnight. The precipitate was then washed in 70% ethanol and dissolved in water. RNA samples were purified with the RNeasy Mini Kit (74104; Qiagen), and residual DNA was digested with DNase I (79254; Qiagen). 900 nanograms of RNA was reverse transcribed using random nonamers and Maxima H Minus Reverse Transcriptase (EP0753; Thermo Fisher). The cDNA product was isolated by alkaline hydrolysis and treated with RNase A. Subsequently, qPCR was performed on 1/20 dilutions of cDNA product with the SensiFAST SYBR Hi-ROX Kit (BIO-92005; Meridian Bioscience) on the CFX384 platform (BioRad). The data were plotted using R studio ggplot2.

Spot analysis

Yeast strains were grown for 7 days at 30°C to the stationary phase. Each strain was diluted to an OD600= 0.4, serial diluted four times by 10-fold and spotted onto agar plates containing synthetic complete media supplemented with 2% glucose with or without 0.1mg/mL azetidine-2-carboxylic acid (AZC), as indicated.

Metabolite extractions and measurements by LC-MS/MS

Strains were grown overnight in YPAD. Saturated cultures were diluted to 0.02 and 0.05 OD for wild type andpor1Δ, respectively, and 5 ODs of cells were collected at the 24-hour time point. Intracellular metabolites were extracted from yeast cells, and quantitatively estimated using targeted, liquid chromatography–tandem mass spectrometry (LC-MS/MS) approaches described earlier, with samples collected from the respective genotype and at the indicated time point ([WALVEKARet al. 2018](#ref-WALVEKARet al. 2018);[RASHIDAet al. 2021](#ref-RASHIDAet al. 2021)). Specifically, equal numbers of cells (~2×10^7) were quenched in extraction buffer (60% methanol), extracted in 75% ethanol and dried down using a speed vacuum (rotatory evaporator). Metabolites were resuspended in mass spectrometry grade water and 10µl sample was injected for LC-MS/MS and separated using Synergi 4-µm Fusion-RP 80 Å (150 × 4.6 mm) LC column (Phenomenex, 00F-4424-E0). Solvents used: 0.1% formic acid in water (Solvent A) and 0.1% formic acid in methanol (Solvent B). An AB Sciex QTRAP 5500 with Shimadzu Nexera series UPLC system was used. Mass spectrometry data were acquired using analyst 1.6.2 software (Sciex). For analysis, Multi-Quant version 3.0.1 and Peak View version 2.0 were used.

Results

POR1represses Snf1/AMPK signaling in stationary phase to prevent L-A replication

Dihanich et al found thatPOR1repressed L-A after cells had adapted to respiratory metabolism over several days ([DIHANICHet al. 1987](#ref-DIHANICHet al. 1987)). To confirm these findings in the reference S288c strain background that is naturally infected with L-A we first characterized growth and metabolism of isogenic wild-type andpor1Δstrains through seven-day batch cultures. As expected, logarithmically growing wild-type cells vigorously fermented glucose to produce ethanol and ceased growth when glucose was exhausted (Figure 1A and 1B). Whilepor1Δdisplayed slower logarithmic growth and an according reduced rate of glucose consumption and ethanol production, both wild-type andpor1Δ expended all the glucose by 20 hours of culture (Figure 1A and 1B). Over the course of the following six days both wild-type andpor1Δ consumed all the ethanol indicating robust respiratory metabolism (Figure 1B).

*Por1 represses L-A replication in stationary phase cells.(a)Growth curves and media glucose concentration of wild type andpor1Δstrains is shown. Cells were grown in YPAD at 30°C for 24 hours. Density measurements of cell cultures were taken every 15 minutes, and glucose concentration in the media was measured at indicated time points. n = 3.(b)Media ethanol concentration of these exact cultures was measured.(c)Western blotting of L-A gag and Pgk1 protein levels in the indicated strains. Samples were collected from cultures grown at the indicated time points in SC media. Molecular weight markers are indicated on the right.(d)Quantification of L-A Gag levels normalized to Pgk1 protein level in indicated strains cultured for 7 days. Relative fold change to wild type and standard deviation are shown. n = 8. **p< 0.01 using unpaired student’s t-test.(e)RT-qPCR quantification of L-A RNA normalized to endogenousACT1RNA in the indicated strains cultured for 7 days. Mean RNA level and standard deviation are shown. n = 3. p< 0.05 using unpaired student’s t-test.

To investigatePOR1repression of L-A we measured L-A Gag levels using western blots in wild type andpor1Δstrains at intervals using these same culture conditions. We found a large increase of Gag levels inpor1Δ cells by 7 days of incubation (Figure 1C). In many replicates shown below, wild type Gag levels were undetectable in comparison to the large amount observed inpor1Δ. We measured relative Gag levels in 8 replicates in which Gag was detectable in the wild type strain and found a 20-fold increase inpor1Δ, though this seems likely to be an underestimate (Sure 1D). To confirm that this increase in Gag protein reflects higher viral copy number we measured L-A RNA levels using RT-qPCR, which revealed a 60-fold increase inpor1Δ compared with wild type (Figure 1E). S288c is infected with another totivirus called L-BC that is maintained at a significantly reduced copy number compared with L-A ([SOMMER ANDWICKNER1982](#ref-SOMMER ANDWICKNER1982)). RT-qPCR quantification of L-BC did not reveal any differences, perhaps suggesting that L-A outcompetes L-BC (Figure S1). These findings confirm that Por1 represses L-A viral replication in respiratory cells that have exhausted nutrients. These are typically referred to as stationary phase cells, known for their metabolic quiescence and enhanced stress resistance ([WERNER-WASHBURNEet al. 1993](#ref-WERNER-WASHBURNEet al. 1993)).

Progression into stationary phase involves massive gene expression reprogramming characterized by repression of nearly all transcription but activation of catabolic genes ([RADONJICet al. 2005](#ref-RADONJICet al. 2005)). The PAS family kinase Rim15 and the AMP-activated protein kinase Snf1 govern much of this gene expression program through phosphorylation of downstream transcriptional regulators ([GALDIERIet al. 2010](#ref-GALDIERIet al. 2010)). To investigate the roles ofSNF1andRIM15for viral replication in stationary phase, we compared the levels of L-A Gag in deletion mutants of these genes by themselves or in combination withpor1Δ. Deletion ofSNF1completely reversed the Gag accumulation phenotype ofpor1Δwhilerim15Δhad a comparatively minor effect (Figure 2A). This epistatic relationship suggests that Por1 represses Snf1 and that de-repressed Snf1 promotes L-A replication. To test this, we measured the accumulation of the active isoform of Snf1 phosphorylated on threonine-210 (Snf1-pT210) using western blotting experiments with an antibody that recognizes it ([MCCARTNEY ANDSCHMIDT2001](#ref-MCCARTNEY ANDSCHMIDT2001)). Supporting our model, we detected significantly higher levels of Snf1-pT210 throughout stationary phase inpor1Δcells compared with wild-type (Figure 2B, S3A-C). Moreover, Por1 levels were strongly induced in stationary phase exactly when our genetic results show that it represses Snf1-pT210 (Figure 2B, S3B). These findings demonstrate that Por1 represses Snf1 activation in post-diauxic stationary phase cells and thatSNF1function is required for L-A hyper-activation.

Por1 negatively regulates Snf1 activity to control L-A replication.(a)Western blotting of L-A Gag and Pgk1 protein levels in the indicated strains. Samples were collected from 7-day cultures grown in SC media. Molecular weight markers are indicated on the right.(b)Western blotting of phosphorylated Snf1, Snf1-FLAG, Pgk1, and Por1 protein levels in the indicated strains. Samples were collected from cultures grown for the indicated time points in YPAD media. Molecular weight markers are indicated on the right.(c)Schematic of Snf1 pathway. The Snf1 complex contains the alpha subunit Snf1, one of the three beta subunits Sip1/2 and Gal83, and the gamma subunit Snf4. The Snf1 complex regulates expression of target genes Pck1, Fbp1, and Icl1 by phosphorylating downstream transcription factors Cat8 and Sip4.(d)Western blotting of L-A Gag and Pgk1 protein levels in the indicated strains. Samples were collected from 7-day cultures grown in SC media. Molecular weight markers are indicated on the right.(e)Western blotting of L-A Gag and Pgk1 protein levels in the indicated strains. Samples were collected from 7-day cultures grown in SC media. Molecular weight markers are indicated on the right.

In addition to its activating phosphorylation, Snf1 is controlled through its association with co-factors in a conserved hetero-trimeric complex (Figure 2C). The gamma subunit Snf4 binds to Snf1’s autoinhibitory domain to relieve Snf1’s autoinhibition, while the beta subunits, Sip1, Sip2, and Gal83, control Snf1 activity in different subcellular localizations ([HEDBACKER ANDCARLSON2008](#ref-HEDBACKER ANDCARLSON2008)). We assessed the levels of L-A Gag inpor1Δstrains lacking these proteins to discern the contribution of Snf1 subunits for viral control. Like withsnf1Δ, snf4Δcompletely reversed the accumulation of high Gag levels caused bypor1Δ(Figure 2D). While none of the beta subunit deletions similarly reversed thepor1Δphenotype,gal83Δcaused a visibly decreased abundance of Gag (Figure 2D). Gal83 controls Snf1 function in the nucleus ([VINCENTet al. 2001](#ref-VINCENTet al. 2001)). These findings show that Snf1 cofactors are required for its proviral function when it is hyper-activated in cells lackingPOR1.

Snf1 promotes L-A replication through its glyoxylate cycle targets

To elucidate how hyper-activated Snf1 promotes L-A replication we investigated its well-characterized targets, the transcription factors Adr1, Cat8, and Sip4, which drive the expression of genes involved in alternative carbon utilization in glucose starved cells (Figure 2C) ([HEDBACKER ANDCARLSON2008](#ref-HEDBACKER ANDCARLSON2008)). To determine if these transcription factors mediate Snf1’s proviral activity, we assessed L-A Gag levels in double mutants combiningpor1Δ withcat8Δ,sip4Δ oradr1Δ. Whileadr1Δ had no consequence, the high Gag levels ofpor1Δ cells were reduced whenCAT8orSIP4were deleted with a marked effect caused bycat8Δ (Figure 2E). Cat8 and Sip4 regulate overlapping sets of genes involved in gluconeogenesis and the glyoxylate cycle (Figure 2C) ([HIESINGERet al. 2001](#ref-HIESINGERet al. 2001);[ROTHet al. 2004](#ref-ROTHet al. 2004)). To test if Cat8 and Sip4 redundantly mediate the proviral function of Snf1 we combined deletions of their shared targetsFBP1, PCK1, andICL1, withpor1Δ. FBP1andPCK1encode gluconeogenic proteins whileICL1encodes a key protein of the glyoxylate cycle. We found that the high Gag levels ofpor1Δ cells were completely reverted byicl1Δ with only weak or no consequence offbp1Δ orpck1Δ (Figure 3A). Collectively, these findings show thatPOR1prevents hyperactivation of Snf1 kinase in stationary phase cells, and that activated Snf1 promotes L-A replication in a manner dependent on its downstream target geneICL1.

L-A replication inpor1Δstationary phase requires the glyoxylate cycle.(a)Western blotting of L-A Gag and Pgk1 protein levels in the indicated strains. Samples were collected from 7-day cultures grown in SC media. Molecular weight markers are indicated on the right.(b)Schematic of the glyoxylate and TCA cycles. The intermediates and flow of carbon through both cycles are indicated. Enzymes tested to be required for viral replication are shown in red, while enzymes dispensable for viral replication are shown in blue.(c)Heat map of normalized fold change amino acid levels measured with LC-MS/MS (n=3). Prototrophic strains of the indicated genotype were used for the metabolic studies. Samples were collected from 24-hour cultures grown in SC media.

Icl1 catalyzes the conversion of isocitrate to succinate and glyoxylate, a key intermediate of a TCA-cycle shunt known as the glyoxylate cycle found in bacteria, fungi, plants, and some invertebrates (Figure 3B) ([DOLAN ANDWELCH2018](#ref-DOLAN ANDWELCH2018);[CHEWet al. 2019](#ref-CHEWet al. 2019)). The requirement ofICL1for L-A hyper-replication thus implicates the glyoxylate cycle in this process. We tested this through double mutant analysis combiningpor1Δ with other glyoxylate cycle gene deletions. As withicl1Δ, we found thatmls1Δ,mdh2Δ, orcit2Δ reversed thepor1Δ hyper-L-A phenotype (Figure 3Band S3A; Table S1). While Icl1, Mls1, and Cit2 all act in the cytosol, glyoxylate metabolism also occurs within peroxisomes through Dal7 and Mdh3 as well as the cytoplasmic/peroxisomal dual-localized Mdh2 ([KUNZEet al. 2006](#ref-KUNZEet al. 2006);[GABAY-MASKITet al. 2020](#ref-GABAY-MASKITet al. 2020)). Deletion ofMDH2, but not ofDAL7orMDH3, reversed L-A hyper-replication caused bypor1Δ (Figure 3Band S3A; Table S1).MDH2andMDH3encode malate dehydrogenases, and a third, Mdh1, localizes to the mitochondria where it functions in the TCA cycle. Deletion ofmdh1Δ similarly failed to revert thepor1Δ phenotype (Figure 3Band S3B; Table S1). These findings show that genes encoding cytosolic glyoxylate cycle proteins are crucial for L-A hyper-replication inpor1Δ cells.

Succinate produced by the glyoxylate cycle can be funneled to mitochondrial TCA cycle metabolism (Figure 3B). As the glyoxylate and TCA cycles are thus intertwined, we further tested the role of TCA cycle genes for L-A Gag accumulation caused bypor1Δ. Like withmdh1Δ, deletion of most TCA cycle genes failed to revert thepor1Δ phenotype (Figure 3B, S3B, S3C; Table S1). However, the TCA cycle mutantsfum1Δandsdh1Δdid revertpor1Δ(Figure 3B, S3B, S3C; Table S1). As Fum1 and Sdh1 act early in the TCA cycle to metabolize succinate, a plausible explanation for these findings may be through succinate buildup causing inhibition of Icl1 and/or other glyoxylate cycle enzymes ([HONERZU BENTRUPet al. 1999](#ref-HONERZU BENTRUPet al. 1999)). These findings refine a model in which Por1 repression of Snf1 prevents glyoxylate cycle activation that promotes L-A replication.

ThePOR1-SNF1regulatory system prevents L-A hyper-replication by limiting amino acid availability

How might the glyoxylate cycle promote L-A replication inpor1Δ cells? The glyoxylate cycle enables utilization of two-carbon units during glucose starvation, producing metabolic intermediates that support gluconeogenesis and amino acid biosynthesis ([CHEWet al. 2019](#ref-CHEWet al. 2019)). An increased glyoxylate cycle will lead to more oxaloacetate, which can directly be converted to aspartic acid (which in turn supports the biosynthesis of multiple amino acids, as well as sustaining alpha-ketoglutarate production leading to glutamate/glutamine synthesis). We therefore hypothesized that enhanced amino acid availability caused by increased glyoxylate cycle flux may fuel L-A replication inpor1Δ stationary phase cells. To test this, we first used LC-MS/MS to compare the levels of amino acids in post-diauxic wild type andpor1Δ strains 24 hours post-inoculation. Consistent with our hypothesis, we observed substantially increased steady-state pools of multiple amino acids inpor1Δ (Figure 3C).

We further investigated amino acid synthesis enzymes that utilize glyoxylate cycle produced precursors. Glyoxylate can be converted to glycine by the alanine-glyoxylate aminotransferase Agx1 while oxaloacetate is converted to aspartate through the mitochondrial and cytosolic localized aspartate aminotransferases Aat1 and Aat2, respectively (Figure 4A) ([MORINet al. 1992](#ref-MORINet al. 1992);[VERLEURet al. 1997](#ref-VERLEURet al. 1997);[Schlosseret al. 2004](#ref-Schlosseret al. 2004)). In contrast to glycine, aspartate is a versatile amino acid that can be used to produce numerous additional amino acids (Figure 4A). To test if glyoxylate cycle intermediates promote amino acid synthesis that enhance L-A replication, we combinedpor1Δ with deletions ofAAT1, AAT2, andAGX1to test for reversion of thepor1Δ phenotype. While deletion ofAAT1orAGX1had little effect on the levels of L-A Gag,aat2Δ reverted the accumulation of Gag inpor1Δ (Figure 4B). Like the required glyoxylate cycle proteins, Aat2 is cytosolic, suggesting that L-A replication in stationary phase requires the synthesis of aspartate from oxaloacetate in the cytosol.

Amino acid synthesis produces the biomolecules necessary for viral replication in a stationary phase.(a)Schematic of amino acids synthesis pathways from the glyoxylate cycle. Enzymes tested to be required for viral replication are shown in red, while enzymes dispensable for viral replication are shown in blue.(b-d)Western blotting of L-A Gag and Pgk1 protein levels in the indicated strains. Samples were collected from 7-day cultures grown in SC media. Molecular weight markers are indicated on the right. Additionally, samples were either supplemented with the indicated amount of(c)additional aspartic acid or(d)amino acids.(d)The ponceau stain of the blot is included below. The protein ladder and corresponding molecular weight are indicated on the left.

If Aat2 promotes L-A replication in stationary phase by promoting the synthesis of aspartate, then aspartate supplementation in the media might restore high L-A viral load inpor1Δ aat2Δ. Confirming this prediction,por1Δ aat2Δcultured in increasing amounts of aspartate showed a dose responsive increase in L-A Gag levels (Figure 4C). Since aspartate supplementation was insufficient to fully restore Gag levels inaat2Δ por1Δ, we tested if supplementing with the full complement of amino acids further enabled L-A replication. Remarkably, we found thataat2Δ por1Δsupplemented with 10x amino acids restored Gag accumulation comparable to as in apor1Δsingle mutant (Figure 4D). By treating these western blot membranes with the generic protein dye ponceau S, we found that Gag is the only protein that accumulates to high levels in these cells (Figure 4D).

High L-A levels are known to cause proteostatic stress that sensitize cells to the amino acid analog azetidine-2-carboxylic acid (AZC) ([CHAUet al. 2023](#ref-CHAUet al. 2023)). To test if high L-A has a similar consequence in stationary phase cells, we assayed them for growth following spotting onto SC plates with or without AZC. Wild type andpor1Δ strains exhibited moderate and severe growth defects in the presence of AZC respectively and the severe growth defect ofpor1Δ was reversed in strains cured of L-A (Figure S4). These results suggest that high viral burden inpor1Δ cells cause proteostatic stress.

To summarize, here we identify a new yeast antiviral mechanism controlled through Por1/VDAC mediated inhibition of Snf1/AMP Kinase in stationary phase cells. Consistent with many studies showing that Snf1 is activated by glucose starvation, we only observe accumulation of the activated phosphorylated form of Snf1 in post-diauxic phase cells that have consumed all the glucose. Following this transition to glucose starvation,por1Δ cells exhibit dramatically increased levels of Snf1-pT210 as well as a 60-fold increase in L-A, suggesting that activated Snf1 promotes L-A replication. Supporting this hypothesis, genetic epistasis experiments show that Snf1 acts with its co-factors and Cat8/Sip4 transcription factor targets to elevate L-A replication inpor1Δ cells. Through LC-MS/MS, supplementation and genetic experiments, we identify the glyoxylate cycle and amino acid production as the essential outputs of Snf1 signaling that foster L-A replication (Figure 5).

Model of Por1 repression on Snf1 pathway to limit L-A viral replication.In nutritionally starved cells, the AMPK Snf1 is activated to induce the glyoxylate cycle for the synthesis of amino acids. In wild type cells, Por1 negatively represses Snf1 activity to prevent overproduction of amino acids in stationary phase cells. However, when this process is not properly regulated inpor1Δstrains, the L-A virus hijacks the excess amino acid produced to enhance its replication success.

Discussion

One of the distinguishing features of mycoviruses is their endemic presence in strains once an infection is established. These infections persist on evolutionary timescales and can adaptively shape fungal physiology raising questions about how these long-term host-virus interactions are mediated in fungi ([WU ANDLI2025](#ref-WU ANDLI2025)). Here we identifyPOR1-SNF1signaling as a new yeast mechanism that prevents hyper-replication of the L-A totivirus through its control of cellular metabolism. Of note, we only observePOR1-SNF1signaling in nutrient exhausted stationary phase cells that have exited the cell cycle. In nature, fungal cells likely encounter such conditions frequently, representing a point of vulnerability where mycoviruses could opportunistically replicate in their dormant hosts. Physiological control mechanisms that limit bio-molecules essential for viral replication may thus represent a broadly useful strategy. Indeed, amino acid restriction acts as an antiviral strategy for bacterial phage and human HIV infections ([JIANGet al. 2017](#ref-JIANGet al. 2017);[FITZPATRICKet al. 2025](#ref-FITZPATRICKet al. 2025)). Given that Snf1 controls allocations towards amino acids after glucose is depleted, any mechanism that can modulate this might provide advantages to the virus during infections ([RASHIDAet al. 2021](#ref-RASHIDAet al. 2021)). Despite these connections however, our studies do not necessarily indicate that the sole function ofPOR1-SNF1signaling is antiviral and it is easy to envision other potential roles of this metabolic control system.

A pressing question concerns the molecular mechanisms by which Por1 represses Snf1. Two studies showed Snf1 mitochondrial localization and physical association of Snf1 with Por1 in cells undergoing acute glucose withdrawal ([STROGOLOVAet al. 2012](#ref-STROGOLOVAet al. 2012);[YIet al. 2017](#ref-YIet al. 2017)). Although neither study addressed Snf1 activation and signaling such as what we show here, they nevertheless are consistent with a proximal regulatory role of Por1 for Snf1.

Recent findings support a more vivid hypothesis ofPOR1-SNF1signaling and point to Por1’s control of lipid transport/metabolism as the underlying mechanism by which it represses Snf1. Thin layer chromatography and shotgun lipidomics experiments showed that deletion ofPOR1caused myriad defects in bulk phospholipid levels, including reductions in phosphatidylethanolamine (PE) and cardiolipin (CL) along with increased phosphatidic acid (PA) and several others ([MIYATAet al. 2018](#ref-MIYATAet al. 2018);[BROESKAMPet al. 2021](#ref-BROESKAMPet al. 2021);[DAVISet al. 2023](#ref-DAVISet al. 2023)). Por1 controls these processes in part through its physical interactions with the Ups1/2-Mdm35 intra-mitochondria shuttles that control transport of PA and phosphatidylserine from the outer mitochondrial membrane to the inner membrane ([MIYATAet al. 2018](#ref-MIYATAet al. 2018)). Notably, these studies investigated dividing cells, and our findings show Por1 function in stationary phase, a context known to involve massive lipid dynamics ([HOMANNet al. 1987](#ref-HOMANNet al. 1987);[KLOSEet al. 2012](#ref-KLOSEet al. 2012);[REINHARDet al. 2023](#ref-REINHARDet al. 2023)). A key insight comes from a study of Ups2-Mdm35 in stationary phase cells showing that its mutation caused PE accumulation and was associated with a modest increase in Snf1-pT210 levels, though not to the degree caused bypor1Δthat we report here ([MIYATAet al. 2022](#ref-MIYATAet al. 2022)). It was further shown that Snf1 binds to several phospholipids including PA and PEin vitrowith the implied hypothesis being that Snf1 activity is controlled through sensing of lipids differentially controlled by Ups2-Mdm35 ([MIYATAet al. 2022](#ref-MIYATAet al. 2022)).

All the above-mentioned studies preceded the discovery that Por1 possesses potent lipid scramblase activity in its oligomerized form and howPOR1influences lipid dynamics in stationary phase remains unknown ([JAHNet al. 2023](#ref-JAHNet al. 2023);[TAKEDAet al. 2025](#ref-TAKEDAet al. 2025)). We report here that Por1 levels are strongly increased in stationary phase, which may promote the formation of Por1 oligomers that activate its lipid scramblase activity. Pharmacological inhibition of human VDAC has been shown to cause increased Snf1 activity and the human Snf1 homolog AMPK is known to be allosterically activated by long-chain fatty acyl-CoA esters ([HEADet al. 2015](#ref-HEADet al. 2015);[PINKOSKYet al. 2020](#ref-PINKOSKYet al. 2020);[KOet al. 2024](#ref-KOet al. 2024)). Evidence from both yeast and human thus suggest an underlying universality ofPOR1-SNF1signaling.

Acknowledgments

We thank Dr. Alex Ensminger for use of the S&P growth curve robot.

Funding

This study was supported by grants from the Canadian Institutes of Health Research (PJT-18592) and Natural Sciences and Engineering Research Council (RGPIN-201) to M.D.M. S.L. is supported through a DBT-Wellcome Trust India Alliance Senior fellowship (IA/S/21/2/505922).

Data Availability Statement

Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article figures and tables. Supplemental material available at GENETICS online.

Associated Data

Data Availability Statement

Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article figures and tables. Supplemental material available at GENETICS online.

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Republished from the open web under CC-BY. Authors: Chau S, Marek S, Khanna A, Sathe J, Laxman S, Meneghini MD. Read the original.