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Recent studies on recombinant adeno-associated viral (rAAV) vector production demonstrated the generation of infectious viral particles in Saccharomyces cerevisiae. Proof-of-concept results showed low vector yields that correlated with low AAV DNA encapsidation rates. In an attempt to understand the host cell response to rAAV production, we profiled proteomic changes throughout the fermentation process by mass spectrometry. By comparing an rAAV-producing yeast strain with a respective non-producer control, we identified a subset of yeast host proteins with significantly different expression patterns during the rAAV induction period. Gene ontology enrichment and network interaction analyses identified changes in expression patterns associated mainly with protein folding, as well as amino acid metabolism, gluconeogenesis, and stress response. Specific fold change patterns of heat shock proteins and other stress protein markers suggested the occurrence of a cytosolic unfolded protein response during rAAV protein expression. Also, a correlative increase in proteins involved in response to oxidative stress suggested cellular activities to ameliorate the effects of reactive oxygen species or other oxidants. We tested the functional relevance of the identified host proteins by overexpressing selected protein leads using low- and high-copy number plasmids. Increased vector yields up to threefold were observed in clones where proteins SSA1, SSE1, SSE2, CCP1, GTT1, and RVB2 were overexpressed. Recombinant expression of SSA1 and YDJ insect homologues (HSP40 and HSC70, respectively) in Sf9 cells led to a volumetric vector yield increase of 50% relative to control, which validated the importance of chaperone proteins in rAAV-producing systems. Overall, these results highlight the utility of proteomic-based tools for the understanding and optimization of rAAV-producing recombinant strains., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The yeast Saccharomyces cerevisiae 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 S. cerevisiae 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.

Unlabelled: RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs., Importance: RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.

To identify new genes affecting Tomato bushy stunt virus (TBSV) replication in yeast model host, we are studying protein families, whose members have been identified during previous high throughput screening. In this paper, we have characterized the WW domain-containing protein family from yeast and plants. We find that, in addition to Rsp5 E3 ubiquitin ligase, yeast Wwm1 and Prp40 and three Arabidopsis WW domain-containing proteins are strong inhibitors of TBSV replication. The tombusvirus replicase complex isolated from yeast with down-regulated Wwm1 protein level was more active. Accumulation of viral p92(pol) was reduced when Wwm1 was over-expressed, suggesting that the stability of p92(pol) might be reduced, as observed with Rsp5. Moreover, replication of two insect RNA viruses is also inhibited by Wwm1 and Rsp5, suggesting that WW domain-containing proteins might have broad regulatory effects on RNA viruses. Thus, artificial antiviral proteins with WW domains could be useful antiviral strategy., (Copyright © 2012. Published by Elsevier Inc.)

To identify host proteins interacting with the membrane-bound replication proteins of tombusviruses, we performed membrane yeast two-hybrid (MYTH) screens based on yeast cDNA libraries. The screens led to the identification of 57 yeast proteins interacting with replication proteins of two tombusviruses. Results from a split ubiquitin assay with 12 full-length yeast proteins and the viral replication proteins suggested that the replication proteins of two tombusviruses interact with a similar set of host proteins. Follow-up experiments with the yeast Cpr1p cyclophilin, which has prolyl isomerase activity that catalyzes cis-trans isomerization of peptidyl-prolyl bonds, confirmed that Cpr1p interacted with the viral p33 replication protein in yeast and in vitro. Replication of Tomato bushy stunt virus replicon RNA increased in cpr1Δ yeast, while over-expression of Cpr1p decreased viral replication. We also show that the Ess1p parvulin prolyl isomerase partly complements Cpr1p function as an inhibitor of tombusvirus replication., (Copyright © 2010. Published by Elsevier Inc.)

To identify host proteins interacting with Tomato bushy stunt virus (TBSV) replication proteins in a genome-wide scale, we have used a yeast (Saccharomyces cerevisiae) proteome microarray carrying 4,088 purified proteins. This approach led to the identification of 58 yeast proteins that interacted with p33 replication protein. The identified host proteins included protein chaperones, ubiquitin-associated proteins, translation factors, RNA-modifying enzymes, and other proteins with yet-unknown functions. We confirmed that 19 of the identified host proteins bound to p33 in vitro or in a split-ubiquitin-based two-hybrid assay. Further analysis of Cdc34p E2 ubiquitin-conjugating enzyme, which is one of the host proteins interacting with p33, revealed that Cdc34p is a novel component of the purified viral replicase. Downregulation of Cdc34p expression in yeast, which supports replication of a TBSV replicon RNA (repRNA), reduced repRNA accumulation and the activity of the tombusvirus replicase by up to fivefold. Overexpression of wild-type Cdc34p, but not that of an E2-defective mutant of Cdc34p, increased repRNA accumulation, suggesting a significant role for the ubiquitin-conjugating enzyme function of Cdc34p in TBSV replication. Also, Cdc34p was able to ubiquitinate p33 in vitro. In addition, we have shown that p33 becomes ubiquitinated in vivo. We propose that ubiquitination of p33 likely alters its function or affects the recruitment of host factors during TBSV replication. [ABSTRACT FROM AUTHOR]

Recombinant adeno‐associated viral vectors (rAAV) are promising therapies for genetic diseases. Although current platforms for recombinant vector production can generate drug material for pre‐clinical and clinical studies, rAAV biomanufacturing will eventually face commercial supply challenges if per cell vector productivity and process scalability are not improved. Because considerable efforts have traditionally focused on optimizing rAAV plasmid design, herein we investigate the impact of host cell proteins on vector production to identify proteins that may enhance rAAV yield. Using a rAAV2‐GFP‐producing Saccharomyces cerevisiae model in combination with the yeast Tet Hughes Collection screening library, we identified 22 gene candidates that improved rAAV DNA replication (rAAV‐GFP/18s rDNA ratio) and vector yield (benzonase‐resistant rAAV DNA vector genome titer) as high as 6‐fold and 15‐fold relative to control, respectively. The candidate proteins participate in biological processes such as DNA replication, ribosome biogenesis, and RNA and protein processing. The best five candidates (PRE4, HEM4, TOP2, GPN3, and SDO1) were further screened by generating overexpression mutants in the YPH500 yeast strain. Subsequent clone evaluation was performed to confirm the rAAV‐promoting activity of selected candidates under plate‐based and bioreactor‐controlled fermentation conditions. Digital droplet PCR analysis of cell lysate and AVB resin‐purified material confirmed HEM4 and TOP2 overexpression mutants displayed the highest per cell total rAAV DNA productivity (1.6 and 1.7‐fold increase over control, respectively) and per cell vector productivity (3 and 4‐fold over control, respectively). This evaluation confirmed that overexpression of HEM4 and TOP2 proteins enhanced total and benzonase‐resistant rAAV DNA yield. Further studies are needed to understand their mechanism of action and to assess their potential application in molecular strategies for rAAV production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2725, 2019 [ABSTRACT FROM AUTHOR]

Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant- negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ~600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a "measuring string" during VRC assembly and spherule formation. [ABSTRACT FROM AUTHOR]