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To further our understanding of the pro-viral roles of the host cytosolic heat shock protein 70 (Hsp70) family, we chose the conserved Arabidopsis thaliana Hsp70-2 and the unique Erd2 (early response to dehydration 2), which contain Hsp70 domains. Based on in vitro studies with purified components, we show that AtHsp70-2 and AtErd2 perform pro-viral functions equivalent to that of the yeast Ssa1 Hsp70. These functions include activation of the tombusvirus RdRp, and stimulation of replicase assembly. Yeast-based complementation studies demonstrate that AtHsp70-2 or AtErd2 are present in the purified tombusvirus replicase. RNA silencing and over-expression studies in Nicotiana benthamiana suggest that both Hsp70-2 and Erd2 are co-opted by tomato bushy stunt virus (TBSV). Moreover, we used allosteric inhibitors of Hsp70s to inhibit replication of TBSV and related plant viruses in plants. Altogether, interfering with the functions of the co-opted Hsp70s could be an effective antiviral approach against tombusviruses in plants.

The genus Flavivirus encompasses several worldwide-distributed arthropod-borne viruses including, dengue virus, Japanese encephalitis virus, West Nile virus, yellow fever virus, Zika virus, and tick-borne encephalitis virus. Infection with these viruses manifest with symptoms ranging from febrile illness to life- threatening hypotensive shock and encephalitis. Therefore, flaviviruses pose a great risk to public health. Currently, preventive measures are falling short to control epidemics and there are no antivirals against any Flavivirus.Flaviviruses carry a single stranded positive-sense RNA genome that plays multiple roles in infected cells: it is translated into viral proteins, used as template for genome replication, it is the precursor of the subgenomic flaviviral RNA and it is assembled into new virions. Furthermore, viral RNA genomes are also packaged into extracellular vesicles, e.g. exosomes, which represent an alternate mode of virus dissemination.Because RNA molecules are at the center of Flavivirus replication cycle, viral and host RNA-binding proteins (RBPs) are critical determinants of infection. Numerous studies have revealed the function of RBPs during Flavivirus infection, particularly at the level of RNA translation and replication. These proteins, however, are also critical participants at the late stages of the replication cycle. Here we revise the function of host RBPs and the viral proteins capsid, NS2A and NS3, during the packaging of viral RNA and the assembly of new virus particles. Furthermore, we go through the evidence pointing towards the importance of host RBPs in mediating cellular RNA export with the idea that the biogenesis of exosomes harboring Flavivirus RNA would follow an analogous pathway.

RNAs that are 5′-truncated versions of a longer RNA but share the same 3′ terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to—and amplify—the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost, and specific detection of these truncated RNAs, we report detection of smaller coterminal RNA by PCR (DeSCo-PCR). DeSCo-PCR uses a nonextendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP, and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5′-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA.

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target., Highlights • Topoisomerase III-ß (TOP3B), which is the only human topoisomerase that can act on both DNA and RNA, is a host factor for all single stranded positive strand RNA viruses. • TOP3B is not a host factor for Ebola and influenza viruses, which are negative strand RNA viruses. • TOP3B acts directly on the viral genome of DENV2. • TOP3B could be a target for antiviral development.

Plus-stranded RNA viruses have limited coding capacity and have to co-opt numerous pro-viral host factors to support their replication. Many of the co-opted host factors support the biogenesis of the viral replication compartments and the formation of viral replicase complexes on subverted subcellular membrane surfaces. Tomato bushy stunt virus (TBSV) exploits peroxisomal membranes, whereas the closely-related carnation Italian ringspot virus (CIRV) hijacks the outer membranes of mitochondria. How these organellar membranes can be recruited into pro-viral roles is not completely understood. Here, we show that the highly conserved Fis1 mitochondrial fission protein is co-opted by both TBSV and CIRV via direct interactions with the p33/p36 replication proteins. Deletion of FIS1 in yeast or knockdown of the homologous Fis1 in plants inhibits tombusvirus replication. Instead of the canonical function in mitochondrial fission and peroxisome division, the tethering function of Fis1 is exploited by tombusviruses to facilitate the subversion of membrane contact site (MCS) proteins and peroxisomal/mitochondrial membranes for the biogenesis of the replication compartment. We propose that the dynamic interactions of Fis1 with MCS proteins, such as the ER resident VAP tethering proteins, Sac1 PI4P phosphatase and the cytosolic OSBP-like oxysterol-binding proteins, promote the formation and facilitate the stabilization of virus-induced vMCSs, which enrich sterols within the replication compartment. We show that this novel function of Fis1 is exploited by tombusviruses to build nuclease-insensitive viral replication compartment., Author summary Positive-strand RNA viruses, which include many dangerous pathogens of animals and plants, require the biogenesis of viral replication compartments. Utilizing TBSV and the model host yeast, we found a critical and novel role for the Fis1 mitochondrial fission protein. The pro-viral function of Fis1 is manifested through binding to the TBSV p33 replication protein that results in recruitment into the replication compartment formed from aggregated peroxisomes. Fis1 also binds to the related carnation Italian ringspot virus (CIRV) p36 replication protein in the replication compartment formed from aggregated mitochondria. Instead of the canonical function in mitochondria fission or peroxisome division, the tethering function of Fis1 is exploited by TBSV in facilitating the recruitment of cellular membrane contact site proteins into the viral replication compartment. Altogether, co-opting Fis1 is critical for tombusviruses to build their replication compartments in infected cells. Targeting these organelle-tethering proteins by inhibitors or other ways could open up new antiviral strategies.

The Zika emergency calls for urgent countermeasures. Recently, Barrows et al. (2016) and Xu et al. (2016) conducted in vitro anti-ZIKV screens to identify potential therapeutics. The off-label use of drugs that may protect against Zika virus-induced brain damage has, however, to be balanced with their risk during pregnancy.

International audience; Flaviviruses, such as dengue, Japanese encephalitis, tick-borne encephalitis, West Nile, yellow fever, and Zika viruses, are critically important human pathogens that sicken a staggeringly high number of humans every year. Most of these pathogens are transmitted by mosquitos, and not surprisingly, as the earth warms and human populations grow and move, their geographic reach is increasing. Flaviviruses are simple RNA–protein machines that carry out protein synthesis, genome replication, and virion packaging in close association with cellular lipid membranes. In this review, we examine the molecular biology of flaviviruses touching on the structure and function of viral components and how these interact with host factors. The latter are functionally divided into pro-viral and antiviral factors, both of which, not surprisingly, include many RNA binding proteins. In the interface between the virus and the hosts we highlight the role of a noncoding RNA produced by flaviviruses to impair antiviral host immune responses. Throughout the review, we highlight areas of intense investigation, or a need for it, and potential targets and tools to consider in the important battle against pathogenic flaviviruses.

RNA viruses exploit host cells by altering cellular pathways, recruiting host factors, remodeling intracellular membranes and escaping host antiviral responses. Model hosts, such as Saccharomyces cerevisiae (yeast), are valuable to identify host factors involved in viral RNA replication. The many advantages of using yeast include the availability of various yeast mutant libraries, such as (i) single gene-deletion library; (ii) the essential gene library (yTHC); and (iii) the yeast ORF over-expression library. Here, we have used a novel temperature-sensitive (ts) mutant library of essential yeast genes to identify 118 host proteins affecting replication of Tomato bushy stunt virus, in yeast model host. Testing 787 ts mutants led to the identification of host factors, of which 72 proteins facilitated TBSV replication in yeast and 46 proteins were inhibitory. Altogether, ~85% of the identified proteins are novel host factors affecting tombusvirus replication. The ts mutant library screen also led to the identification of 17 essential genes, which have been documented before, thus confirming the importance of these genomic screens. Overall, we show the power of ts mutant library in identification of host factors for RNA virus replication.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions., Author Summary The actin network, which is a central node in cellular pathways, is frequently targeted by various pathogens to modulate cellular responses. In this paper, the authors show that TBSV interacts with cofilin actin depolymerization factor leading to inhibition of the dynamic function of the actin network in infected cells. This allows TBSV to utilize the existing actin filaments to efficiently recruit host proteins and lipids for viral replication and to build viral replication compartments for robust viral replication. Altogether, subversion of the actin network by TBSV is a key step for the virus to gain access to cellular resources required for virus replication.

The identification of cellular proteins associated with virus replicase complexes is crucial to our understanding of virus-host interactions, influencing the host range, replication, and virulence of viruses. A previous in vitro study has demonstrated that partially purified Bamboo mosaic virus (BaMV) replicase complexes can be employed for the replication of both BaMV genomic and satellite BaMV (satBaMV) RNAs. In this study, we investigated the BaMV and satBaMV 3 untranslated region (UTR) binding proteins associated with these replicase complexes. Two cellular proteins with molecular masses of 35 and 55 kDa were specifically cross-linked with RNA elements, whereupon the 35-kDa protein was identified as the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gel mobility shift assays confirmed the direct interaction of GAPDH with the 3 UTR sequences, and competition gel shift analysis revealed that GAPDH binds preferentially to the positive-strand BaMV and satBaMV RNAs over the negative-strand RNAs. It was observed that the GAPDH protein binds to the pseudoknot poly(A) tail of BaMV and stem-loop-C poly(A) tail of satBaMV 3 UTR RNAs. It is important to note that knockdown of GAPDH in Nicotiana benthamiana enhances the accumulation of BaMV and satBaMV RNA; conversely, transient overexpression of GAPDH reduces the accumulation of BaMV and satBaMV RNA. The recombinant GAPDH principally inhibits the synthesis of negative-strand RNA in exogenous RdRp assays. These observations support the contention that cytosolic GAPDH participates in the negative regulation of BaMV and satBaMV RNA replication. The replication of plus-strand RNA viruses is arbitrated by virus-specific replicase complexes (RC) (3), which are membrane-associated replication complexes derived from cell organelles. These membrane structures recruit several host proteins, providing a suitable environment for the synthesis of viral RNA. Unraveling the interactions between viruses and their host cells as a function of time could make considerable contributions to our understanding of the dynamics of viral infections. In vitro RNA-dependent RNA polymerase (RdRp) systems are commonly used to analyze the components of host and viral proteins associated with RdRp complexes, as well as for identifying the putative cis-acting elements of the RNA templates (3, 24). Such systems have been established for sev

Viruses accomplish their replication by exploiting many cellular resources, including metabolites and energy. Similarly to other (+)RNA viruses, tomato bushy stunt virus (TBSV) induces major changes in infected cells. However, the source of energy required to fuel TBSV replication is unknown. We find that TBSV co-opts the cellular glycolytic ATP-generating pyruvate kinase (PK) directly into the viral replicase complex to boost progeny RNA synthesis. The co-opted PK generates high levels of ATP within the viral replication compartment at the expense of a reduction in cytosolic ATP pools. The ATP generated by the co-opted PK is used to promote the helicase activity of recruited cellular DEAD-box helicases, which are involved in the production of excess viral (+)RNA progeny. Altogether, recruitment of PK and local production of ATP within the replication compartment allow the virus replication machinery an access to plentiful ATP, facilitating robust virus replication.

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.

The intricate interactions between viruses and hosts include an evolutionary arms race and adaptation that is facilitated by the ability of RNA viruses to evolve rapidly due to high frequency mutations and genetic RNA recombination. In this paper, we show evidence that the co-opted cellular DDX3-like Ded1 DEAD-box helicase suppresses tombusviral RNA recombination in yeast model host, and the orthologous RH20 helicase functions in a similar way in plants. In vitro replication and recombination assays confirm the direct role of the ATPase function of Ded1p in suppression of viral recombination. We also present data supporting a role for Ded1 in facilitating the switch from minus- to plus-strand synthesis. Interestingly, another co-opted cellular helicase, the eIF4AIII-like AtRH2, enhances TBSV recombination in the absence of Ded1/RH20, suggesting that the coordinated actions of these helicases control viral RNA recombination events. Altogether, these helicases are the first co-opted cellular factors in the viral replicase complex that directly affect viral RNA recombination. Ded1 helicase seems to be a key factor maintaining viral genome integrity by promoting the replication of viral RNAs with correct termini, but inhibiting the replication of defective RNAs lacking correct 5’ end sequences. Altogether, a co-opted cellular DEAD-box helicase facilitates the maintenance of full-length viral genome and suppresses viral recombination, thus limiting the appearance of defective viral RNAs during replication., Author Summary A major force in virus evolution is the ability of viruses to recombine and change their genomes rapidly. Similar to viral replication that greatly depends on subverted cellular proteins, viral genetic recombination is also affected by host factors based on genome-wide screens with tomato bushy stunt virus (TBSV) in yeast model host. However, the roles of host factors in the viral genomic RNA recombination process remain elusive. In this paper, we show evidence, in yeast, plants and in vitro, that co-opted cellular helicases by TBSV affect viral recombination through suppressing template-switching and replication of the new recombinant viral RNAs. Based on the presented data, a new concept emerges on the roles of co-opted cellular helicases in maintaining viral genome integrity. Altogether, the hijacked cellular DEAD-box helicases are involved in maintenance of full-length viral RNA genome and suppression of viral RNA recombination, thus blocking the appearance of defective or recombinant viral RNAs during replication.

Genetic recombination in RNA viruses drives the evolutionary arms race with host׳s antiviral strategies and recombination also facilitates adaptation of viruses to new hosts. In this paper, the authors used tombusvirus and a temperature-sensitive (ts) mutant library of yeast to identify 40 host proteins affecting viral recombination in yeast model host. Subsequent detailed analysis with two identified actin-related proteins, Act1p and Arp3p, has revealed that the wt actin network helps TBSV to maintain low level viral recombination. Pharmacological inhibition of actin in plant protoplasts confirmed the role of the actin network in tombusvirus recombination. An in vitro approach revealed the altered activity of the tombusvirus replicase in the presence of mutated Act1p. The authors show more efficient recruitment of a cellular DEAD-box helicase, which enhances tombusvirus recombination, into the membrane-bound replicase in Act1p mutant yeast. Overall, this work shows that the actin network affects tombusvirus recombination in yeast and plant cells.