Plus-stranded RNA viruses replicate in infected cells by assembling viral replicase complexes consisting of viral- and host-coded proteins. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of seven ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. In this paper, we show that the expression of dominant negative Vps23p, Vps24p, Snf7p, and Vps4p ESCRT factors inhibited virus replication in the plant host, suggesting that tombusviruses co-opt selected ESCRT proteins for the assembly of the viral replicase complex. We also show that TBSV p33 replication protein interacts with Vps23p ESCRT-I and Bro1p accessory ESCRT factors. The interaction with p33 leads to the recruitment of Vps23p to the peroxisomes, the sites of TBSV replication. The viral replicase showed reduced activity and the minus-stranded viral RNA in the replicase became more accessible to ribonuclease when derived from vps23Δ or vps24Δ yeast, suggesting that the protection of the viral RNA is compromised within the replicase complex assembled in the absence of ESCRT proteins. The recruitment of ESCRT proteins is needed for the precise assembly of the replicase complex, which might help the virus evade recognition by the host defense surveillance system and/or prevent viral RNA destruction by the gene silencing machinery.

Replication of plus-stranded RNA viruses requires many components of the host cells, including host proteins and intracellular membranes, which serve as sites of virus replication in infected cells (1,3,38). Accordingly, the virus-specific replicase complex (RC) consists of virus-and host-coded proteins and the viral RNA, which assemble on intracellular membranes into functional complexes. In addition, the viral replication proteins and host factors likely play roles in template selection for replication and recruitment (intracellular transport/targeting) of the viral RNA into replication (2,20). Host factors could also affect the stability/degradation of viral proteins and the viral RNA (4, 40, 41). Overall, viruses utilize/ depend on many diverse resources of the host cells.To identify the roles and/or effects of host genes on virus replication, systematic genome-wide screens were conducted in yeast, a model host, using the single-gene deletion library (YKO) with two distantly related plus-strand RNA viruses, namely Brome mosaic virus (BMV) and Tomato bushy stunt virus (TBSV) (12,27). These studies led to the identification of ϳ100 host genes for each virus that either stimulated or inhibited virus replication. Interestingly, most of the identified genes had a virus-specific effect, whereas only a small number of genes affected the replication of both BMV and TBSV. These observations suggest that BMV and TBSV, belonging to different supergroups within plus-stranded RNA viruses, could use and/or be affected by mostly different host factors (12, 27). Altogether, the above systematic screens tested only ϳ80% of all the known genes, which are not essential for yeast growth, whereas the effect of essential yeast genes remained untested.Tombusviruses, such as TBSV and Cucumber necrosis virus (CNV), are single-component RNA viruses of ϳ4,800 nucleotides (nt). Among the five virus-coded proteins, only two, termed p33 and p92 pol , are essential for TBSV replication (44). p92 pol is the viral RNA-dependent RNA polymerase (RdRp), whereas p33 replication cofactor (which overlaps with the Nterminal pre-readthrough segment of p92 pol ) is an RNA-binding protein (28,32,33). Earlier work defined that p33 is involved in template selection and recruitment of viral RNA into replication (18,25,32). These proteins interact with each other, the viral RNA, and the host proteins in cells (25,34,35,39), which leads to the assembly of RC on peroxisomal membranes (22, 25). The CNV replication proteins can support the replication of TBSV defective interfering (DI) RNA, a small deletion derivative of the genomic RNA, as efficiently as TBSV replication proteins can (19,24). A recent genome-wide screen of the YKO library for tombusvirus replication led to the identification of 96 host genes, whose separate deletions affected replication of a TBSV replicon RNA (repRNA), which is based on a DI RNA, in yeast (27). Based on the large number of host genes identified, the emerging picture is that the host likely plays a complex role in virus rep...

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

No abstract

RNA recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing ϳ80% of all genes of yeast, led to the identification of 11 host genes affecting RNA recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV recombination and evolution.RNA viruses are successful pathogens because they are capable of rapid evolution that helps them to overcome host resistance and other antiviral strategies (13,14,17,27,54,55,64). RNA recombination, the joining of two noncontiguous RNA segments together, is an especially powerful tool for viruses to create new resistance-breaking or drug-resistant strains and/or viruses (27,64). Accordingly, the generation of novel recombinant RNAs (recRNAs) has been described for many human, animal, and plant viruses as well as RNA bacteriophages (1,4,5,11,16,21,23,24,27,32,42,59,64,65).Progress in our understanding of viral RNA recombination has been slowed down by the difficulty of detection of new recRNAs, the adverse selection pressure on some recRNAs, and the poor predictability of recombination events. Development of powerful model RNA recombination systems, however, has revealed many unique features of viral RNA recombination. For example, sequencing of numerous recRNAs in Brome mosaic virus (BMV) (3,33,36,53), Turnip crinkle virus (TCV) (6,7,37,38,40), and tombusviruses (61, 62) established that recombination does not occur randomly within the viral RNA genome but rather, there are recombination "hot spots". These include AU-rich sequences (31, 34, 58), inter-or intramolecular secondary structures (19,35,62), and cis-acting RNA elements with high affinity toward the viral replicase (8,10,40). Mutagenesis of the replicase proteins has led to altered recombination frequencies or altered the sites of recombination (15,30,47), suggesting that many recombination events are due to template switching (replicase jumping) by the viral replicase (22,27,39...

Previous genome-wide screens identified >100 host genes affecting tombusvirus replication using yeast model host. One of those factors was Nsr1p (nucleolin), which is an abundant RNA binding shuttle protein involved in rRNA maturation and ribosome assembly. We find that over-expression of Nsr1p in yeast or in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Regulated over-expression of Nsr1p revealed that Nsr1p should be present at the beginning of viral replication for efficient inhibition, suggesting that Nsr1p inhibits an early step in the replication process. In vitro experiments revealed that Nsr1p binds preferably to the 3' UTR in the viral RNA. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the assay. These data support the model that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

The SARS-CoV-2 Nucleoprotein (NCAP) functions in RNA packaging during viral replication and assembly. Computational analysis of its amino acid sequence reveals a central low-complexity domain (LCD) having sequence features akin to LCDs in other proteins known to function in liquid-liquid phase separation. Here we show that in the presence of viral RNA, NCAP, and also its LCD segment alone, form amyloid-like fibrils when undergoing liquid-liquid phase separation. Within the LCD we identified three 6-residue segments that drive amyloid fibril formation. We determined atomic structures for fibrils formed by each of the three identified segments. These structures informed our design of peptide inhibitors of NCAP fibril formation and liquid-liquid phase separation, suggesting a therapeutic route for Covid-19.

No abstract