Identification of Essential Host Factors Affecting Tombusvirus RNA Replication Based on the Yeast Tet Promoters Hughes Collection

Abstract

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...

Inhibition of Sterol Biosynthesis Reduces Tombusvirus Replication in Yeast and Plants

“…These screens covered 95% of the host genes (16,38,50,51). The 14 identified host genes involved in lipid biosynthesis/metabolism included 8 genes affecting phospholipid biosynthesis, 4 genes affecting fatty acid biosynthesis/metabolism, and 2 genes affecting ergosterol synthesis.…”

Host Factors in Viral Life Cycles

“…To extend these studies to essential genes, additional genome-wide screenings were performed in the yeast/BMV and yeast/TBSV systems using an essential-gene library that covers 80% of the essential genes and in which each essential gene can be specifically turned off. With this approach 24 and 30 genes that affect the replication of BMV and TBSV were identified, respectively [23,37]. Taken together, these studies uncovered previously unconsidered cellular pathways, such as mRNA turnover, stress response, ribosome biosynthesis and the ubiquitin pathway of protein degradation, that either enhanced or inhibited viral replication.…”

Lipid Involvement in Viral Infections: Present and Future Perspectives for the Design of Antiviral Strategies

“…For instance, a marked alteration of cellular metabolism and an increase in fatty acid biosynthetic pathway have been described upon human cytomegalovirus (HCMV), DENV or HCV infection [1, 5,6,11]. The association of viral multiplication with modulation of host cell factors involved in lipid metabolism is not an exclusive feature of animal viruses, thus, replication and recombination of the plant pathogens tombusviruses has been revealed to rely on host genes involved in lipid metabolism [96][97][98][99]. Representative examples of cellular factors related to lipid metabolism and associated to viral replication are summarized in Table 2.…”