The group of positive-strand RNA [(+)RNA] viruses includes many plant, animal, and human pathogens of economical and medical importance. Despite their great diversity, all positive-strand RNA viruses share some fundamental common features in their replication processes. First, upon infection, their genomes act as mRNAs to express the viral proteins and then, at later steps, as templates for replication and encapsidation. Since these functions are mutually exclusive, profound rearrangements in the viral genome RNA-protein (RNP) composition are required to regulate such transitions. The nature and control of these rearrangements are not well defined. Second, replication of (+)RNA viruses universally occurs on virus-induced rearranged host intracellular membranes (1
). Third, because (+)RNA viruses have genomes with limited coding capacity, they greatly depend on the cellular machinery to multiply. Host factors, therefore, play crucial roles in regulating all steps of their life cycles (2
). The identification of common required host factors will expand our knowledge of key steps of (+)RNA life cycles and might provide novel and stable targets for the generation of broad-spectrum antiviral drugs.
The cellular decapping activators LSm1-7 heptameric ring, PatL1, and DDX6 (also designated Rck/p54) are examples of host proteins required for the replication of a wide range of (+)RNA viruses. These proteins are highly conserved from yeast (designated LSm1-7, Pat1, and Dhh1) to humans and can be found in cytoplasmic foci, named P-bodies. These foci are highly dynamic granules, with unclear functions, that contain translationally repressed mRNAs together with multiple proteins from the mRNA decay and microRNA machineries (4
). All P-body components rapidly cycle in and out of these granules, indicating that there is a constant exchange of molecules with the cytoplasm, where all P-body components are also diffusely present (6
). In noninfected cells, LSm1-7, PatL1, and DDX6 form a complex and promote cellular mRNA decay by accelerating decapping in the 5′-3′deadenylation-dependent mRNA decay pathway. Although their precise way of functioning is not fully understood, they have been suggested to facilitate RNP rearrangements required for the transition of cellular mRNAs from an active translatable state to a translationally repressed state that allows the assembly of the decapping complex (9
). The LSm1-7 ring is constituted by seven LSm proteins that belong to the conserved Sm family of proteins, which are characterized by the presence of the Sm fold. It has been described that proteins containing Sm folds act as chaperones facilitating a variety of RNA-RNA and RNA-protein interactions (11
). PatL1 has emerged as a pivotal protein in mRNA decay. Recent evidence supports its function as a scaffold protein, allowing the sequential binding of repression and decay factors on mRNPs that eventually leads to degradation (12
). Finally, DDX6 belongs to the family of DEAD box helicases. These highly conserved enzymes accelerate structural transitions of RNAs and RNPs in an ATP-dependent manner that resembles the activities of certain groups of protein chaperones (13
). Interestingly, in contrast to their decay function in cellular mRNAs, PatL1, LSm1-7, and DDX6 have all or in part been shown to be required for replication of diverse (+)RNA viruses, including human hepatitis C virus (HCV), West Nile virus (WNV), and Dengue virus (DNV), as well as the plant brome mosaic virus (BMV) (15
). Remarkably, Hfq, a homolog of LSm1 in bacteria, is also required for the replication of the (+)RNA phage Qβ (23
). The direct interaction of PatL1, LSm1-7, and DDX6 with essential regulatory signals in the viral genomes and with viral proteins support that this positive regulation is mediated by a direct and specific effect (18
). The conserved dependence on LSm1-7, PatL1, and DDX6 of viruses that infect different kingdoms of life underlines a remarkable robustness in the use of proteins from the decapping pathway to regulate (+)RNA viral life cycles; however, the underlying mechanisms involved remain to be elucidated.
To further explore how wide spread the use of LSm1-7, PatL1, and DDX6 in (+)RNA virus life cycle is and to gain further insights into their mechanism of action, here we tested their function on the replication of Flock House virus (FHV), a natural insect pathogen and well-studied member of the Nodaviridae
family (reviewed in reference 26
). The simplicity of its genome makes FHV a highly tractable system to study basic aspects of (+)RNA biology. The FHV bipartite genome consists of two capped but nonpolyadenylated RNA segments (). RNA1 (3.1 kb) encodes protein A, the only FHV protein required for replication (27
), which occurs in outer mitochondrial membranes (28
). RNA2 (1.4 kb) encodes the capsid precursor α, which is required for virion formation but is dispensable for virus replication (29
). Hence, RNA1 is capable of autonomous replication in cells (32
). Furthermore, RNA1 templates that do not express a functional protein A can be complemented in trans
). During replication, RNA1 also produces subgenomic RNA3 (387 nucleotides). This RNA3 corresponds to the 3′ end of RNA1, and its synthesis requires a long-distance base pairing between two cis
-acting elements in RNA1 (35
). Thus, different RNP rearrangements within genomic RNA1 are predicted to lead to the synthesis of either negative-stranded RNA1 [(−)RNA1), when the polymerase synthesizes a complete cRNA, or (−)RNA3, when the polymerase prematurely stops due to the secondary or tertiary structures formed by the long-distance base pairing. RNA3 encodes protein B2, required for suppression of RNA silencing in infected hosts (36
), and B1, a protein of unknown function. In addition, RNA3 fulfills an important role in the regulation of FHV gene expression by coordinating the production of proper RNA1 and RNA2 levels. RNA3 transactivates RNA2 replication, while high levels of RNA2 inhibit RNA3 synthesis (35
). The downregulation of RNA3 most probably disallows disproportionately high RNA2/RNA1 ratios within FHV-infected cells. This feedback mechanism is of fundamental importance for viruses with segmented genomes, because it would ensure the timely and optimal expression of different viral proteins for the different stages of the viral life cycle, giving, for example, to RNA1 replication an advantage over RNA2 replication during the initial stages of infection, when RNA1 translation products are required for the establishment of viral replication.
Fig 1 Schematic representation of FHV genome and trans-replication of FHV RNA1 in yeast. (A) Schematic diagram of the FHV genome showing ORFs (open boxes), untranslated regions (UTRs) (single lines), the subgenomic RNA3 start site (bent arrow), and the subgenomic (more ...)
A remarkable feature of FHV replication is its ability to replicate in a wide variety of eukaryotic cell types, including Drosophila
, Caenorhabditis elegans
, plants, mammals, and the budding yeast Saccharomyces cerevisiae
). Thus, host factors required for FHV replication must be highly conserved. Because S. cerevisiae
is easy to manipulate genetically, the FHV/yeast system is an excellent model system to study the effect of host factors in (+)RNA virus replication. By using this system, we show here that depletion of the LSM1
, or DHH1
gene dramatically increases RNA3 accumulation, altering RNA3/RNA1 ratios. This effect was not explained by differences between RNA1 or RNA3 steady-state levels in the absence of replication. Importantly, coimmunoprecipitation studies indicated that LSm1-7, Pat1, and Dhh1 interact with FHV protein A and the viral genome. Together, these results are consistent with a replication-dependent way of action of LSm1-7, Pat1, and Dhh1 to control proper RNA3/RNA1 ratios. Since no effects were observed on the early replication steps of RNA1 recruitment to the replication complex and RNA1 synthesis, a direct role on RNA3 synthesis was suggested. Moreover, RNA3/RNA1 ratio alterations were independent of the membrane compartment, where replication occurs, and require the ATPase activity of Dhh1, an essential feature for Dhh1-dependent RNP rearrangements. These findings highlight the role of LSm1-7, Pat1, and Dhh1 as master regulators of (+)RNA virus replication. Their described function on cellular mRNP rearrangements would support a parallel role in mediating key viral RNP transitions, such as the one required to control RNA3 synthesis.