The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms

The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.     

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.     

Host ESCRT Proteins Are Required for Bromovirus RNA Replication Compartment Assembly and Function

Positive-strand RNA viruses genome replication invariably is associated with vesicles or other rearranged cellular membranes. Brome mosaic virus (BMV) RNA replication occurs on perinuclear endoplasmic reticulum (ER) membranes in ~70 nm vesicular invaginations (spherules). BMV RNA replication vesicles show multiple parallels with membrane-enveloped, budding retrovirus virions, whose envelopment and release depend on the host ESCRT (endosomal sorting complexes required for transport) membrane-remodeling machinery. We now find that deleting components of the ESCRT pathway results in at least two distinct BMV phenotypes. One group of genes regulate RNA replication and the frequency of viral replication complex formation, but had no effect on spherule size, while a second group of genes regulate RNA replication in a way or ways independent of spherule formation. In particular, deleting SNF7 inhibits BMV RNA replication > 25-fold and abolishes detectable BMV spherule formation, even though the BMV RNA replication proteins accumulate and localize normally on perinuclear ER membranes. Moreover, BMV ESCRT recruitment and spherule assembly depend on different sets of protein-protein interactions from those used by multivesicular body vesicles, HIV-1 virion budding, or tomato bushy stunt virus (TBSV) spherule formation. These and other data demonstrate that BMV requires cellular ESCRT components for proper formation and function of its vesicular RNA replication compartments. The results highlight growing but diverse interactions of ESCRT factors with many viruses and viral processes, and potential value of the ESCRT pathway as a target for broad-spectrum antiviral resistance.     

Yeast Lsm1p-7p/Pat1p deadenylation-dependent mRNA-decapping factors are required for brome mosaic virus genomic RNA translation

Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5' and 3' noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.     

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.     

Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies

Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.     

Reconstitution of two recombinant LSm protein complexes reveals aspects of their architecture, assembly, and function

Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1-7 and LSm2-8 complexes from their constituent components in vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1-7 and LSm2-8. Isolated sub-complexes form ring-like higher order structures. LSm1-7 is assembled and stable in the absence of RNA. LSm1-7 forms ring-like structures very similar to LSm2-8 at the EM level. Our in vitro reconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and an in vivo cellular transport assay.     

Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication

The nucleotide sequences of brome mosaic virus (BMV) RNAs 1 (3234 bases) and 2 (2865 bases) have been determined, completing the primary structure of the 8200 base tripartite BMV genome. cDNA clones covering 99% of BMV RNA1 and a full-length cDNA clone of BMV RNA2 were isolated in the course of this work. Extensive sequence homology and known interaction with several proteins suggest that the 3' ends of the BMV RNAs are the major regulatory regions of the genome. Smaller regions at the 5' ends of RNAs 1 and 2 show strong homology to each other and lesser homology to RNA3. These and other features of the sequences are discussed in relation to replication, regulation and evolution of the BMV genome.     

Xenopus Xp54 and human RCK/p54 helicases functionally replace yeast Dhh1p in brome mosaic virus RNA replication

By using a Brome mosaic virus (BMV)-Saccharomyces cerevisiae system, we previously showed that the cellular Lsm1p-7p/Pat1p/Dhh1p decapping-activator complex functions in BMV RNA translation and replication. As a first approach in investigating whether the corresponding human homologues play a similar role, we expressed human Lsm1p (hLsm1p) and RCK/p54 in yeast. Expression of RCK/p54 but not hLsm1p restored the defect in BMV RNA translation and replication observed in the dhh1Delta and lsm1Delta strains, respectively. This functional conservation, together with the common replication strategies of positive-stranded RNA viruses, suggests that RCK/p54 may also play a role in the replication of positive-stranded RNA viruses that infect humans.     

Selective repression of translation by the brome mosaic virus 1a RNA replication protein

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.     

Structure of the LSm657 complex: an assembly intermediate of the LSm1-7 and LSm2-8 rings

The nuclear LSm2-8 (like Sm) complex and the cytoplasmic LSm1-7 complex play a central role in mRNA splicing and degradation, respectively. The LSm proteins are related to the spliceosomal Sm proteins that form a heteroheptameric ring around small nuclear RNA. The assembly process of the heptameric Sm complex is well established and involves several smaller Sm assembly intermediates. The assembly of the LSm complex, however, is less well studied. Here, we solved the 2.5 Å-resolution structure of the LSm assembly intermediate that contains LSm5, LSm6, and LSm7. The three monomers display the canonical Sm fold and arrange into a hexameric LSm657-657 ring. We show that the order of the LSm proteins within the ring is consistent with the order of the related SmE, SmF, and SmG proteins in the heptameric Sm ring. Nonetheless, differences in RNA binding pockets prevent the prediction of the nucleotide binding preferences of the LSm complexes. Using high-resolution NMR spectroscopy, we confirm that LSm5, LSm6, and LSm7 also assemble into a  60-kDa hexameric ring in solution. With a combination of pull-down and NMR experiments, we show that the LSm657 complex can incorporate LSm23 in order to assemble further towards native LSm rings. Interestingly, we find that the NMR spectra of the LSm57, LSm657-657, and LSm23-657 complexes differ significantly, suggesting that the angles between the LSm building blocks change depending on the ring size of the complex. In summary, our results identify LSm657 as a plastic and functional building block on the assembly route towards the LSm1-7 and LSm2-8 complexes.     

Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum

The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis

Brome mosaic virus (BMV) belongs to a "superfamily" of plant and animal positive-strand RNA viruses that share, among other features, three large domains of conserved sequence in nonstructural proteins involved in RNA replication. Two of these domains reside in the 109-kDa BMV 1a protein. To examine the role of 1a, we used biologically active cDNA clones of BMV RNA1 to construct a series of linker insertion mutants bearing two-codon insertions dispersed throughout the 1a gene. The majority of these mutations blocked BMV RNA replication in protoplasts, indicating that both intervirally conserved domains function in RNA replication. Coinoculation tests with a large number of mutant combinations failed to reveal detectable complementation between mutations in the N- and C-terminal conserved domains, implying that these two domains either function in some directly interdependent fashion or must be present in the same protein. Four widely spaced mutations with temperature-sensitive (ts) defects in RNA replication were identified, including a strongly ts insertion near the nucleotide-binding consensus of the helicaselike C-terminal domain. Temperature shift experiments with this mutant show that 1a protein is required for continued accumulation of all classes of viral RNA (positive strand, negative strand, and subgenomic) and is required for at least the first 10 h of infection. ts mutations were also identified in the 3' noncoding region of RNA1, 5' to conserved sequences previously implicated in cis for replication. Under nonpermissive conditions, the cis-acting partial inhibition of RNA1 accumulation caused by these noncoding mutations was also associated with reduced levels of the other BMV genomic RNAs. Comparison with previous BMV mutant results suggests that RNA replication is more sensitive to reductions in expression of 1a than of 2a, the other BMV-encoded protein involved in replication.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNA synthesis

All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding delta9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.     

Host acyl coenzyme A binding protein regulates replication complex assembly and activity of a positive-strand RNA virus

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ～50% smaller but ～4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.