Apolipoprotein B-100 after lipidation is dislocated from the ER lumen to the cytoplasmic surface of lipid droplets for proteasomal degradation. UBXD8 in lipid droplets and Derlin-1 in the ER membrane interact with each other and with ApoB and are engaged in the pre- and postdislocation steps, respectively.

Replication of positive-strand RNA viruses occurs through the assembly of membrane-associated viral RNA replication complexes that include viral replicase proteins, viral RNA templates, and host proteins. Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA plant virus with a genome consisting of RNA1 and RNA2. The two proteins encoded by RNA1, a 27-kDa protein (p27) and an 88-kDa protein containing an RNA-dependent RNA polymerase (RdRP) motif (p88), are essential for RCNMV RNA replication. To analyze RCNMV RNA replication complexes, we used blue-native polyacrylamide gel electrophoresis (BN/PAGE), which enabled us to analyze detergent-solubilized large membrane protein complexes. p27 and p88 formed a complex of 480 kDa in RCNMV-infected plants. As a result of sucrose gradient sedimentation, the 480-kDa complex cofractionated with both endogenous template-bound and exogenous template-dependent RdRP activities. The amount of the 480-kDa complex corresponded to the activity of exogenous template-dependent RdRP, which produced RNA fragments by specifically recognizing the 3-terminal core promoter sequences of RCNMV RNAs, but did not correspond to the activity of endogenous template-bound RdRP, which produced genome-sized RNAs without the addition of RNA templates. These results suggest that the 480-kDa complex contributes to template-dependent RdRP activities. We subjected those RdRP complexes to affinity purification and analyzed their components using two-dimensional BN/sodium dodecyl sulfate-PAGE (BN/SDS-PAGE) and mass spectrometry. The 480-kDa complex contained p27, p88, and possible host proteins, and the original affinity-purified RdRP preparation contained HSP70, HSP90, and several ribosomal proteins that were not detected in the 480-kDa complex. A model for the formation of RCNMV RNA replication complexes is proposed.Positive-strand RNA viruses replicate their RNA on intracellular membranes (5). One or more viral nonstructural proteins, including RNA-dependent RNA polymerase (RdRP) and accessory proteins, play essential roles in RNA replication as integral membrane proteins (13,25,28) or as peripheral membrane proteins (4, 6). These viral nonstructural proteins interact with each other and with host proteins and viral RNA templates to form the membrane-associated viral RNA replication complexes. The membrane-associated RNA replication complexes of positive-strand RNA viruses have been isolated from the membrane fraction of virally infected tissues by the use of detergents (2,11,29,32,40,49,50). The purified fractions contain viral and host proteins and retain two types of RdRP activities. One is an endogenous template-bound RdRP activity that synthesizes virus-related RNAs without adding RNA templates. The other is an exogenous template-dependent RdRP activity capable of de novo initiation of cRNA synthesis from selected RNA templates. Although there has been intensive work performed on viral RdRP complexes, their organizations, properties, and functions have not been fully characterized.Red cl...

Japan bAssembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex. Most plant and animal viruses are positive-strand RNA viruses, which have single-stranded messenger-sense genomic RNAs. These viruses often induce host membrane rearrangements to form organelle-like compartments in which viral genomic RNAs are replicated via negative-strand RNA intermediates by the viral replicase complexes (10). Viral replicase complexes comprise multiple proteins, including viral auxiliary proteins, viral RNAdependent RNA polymerase (RdRP), and host proteins (61). Viral replicase complexes have been studied extensively by characterizing their RdRP activities and the functions of the viral and host components of the complexes. These studies have provided important information about the mechanisms regulating genome replication (15, 19, 47, 89), viral pathogenicity (68, 69), and virushost interactions (24,25,32,33). However, an important question remains: how do multiple viral and host components assemble properly into the replicase complex?Molecular chaperones are essential for cell viability by ensuring folding of newly synthesized proteins, refolding of misfolded or aggregated proteins, protein complex assembly and disassembly, membrane translocation of organellar and secretory proteins, protein degradation, and activities of regulatory proteins in signal transduction pathways (12,18,51). In eukaryotic cells, the abundant and highly conserved molecular chaperones heat shock proteins Hsp70 and Hsp90 play central roles in the biological processes mentioned above, and the activities of Hsp70 and Hsp90 are modulated by a variety of cochaperones (37,80). Considering their pivotal roles i...

bEukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery. E ukaryotic positive-strand RNA [(ϩ)RNA] viruses replicate their genomes using membrane-bound replicase complexes, which contain multiple viral and host components. A growing number of host proteins that affect viral RNA replication have been identified using genome-wide and proteomics analyses in several animal and plant viruses (1-13). These host proteins are involved in translation, template selection, and the assembly of the viral replication complex (VRC) on intracellular membranes, which serve as the site of viral RNA replication (14). However, the functions of host proteins remain largely unknown.The replication compartments of (ϩ)RNA viruses are derived from various cellular organelle membranes, such as the endoplasmic reticulum (ER), mitochondria, chloroplasts, peroxisomes, and the Golgi apparatus (15-17). The formation of viral replication compartments generally involves the emergence of spherules, vesicles, and multivesicular bodies associated with various organelles (15, 17). Although viral proteins play an essential role in the formation of replication compartments containing VRCs, host factors also regulate this process (14,15,18). Tomato bushy stunt virus (TBSV) coopts the proteins of the endosomal sorting compl...

Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.