Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl -cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4°C but shifted to lipid rafts at 37°C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.
The Ro autoantigen is a ring-shaped RNA-binding protein that binds misfolded RNAs in nuclei and is proposed to function in quality control. In the cytoplasm, Ro binds noncoding RNAs, called Y RNAs, that inhibit access of Ro to other RNAs. Ro also assists survival of mammalian cells and at least one bacterium after UV irradiation. In mammals, Ro undergoes dramatic localization changes after UV irradiation, changing from mostly cytoplasmic to predominantly nuclear. Here, we report that a second role of Y RNAs is to regulate the subcellular distribution of Ro. A mutant Ro protein that does not bind Y RNAs accumulates in nuclei. Ro also localizes to nuclei when Y RNAs are depleted. By assaying chimeric proteins in which portions of mouse Ro were replaced with bacterial Ro sequences, we show that nuclear accumulation of Ro after irradiation requires sequences that overlap the Y RNA binding site. Ro also accumulates in nuclei after oxidative stress, and similar sequences are required. Together, these data reveal that Ro contains a signal for nuclear accumulation that is masked by a bound Y RNA and suggest that Y RNA binding may be modulated during cell stress.
A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method. Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs revealed that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and is related to members of the UBP family containing highly conserved Cys and His domains. Chick UBP41 was expressed in E. coli and purified from the cells to apparent homogeneity, using 125 I-labeled ubiquitin-␣NH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide. The UBP41 protease cleaved at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes; thus, it is active against ubiquitin--galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids. UBP41 also released free ubiquitin from poly-His-tagged diubiquitin. Moreover, it converted poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules were not further degraded to free ubiquitin and lysozyme. These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins. Ubiquitin (Ub)1 is a highly conserved 76-amino acid polypeptide involved in a variety of cellular functions, including regulation of intracellular protein breakdown, cell cycle regulation, and stress response (1-6). This small protein is covalently ligated to target proteins by a family of Ub-conjugating enzymes, called E2s (7,8), through an isopeptide linkage between the C-terminal Gly residue of Ub and the ⑀-amino group of Lys residue(s) of the proteins. Ubs by themselves or in conjugation to proteins may also be ligated to additional Ub molecules to form branched poly-Ub by the linkage between the ⑀-amino group of Lys-48 of one Ub and the C terminus of the other. Proteins ligated to multiple units of Ub are degraded by the ATP-dependent 26 S proteasome (1-3, 9).Ubs are encoded by two distinct classes of genes, neither of which encodes a monomeric form of Ub (10, 11). One is a poly-Ub gene, which encodes a linear polymer of Ubs that are linked through peptide bonds between the C-terminal Gly and N-terminal Met of contiguous Ub molecules. The other encodes a fusion protein in which a single Ub is linked to a ribosomal protein consisting of 52 or 76 -80 amino acids (12). Thus, generation of free Ub from the linear poly-Ub and Ub fusion prot...
Polypyrimidine-tract-binding protein (PTB) has been shown to bind specifically to the 5' ends of mouse hepatitis virus (MHV) RNA and its complementary strand. To further characterize the function of PTB in MHV replication, we generated dominant-negative mutant cell lines that express a full-length PTB or a truncated form of PTB, which includes only the N-terminal half of the protein, retaining its protein-dimerization domain. The truncated form of PTB was localized in the cytoplasm, whereas the full-length PTB was present mainly in the nucleus. The truncated form can interact with the full-length PTB in vitro. We observed that both the full-length and the truncated PTB, when overexpressed, functioned in a dominant-negative manner in MHV replication. However, the truncated form exhibited more severe effects on syncytia formation, virus production, and synthesis of viral RNA and viral proteins. To clarify the precise function of PTB in MHV replication, we dissociated the processes of viral transcription from translation by transfecting different types of MHV defective-interfering (DI) RNA that contain various reporter genes into these stable cell lines. Transcription of the DI RNA during MHV infection was greatly inhibited in these cell lines, indicating that PTB modulates MHV transcription. In contrast, translation of the DI RNA was not affected by PTB depletion in in vitro translation in rabbit reticulocyte lysate or by PTB overexpression in in vivo translation experiments in MHV-infected cells. Given that PTB interacts with the viral N protein, which is one of the components of the MHV replication complex, PTB may exert its function on viral replication/transcription by association with viral RNA as well as other viral and cellular factors in the replication complex.
Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5 and 3 untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive-and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive-or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5-UTR of both strands. Mouse hepatitis virus (MHV) belongs to the Coronaviridae family and contains a single-stranded, 31-kb, positive-sense RNA (15). The viral genome is composed of a series of open reading frames (ORFs 1 to 7), flanked by untranslated regions (UTRs) at the 5Ј and 3Ј ends. MHV RNA replication and transcription take place in the cytoplasm and are mediated by its own RNA-dependent RNA polymerase and other viral and cellular proteins. Six to seven subgenomic mRNAs share 5Ј and 3Ј ends with the genomic RNA and are translated through a cap-dependent mechanism. Regulation of transcription, replication, and translation of viral RNA involves several cis-and trans-acting RNA elements, including intergenic sequence, leader sequence, and the 3Ј-UTR of viral RNA (19,20,36,37) and viral and cellular proteins. The leader RNA can function in viral RNA synthesis both in cis and in trans in virus-infected cells (36). Several trans-acting factors, including viral and cellular proteins, have been shown to bind to this region (6).Besides the cis-and trans-acting RNA elements, cellular proteins have been increasingly recognized to play important roles in virus replication, transcription, and translation, as well as virus entry, assembly, and release (16). For example, poliovirus translation and replication are coordinated by the interaction of host factors with viral factors at the 5Ј and/or 3Ј end of viral RNA. These factors include host poly(C)-binding protein, poly(A)-binding protein and the viral polymerase precursor 3CD. Poly(C)-binding protein and poly(A)-binding protein bind to the 5Ј and 3Ј ends, respectively, thus promoting translation early in the infection. As 3CD accumulates later in infection and binds to the cloverleaf structure in the 5Ј end of viral RNA, poly(C)-binding protein, poly(A)-binding protein, and 3CD interact with each other to induce the circularization of poliovirus RNA. This circular RNP complex has been shown to be required for positive-strand RNA synthesis, thus affecting viral replication (1, 10). In addition, it has been reported that La autoantigen (22), poly...
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