The mRNA Codon Environment at the P and E Sites of Human Ribosomes Deduced from Photocrosslinking with pUUUGUU Derivatives

Three mRNA analogs—derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position—were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA^Phe was used, while tRNA^Val was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions –3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with mild UV light ( > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions –1 to +3 binds to G1207, while that in positions –2 or –3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the –3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the –3 position, this crosslinking was not observed in the absence of tRNA.     

Evolutionary Change of Restriction Cleavage Sites and Phylogenetic Inference

Mathematical formulas are developed for the evolutionary change of restriction cleavage sites in a DNA sequence, allowing unequal rates between transitional and transversional types of nucleotide substitution. Formulas are also developed for the probability of having a particular pattern of site changes among evolutionary lineages, such as parallel gains or losses of sites, and for inferring the presence or absence of a restriction site in an ancestral sequence from data on the present-day sequences. The unordered compatibility method is proposed for inferring the phylogenetic relationships among relatively closely related organisms, treating restriction sites as cladistic characters. Formulas are derived for the probability (P+) of obtaining the correct network for a given number (N) of informative sites for the cases of four and five species. These formulas are applied to evaluate the performance of the method and to estimate the N value required for P+ to be 95% or larger. The method performs well when the branches between ancestral nodes and the branches leading to the two most recent species are more or less equal in length, but performs poorly when the latter two branches are considerably longer than the former.     

Protease Cleavage Sites in HIV-1 gp120 Recognized by Antigen Processing Enzymes Are Conserved and Located at Receptor Binding Sites

The identification of vaccine immunogens able to elicit broadly neutralizing antibodies (bNAbs) is a major goal in HIV vaccine research. Although it has been possible to produce recombinant envelope glycoproteins able to adsorb bNAbs from HIV-positive sera, immunization with these proteins has failed to elicit antibody responses effective against clinical isolates of HIV-1. Thus, the epitopes recognized by bNAbs are present on recombinant proteins, but they are not immunogenic. These results led us to consider the possibility that changes in the pattern of antigen processing might alter the immune response to the envelope glycoprotein to better elicit protective immunity. In these studies, we have defined protease cleavage sites on HIV gp120 recognized by three major human proteases (cathepsins L, S, and D) important for antigen processing and presentation. Remarkably, six of the eight sites identified in gp120 were highly conserved and clustered in regions of the molecule associated with receptor binding and/or the binding of neutralizing antibodies. These results suggested that HIV may have evolved to take advantage of major histocompatibility complex (MHC) class II antigen processing enzymes in order to evade or direct the antiviral immune response.A major goal of HIV vaccine development is the development of immunogens that elicit protective antiviral antibody and cellular immune responses. However, after more than 25 years of research, vaccine immunogens able to elicit protective immunity in humans have yet to be described (11, 31). Although it has been possible to produce recombinant envelope proteins (gp120 and gp140) with many of the features of native virus proteins (e.g., complex glycosylation and the ability to bind CD4, chemokine receptors, and neutralizing antibodies), these antigens have not been able to elicit broadly neutralizing antibodies (bNAbs) or protective immune responses when used as immunogens (11, 32, 43, 50, 56, 74, 79). The fact that recombinant proteins can adsorb virus bNAbs from HIV-1-positive sera (59, 91) indicates that many recombinant envelope proteins are correctly folded but that the epitopes recognized by bNAbs are simply not immunogenic. Over the last decade, several different approaches have been employed to create immunogens able to elicit broadly neutralizing antibodies. These strategies have included efforts to duplicate and/or stabilize the oligomeric structure of HIV envelope proteins (5, 26, 87), the creation of minimal antigenic structures lacking epitopes that conceal important neutralizing sites (27, 46, 70, 89), and prime/boost strategies combining protein immunization with DNA immunization or infection with recombinant viruses in order to stimulate the endogenous synthesis and presentation of HIV immunogens (15, 29, 30, 83). However, none of these approaches has resulted in a clinically significant improvement in antiviral immunity or HIV vaccine efficacy. Efforts to elicit protective cellular immune responses (e.g., cytotoxic lymphocytes) by use of recombinant virus vaccines have likewise been disappointing (10, 61). In fact, such vaccines may have promoted HIV infection rather than inhibiting it (22, 23).In the present study, we describe the first steps in a new approach to reengineering the immunogenicity of HIV envelope proteins in order to improve the potency and specificity of humoral and cellular immune responses. The approach is based on defining the determinants of antigen processing and presentation of HIV envelope glycoproteins. Both humoral and cellular immune responses depend on proteolytic degradation of protein antigens prior to antigen presentation, mediated by professional antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells (97). Normally, proteins of intracellular origin are processed by the proteasome, a 14- to 17-subunit protein complex located in the cytosol. Proteins of extracellular origin are processed in lysosomes or late endosomes of APCs. The resulting peptide epitopes are then loaded into major histocompatibility complex (MHC) class I or class II molecules and presented on the surfaces of APCs to CD8 or CD4 T cells. Within the endosomes and lysosomes of APCs, there are cathepsins, acid thiol reductase, and aspartyl endopeptidase. The enzymes perform two activities: degrading endocytosed protein antigens to liberate peptides for MHC class II binding (99) and removing the invariant chain chaperone (6, 94). Although all cathepsins can liberate epitopes from a diverse range of antigens (16), only cathepsins S and L have nonredundant roles in antigen processing in vivo (reviewed by Hsing and Rudensky [45]). Cathepsin L is expressed in thymic cortical epithelial cells but not in B cells or dendritic cells, while cathepsin S is found in all three types of APCs. Unlike cathepsins L and S, which are cysteine proteases and active at neutral pH, cathepsin D is an aspartic protease, is active at acidic pH, and participates in proteolysis and antigen presentation in connection with MHC class I and class II antigen presentation pathways established for CD4 and CD8 T cells. In considering the use of envelope proteins as potential vaccines, the route of immunization, formulation (e.g., adjuvants), protein folding, disulfide bonding, and glycosylation pattern all determine which peptides are available for MHC-restricted presentation.Previous studies provided evidence that gp120 was sensitive to digestion by cathepsins B, D, and L, but the specific cleavage sites were not defined (18). In the present study, we (i) describe the locations of eight protease cleavage sites on HIV-1 gp120 recognized by cathepsins L, S, and D, involved in antigen processing; (ii) determine the extent to which they are conserved; and (iii) evaluate the effect of cathepsin cleavage on the binding of gp120 to CD4-IgG and neutralizing antibodies. The results obtained provide new insights into the basis of envelope immunogenicity that may prove to be useful in the development of HIV vaccine antigens.     

Crosslinking of mRNA Analog,pUUAGUAUUUAUU Derivative with Photoactivatable Group at the Guanosine Residue,with Human 80S Ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg²⁺, or 4 mM Mg²⁺ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840–1849 of 18S rRNA, but in the complexes formed with participation of Phe-tRNA^Phe (where the G residue carrying the arylazido group occupied position –3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position –3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg²⁺ concentration and the presence of polyamines.     

Newly Characterized Region of CP190 Associates with Microtubules and Mediates Proper Spindle Morphology in Drosophila Stem Cells

CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.     

Inter-Protein Sequence Co-Evolution Predicts Known Physical Interactions in Bacterial Ribosomes and the Trp Operon

Interaction between proteins is a fundamental mechanism that underlies virtually all biological processes. Many important interactions are conserved across a large variety of species. The need to maintain interaction leads to a high degree of co-evolution between residues in the interface between partner proteins. The inference of protein-protein interaction networks from the rapidly growing sequence databases is one of the most formidable tasks in systems biology today. We propose here a novel approach based on the Direct-Coupling Analysis of the co-evolution between inter-protein residue pairs. We use ribosomal and trp operon proteins as test cases: For the small resp. large ribosomal subunit our approach predicts protein-interaction partners at a true-positive rate of 70% resp. 90% within the first 10 predictions, with areas of 0.69 resp. 0.81 under the ROC curves for all predictions. In the trp operon, it assigns the two largest interaction scores to the only two interactions experimentally known. On the level of residue interactions we show that for both the small and the large ribosomal subunit our approach predicts interacting residues in the system with a true positive rate of 60% and 85% in the first 20 predictions. We use artificial data to show that the performance of our approach depends crucially on the size of the joint multiple sequence alignments and analyze how many sequences would be necessary for a perfect prediction if the sequences were sampled from the same model that we use for prediction. Given the performance of our approach on the test data we speculate that it can be used to detect new interactions, especially in the light of the rapid growth of available sequence data.     

Exchange of the Coronavirus Replicase Polyprotein Cleavage Sites Alters Protease Specificity and Processing

Coronavirus nonstructural proteins 1 to 3 are processed by one or two papain-like proteases (PLP1 and PLP2) at specific cleavage sites (CS1 to -3). Murine hepatitis virus (MHV) PLP2 and orthologs recognize and cleave at a position following a p4-Leu-X-Gly-Gly-p1 tetrapeptide, but it is unknown whether these residues are sufficient to result in processing by PLP2 at sites normally cleaved by PLP1. We demonstrate that exchange of CS1 and/or CS2 with the CS3 p4-p1 amino acids in engineered MHV mutants switches specificity from PLP1 to PLP2 at CS2, but not at CS1, and results in altered protein processing and virus replication. Thus, the p4-p1 residues are necessary for PLP2 processing but require a specific protein or cleavage site context for optimal PLP recognition and cleavage.Coronaviruses are positive-strand RNA viruses that translate their first open reading frames (ORF1a and ORF1b) into polyproteins that are processed by viral proteases into intermediate and mature nonstructural proteins (nsp1 to -16) (Fig. ​(Fig.11 A) (4, 7, 17, 20). nsp1, -2, and -3 are liberated at cleavage sites (CSs) between nsp1-2 (CS1), nsp2-3 (CS2), and nsp3-4 (CS3) by one or two papain-like protease (PLP) activities encoded within nsp3 (1, 2, 12, 13, 15) (Fig. ​(Fig.1B).1B). Murine hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) use two PLPs (PLP1 and PLP2) to process at CS1 to -3, while severe acute respiratory syndrome coronavirus (SARS-CoV) and avian infectious bronchitis virus (IBV) use a single PLP each (PLpro and PLP2, respectively) (10, 20, 25, 26). The factors determining the evolution and use of one versus two PLPs by different coronaviruses for processing of nsp1, -2, and -3 are unknown. Mutations at MHV CSs or within PLP1 alter replication and protein processing in surprising ways (8, 13). Loss of processing at MHV CS1 and CS2 by CS deletion or mutation results in changes in the timing and extent of virus replication. Inactivation of MHV PLP1 is more detrimental for virus replication than deletion of CS1 and CS2 or than inactivation of PLP1 combined with the CS deletions, even though not all of the mutant viruses process at CS1 or CS2 or display similar protein processing phenotypes. In contrast to MHV results, the HCoV-229E PLP1 and PLP2 have both been shown to process at CS1 and CS2, albeit at different efficiencies (Fig. ​(Fig.1B)1B) (24). Finally, the single SARS-CoV PLP2 homolog (PLpro) mediates efficient processing at CS1 to -3, each of which has an upstream position 4-Leu-X-Gly-Gly-position 1 (p4-LXGG-p1) amino acid motif implicated in PLpro processing (10, 16, 18). MHV possesses a p4-LXGG-p1 sequence only at CS3 and is cleaved by PLP2. These results suggest that p4-LXGG-p1 may be the critical determinant of recognition by PLP2/PLpro, but this hypothesis has not been tested in studies of replicating virus. Thus, it remains unknown whether the differences in PLP/CS recognition and processing are determined by the proximal p4-p1 residues (22).Open in a separate windowFIG. 1.MHV replicase organization, coronavirus PLP-mediated processing, and experimental design of cleavage site replacement viruses. (A) ORF1 of MHV genome RNA is shown, with overlapping ORF1a and ORF1b. The ORF1ab polyprotein is shown with nonstructural proteins (nsp1 to -16) indicated by vertical lines and numbers. Viral papain-like protease domains in nsp3 are shown as a white box containing black letters (PLP1) and a black box containing white letters (PLP2), and the nsp5 protease (3CLpro) is indicated as a gray box with a white number. Cleavage sites for PLP1 (CS1 and CS2 [shown as white arrowheads]), PLP2 (CS3 [shown as a black arrowhead]), and nsp5 (CS4 to -14 [shown as gray arrowheads]) are indicated. (B) The organization of nsp1 to nsp4 is shown for representative coronaviruses. PLPs are indicated, with the hatched box in IBV indicating a probable catalytically inactive remnant of PLP1. Processing events that were confirmed as occurring in vitro or during infection are shown by arrows with solid lines and large arrowheads, indicating single or dominant protease activity. The dashed lines and small arrowheads indicate minor or secondary cleavage activities. The CS amino acid sequences from position 4 (p4) to p1′ are shown for each CS, with a space and arrow representing the site of proteolytic processing. (C) The CS substitution viruses were engineered to replace the original CS amino acid sequences at CS1 and/or CS2 with that of the CS3 amino acid sequence p4-LKGG-p1. Both CS substitutions were also engineered into a catalytically inactive PLP1 (P1ko) background. PLPs are shown as numbers in boxes within nsp3. Engineered catalytically inactivated PLP1 is shown as a hatched box. Arrowheads indicate cleavage events of the WT virus and are linked to the enzyme predicted to mediate processing at the CS, as indicated by white boxes containing black characters (PLP1) or black boxes containing white characters (PLP2). The p4 through p1 amino acid residues for each CS are shown below each diagram. White and black vertical bars show the respective predicted PLP1 and PLP2 cleavage sites. Engineered substitutions are indicated in bold characters. Asterisks indicate engineered mutant genomes that could not be recovered as infectious virus.In this study, we used MHV as a model to test whether PLP/CS specificities could be switched by an exchange of CS amino acid sequences and to determine the impact of CS exchange on protein processing and virus replication. Replacement of the CS3 p4-LKGG-p1 at CS2, but not at CS1, was sufficient for a switch in protease specificity from PLP1 to PLP2. Some combinations of CS exchange could not be recovered with inactive PLP1, and recovered mutant viruses had altered protein processing and/or impaired growth compared to the wild type (WT). The results confirm that p4-LXGG-p1 amino acid sequences are necessary determinants of cleavage by PLP2 but also indicate that a larger cleavage site or a different protein context is required for efficient recognition and processing. Finally, the results support the conclusion that complex relationships with respect to the timing and extent of PLP/CS interactions are essential for successful replication and, likely, for virus fitness.     

Synthesis of Protein, Ribonucleic Acid, and Ribosomes by Individual Bacterial Cells in Balanced Growth

Relative rates of protein synthesis in individual cells were determined by allowing random populations to incorporate tritiated leucine for very short periods (pulses) and then examining autoradiographs of these cells to assess the amount of incorporation (grains per cell) as a function of cell size. Relative rates of ribonucleic acid (RNA) synthesis were determined in the same way by using tritiated uracil. Unless the uracil pulse was very short (less than 1/20 generation), the RNA labeled during the pulse was predominantly ribosomal. The rate of protein synthesis in individual cells is directly proportional to cell size. The rate of RNA synthesis also increases linearly with size in larger cells, but there appears to be a slight delay in RNA synthesis immediately after cell division. Total cellular content of protein, RNA, and ribosomes is directly proportional to cell size. Thus, we conclude that, in individual cells during the cell cycle (i) the average rate of protein synthesis per ribosome is constant and (ii) the increase in macromolecular mass of the cell is exponential with age.     

Yeast PalA/AIP1/Alix Homolog Rim20p Associates with a PEST-Like Region and Is Required for Its Proteolytic Cleavage

The Saccharomyces cerevisiae zinc finger protein Rim101p is activated by cleavage of its C-terminal region, which resembles PEST regions that confer susceptibility to proteolysis. Here we report that Rim20p, a member of the broadly conserved PalA/AIP1/Alix family, is required for Rim101p cleavage. Two-hybrid and coimmunoprecipitation assays indicate that Rim20p binds to Rim101p, and a two-hybrid assay shows that the Rim101p PEST-like region is sufficient for Rim20p binding. Rim101p-Rim20p interaction is conserved in Candida albicans, supporting the idea that interaction is functionally significant. Analysis of Rim20p mutant proteins indicates that some of its broadly conserved regions are required for processing of Rim101p and for stability of Rim20p itself but are not required for interaction with Rim101p. A recent genome-wide two-hybrid study (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2000) indicates that Rim20p interacts with Snf7p and that Snf7p interacts with Rim13p, a cysteine protease required for Rim101p proteolysis. We suggest that Rim20p may serve as part of a scaffold that places Rim101p and Rim13p in close proximity.     

Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.