Construction and characterization of murine cytomegaloviruses that contain transposon insertions at open reading frames m09 and M83

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.     

The proteome of the bacterium Mycoplasma pneumoniae: comparing predicted open reading frames to identified gene products

An existing proteome map of the bacterium Mycoplasma pneumoniae comprising proteins from 224 genes was extended to 305 genes. This corresponds to about 44% of the 688 proposed genome sequence derived open reading frames (ORFs). The newly assigned gene products were enriched, separated by one-dimensional or two-dimensional (2-D) gel electrophoresis and identified by mass spectrometry. The enrichment procedures included differential centrifugation, anion and cation exchange chromatography, affinity chromatography with heparin as a ligand and isolation of biotinylated proteins by binding to immobilized streptavidin. A comparative analysis of the identified proteins from 305 genes with the as yet unverified 383 ORFs concerning isoelectric point, molecular weight and number of transmembrane segments revealed that proteins with more than three predicted transmembrane segments and an isoelectric point above 10.5 are most likely not to be separated by 2-D gel electrophoresis. The mutual benefits of genomics and proteomics were shown by the identification of a todate unannotated 128 amino acid long protein.     

In vitro and in vivo characterization of a murine cytomegalovirus with a mutation at open reading frame m166

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the mutants, Rvm166, which contained the transposon sequence at open reading frame m166, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. The viral mutant replicated as well as the wild-type Smith strain in vitro in NIH 3T3 cells, whereas the transposon insertion precluded the expression of >65% of the m166 open reading frame. Compared to the wild-type strain and a rescued virus that restored the m166 region, the viral mutant was significantly attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. At 21 days postinfection, the titers of the viral mutant in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice were lower than the titers of the Smith strain and the rescued virus by about 30000-, 10000-, 1000-, 300-, and 800-fold, respectively. Moreover, the virulence of the mutant virus appears to be severely attenuated because no death was found in SCID mice infected with the viral mutant up to 90 days postinfection, whereas all of the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results suggest that m166 probably encodes a virulence factor and is required for MCMV virulence in killing SCID mice and for optimal viral growth in vivo.     

In vitro and in vivo characterization of a murine cytomegalovirus with a transposon insertional mutation at open reading frame M43

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.     

Protein products of the open reading frames encoding nonstructural proteins of human astrovirus serotype 8

Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.     

Translational regulation of human methionine synthase by upstream open reading frames

Methionine synthase is a key enzyme poised at the intersection of folate and sulfur metabolism and functions to reclaim homocysteine to the methionine cycle. The 5' leader sequence in human MS is 394 nucleotides long and harbors two open reading frames (uORFs). In this study, regulation of the main open reading frame by the uORFs has been elucidated. Both uORFs downregulate translation as demonstrated by mutation of the upstream AUG codons (uAUG) either singly or simultaneously. The uAUGs are capable of recruiting the 40S ribosomal complex as revealed by their ability to drive reporter expression in constructs in which the luciferase is fused to the uORFs. uORF2, which is predicted to encode a 30 amino acid long polypeptide, has a clustering of rare codons encoding arginine and proline. Mutation of a tandemly repeated rare codon for arginine at positions 3 and 4 in uORF2 to either common codons for the same amino acid or common codons for alanine results in complete alleviation of translation inhibition. This suggests a mechanism for ribosome stalling and demonstrates that the cis-effects on translation by uORF2 is dependent on the nucleotide sequence but is apparently independent of the sequence of the encoded peptide. This study reveals complex regulation of the essential housekeeping gene, methionine synthase, by the uORFs in its leader sequence.     

Quantitation and characterization of a newly described organic anion binder by radioimmunoassay

As part of a study on the influence of dietary lipids on vitamin transport and metabolism in lactating cows, we have examined the beta-carotene content and other properties of fractions of the high-density lipoprotein (HDL, density 1.05-1.16 g/ml) of bovine blood. Our purpose was primarily to explain previous results indicating that feeding cows polyunsaturated lipids alters the properties of the HDL and increases the concentration of beta-carotene in the blood but not in the milk. Fractions of HDL of different particle size were prepared by gel-filtration chromatography and the particle diameters measured by electron microscopy. We found that large HDL particles contain more beta-carotene per unit weight than small particles. Furthermore the HDL from cows fed lipid-rich diets with a high proportion of linoleic-acid residues, which had been protected against microbial degradation in the rumen, had a high percentage of HDL particles with large diameters. The blood from these cows had a higher concentration of beta-carotene than before feeding polyunsaturated lipids, but their milk had a lower concentration. We suggest that HDL is the main store of beta-carotene in bovine blood. Moreover the concentration of beta-carotene in blood is increased by feeding polyunsaturated lipids largely because of the increase in the percentage of large HDL particles, which contain more beta-carotene. The effect on the concentration of beta-carotene in milk implies that the transfer mechanism is less efficient as a result of feeding polyunsaturated lipids. This lower efficiency may be due in part to the higher percentage of large HDL particles.     

Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations

Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.     

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.     

The mouse synemin gene encodes three intermediate filament proteins generated by alternative exon usage and different open reading frames

We have previously cloned and characterized the human synemin gene, which encodes two intermediate filament proteins (IFPs). We now show that the mouse synemin gene encodes three different synemin isoforms through an alternative splicing mechanism. Two of them, synemin H and M are similar to human alpha and beta synemin, and the third isoform, L synemin, constitutes a new form of IFP. It has a typical rod domain and a short tail (49 residues) with a novel sequence that is produced by a different open reading frame. The synthesis of H/M synemins starts in the embryo, whereas the synemin L isoform is present in adult muscles. The H/M isoforms are bound to desmin or vimentin in the muscle cells of wild-type mice. Using desmin- and vimentin-deficient mice, we have obtained direct evidence that synemin is associated with muscle intermediate filaments in vivo. The organization of the synemin fibril is disrupted in skeletal and cardiac muscle when desmin is absent and in smooth muscle when vimentin is absent. The fact that the three synemin isoforms differ in the sequences of their tail domains as well as in their developmental patterns suggests that they fulfill different functions.     

Regulation of fungal gene expression via short open reading frames in the mRNA 5'untranslated region

We review how the expression of fungal mRNAs can be controlled by ribosome interactions with short upstream open reading frames (uORFs) within the 5'untranslated region. The efficiency of uAUG recognition modulates the impact of a uORF but steps during and after translation of the uORF also influence uORF function. The post-termination behaviour of ribosomes, therefore, plays a major role in determining the expression level of these main ORFs. Translation of a uORF can produce a cis-acting peptide that causes effector molecule-dependent stalling of the ribosomes at the end of the uORF. In other cases it is the length or position, or other features of the uORF, rather than the peptide it encodes, that determine the efficiency with which ribosomes reinitiate translation downstream of it. Whether the form of the ribosome that resumes scanning after termination is the 40S subunit alone or the entire 80S ribosome is not known. Translation of the uORF can also control gene expression by affecting the stability of the mRNA. Finally, trans-acting factors may participate in the regulatory mechanisms. Future work will need not only to provide more information on the mechanisms underlying the known cases of uORF-mediated control but also to define the full complement of uORF-containing mRNAs in at least one fungal organism.     

Physical evidence for distinct mechanisms of translational control by upstream open reading frames.

The Saccharomyces cerevisiae GCN4 mRNA 5'-leader contains four upstream open reading frames (uORFs) and the CPA1 leader contains a single uORF. To determine how these uORFs control translation, we examined mRNAs containing these leaders in cell-free translation extracts to determine where ribosomes were loaded first and where they were loaded during steady-state translation. Ribosomes predominantly loaded first at GCN4 uORF1. Following its translation, but not the translation of uORF4, they efficiently reinitiated protein synthesis at Gcn4p. Adding purified eIF2 increased reinitiation at uORFs 3 or 4 and reduced reinitiation at Gcn4p. This indicates that eIF2 affects the site of reinitiation following translation of GCN4 uORF1 in vitro. In contrast, for mRNA containing the CPA1 uORF, ribosomes reached the downstream start codon by scanning past the uORF. Addition of arginine caused ribosomes that had synthesized the uORF polypeptide to stall at its termination codon, reducing loading at the downstream start codon, apparently by blocking scanning ribosomes, and not by affecting reinitiation. The GCN4 and CPA1 uORFs thus control translation in fundamentally different ways.     

Predicting internal exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames.

A new method which predicts internal exon sequences in human DNA has been developed. The method is based on a splice site prediction algorithm that uses the linear discriminant function to combine information about significant triplet frequencies of various functional parts of splice site regions and preferences of oligonucleotides in protein coding and intron regions. The accuracy of our splice site recognition function is 97% for donor splice sites and 96% for acceptor splice sites. For exon prediction, we combine in a discriminant function the characteristics describing the 5'-intron region, donor splice site, coding region, acceptor splice site and 3'-intron region for each open reading frame flanked by GT and AG base pairs. The accuracy of precise internal exon recognition on a test set of 451 exon and 246693 pseudoexon sequences is 77% with a specificity of 79%. The recognition quality computed at the level of individual nucleotides is 89% for exon sequences and 98% for intron sequences. This corresponds to a correlation coefficient for exon prediction of 0.87. The precision of this approach is better than other methods and has been tested on a larger data set. We have also developed a means for predicting exon-exon junctions in cDNA sequences, which can be useful for selecting optimal PCR primers.