Are There 1031 Virus Particles on Earth, or More, or Fewer?

The number of virus particles on Earth is frequently reported in the scientific literature and in general-interest publications as being on the order of 1031, with some confusion about whether this is a high or low estimate. This number is often given without a source, although it should be attributed to a paper by Hendrix et al. published in 1999 (R. W. Hendrix, M. C. Smith, R. N. Burns, M. E. Ford, and G. F. Hatfull, Proc Natl Acad Sci U S A 96:2192-2197, 1999, https://doi.org/10.1073/pnas.96.5.2192). As with any oft-repeated statistic, it is informative to know how it has been derived and whether it should be revised in the light of new evidence. I review the history of the 1031 estimate and use more recent assessments of the number of bacterial and viral particles in various habitats to conclude that the best estimate of the number of virus particles on Earth ("the Hendrix product") remains close to 1031 and is unlikely to be either much less or much more than that.

Evolutionary perspective on innate immune recognition.

Analysis of human and Drosophila genomes demonstrates an ancient origin of innate immunity and the diversity of the mechanisms of innate immune recognition.

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

The epithelium-specific ETS protein EHF/ESE-3 is a context-dependent transcriptional repressor downstream of MAPK signaling cascades.

Exon trapping and cDNA selection procedures were used to search for novel genes at human chromosome 11p13, a region previously associated with loss of heterozygosity in epithelial carcinomas. Using these approaches, we found the ESE-2 and ESE-3 genes, coding for ETS domain-containing transcription factors. These genes lie in close proximity to the catalase gene within a approximately 200-kilobase genomic interval. ESE-3 mRNA is widely expressed in human tissues with high epithelial content, and immunohistochemical analysis with a newly generated monoclonal antibody revealed that ESE-3 is a nuclear protein expressed exclusively in differentiated epithelial cells and that it is absent in the epithelial carcinomas tested. In transient transfections, ESE-3 behaves as a repressor of the Ras- or phorbol ester-induced transcriptional activation of a subset of promoters that contain ETS and AP-1 binding sites. ESE-3-mediated repression is sequence- and context-dependent and depends both on the presence of high affinity ESE-3 binding sites in combination with AP-1 cis-elements and the arrangement of these sites within a given promoter. We propose that ESE-3 might be an important determinant in the control of epithelial differentiation, as a modulator of the nuclear response to mitogen-activated protein kinase signaling cascades.

Quod erat demonstrandum? The mystery of experimental validation of apparently erroneous computational analyses of protein sequences.

BACKGROUND: Computational predictions are critical for directing the experimental study of protein functions. Therefore it is paradoxical when an apparently erroneous computational prediction seems to be supported by experiment. RESULTS: We analyzed six cases where application of novel or conventional computational methods for protein sequence and structure analysis led to non-trivial predictions that were subsequently supported by direct experiments. We show that, on all six occasions, the original prediction was unjustified, and in at least three cases, an alternative, well-supported computational prediction, incompatible with the original one, could be derived. The most unusual cases involved the identification of an archaeal cysteinyl-tRNA synthetase, a dihydropteroate synthase and a thymidylate synthase, for which experimental verifications of apparently erroneous computational predictions were reported. Using sequence-profile analysis, multiple alignment and secondary-structure prediction, we have identified the unique archaeal 'cysteinyl-tRNA synthetase' as a homolog of extracellular polygalactosaminidases, and the 'dihydropteroate synthase' as a member of the beta-lactamase-like superfamily of metal-dependent hydrolases. CONCLUSIONS: In each of the analyzed cases, the original computational predictions could be refuted and, in some instances, alternative strongly supported predictions were obtained. The nature of the experimental evidence that appears to support these predictions remains an open question. Some of these experiments might signify discovery of extremely unusual forms of the respective enzymes, whereas the results of others could be due to artifacts.

Biosynthesis of isoprenoids via mevalonate in Archaea: the lost pathway.

Isoprenoid compounds are ubiquitous in living species and diverse in biological function. Isoprenoid side chains of the membrane lipids are biochemical markers distinguishing archaea from the rest of living forms. The mevalonate pathway of isoprenoid biosynthesis has been defined completely in yeast, while the alternative, deoxy-D-xylulose phosphate synthase pathway is found in many bacteria. In archaea, some enzymes of the mevalonate pathway are found, but the orthologs of three yeast proteins, accounting for the route from phosphomevalonate to geranyl pyrophosphate, are missing, as are the enzymes from the alternative pathway. To understand the evolution of isoprenoid biosynthesis, as well as the mechanism of lipid biosynthesis in archaea, sequence motifs in the known enzymes of the two pathways of isoprenoid biosynthesis were analyzed. New sequence relationships were detected, including similarities between diphosphomevalonate decarboxylase and kinases of the galactokinase superfamily, between the metazoan phosphomevalonate kinase and the nucleoside monophosphate kinase superfamily, and between isopentenyl pyrophosphate isomerases and MutT pyrophosphohydrolases. Based on these findings, orphan members of the galactokinase, nucleoside monophosphate kinase, and pyrophosphohydrolase families in archaeal genomes were evaluated as candidate enzymes for the three missing steps. Alternative methods of finding these missing links were explored, including physical linkage of open reading frames and patterns of ortholog distribution in different species. Combining these approaches resulted in the generation of a short list of 13 candidate genes for the three missing functions in archaea, whose participation in isoprenoid biosynthesis is amenable to biochemical and genetic investigation.

The alpha subunit of E. coli RNA polymerase activates RNA binding by NusA.

The Escherichia coli NusA protein modulates pausing, termination, and antitermination by associating with the transcribing RNA polymerase core enzyme. NusA can be covalently cross-linked to nascent RNA within a transcription complex, but does not bind RNA on its own. We have found that deletion of the 79 carboxy-terminal amino acids of the 495-amino-acid NusA protein allows NusA to bind RNA in gel mobility shift assays. The carboxy-terminal domain (CTD) of the alpha subunit of RNA polymerase, as well as the bacteriophage lambda N gene antiterminator protein, bind to carboxy-terminal regions of NusA and enable full-length NusA to bind RNA. Binding of NusA to RNA in the presence of alpha or N involves an amino-terminal S1 homology region that is otherwise inactive in full-length NusA. The interaction of the alpha-CTD with full-length NusA stimulates termination. N may prevent termination by inducing NusA to interact with N utilization (nut) site RNA rather than RNA near the 3' end of the nascent transcript. Sequence analysis showed that the alpha-CTD contains a modified helix-hairpin-helix motif (HhH), which is also conserved in the carboxy-terminal regions of some eubacterial NusA proteins. These HhH motifs may mediate protein-protein interactions in NusA and the alpha-CTD.

Conserved phosphoprotein interaction motif is functionally interchangeable between ataxin-7 and arrestins.

Olivopontocerebellar atrophy with retinal degeneration is a hereditary neurodegenerative disorder that belongs to the subtype II of the autosomal dominant cerebellar ataxias and is characterized by early-onset cerebellar and macular degeneration preceded by diagnostically useful tritan colorblindness. The gene mutated in the disease (SCA7) has been mapped to chromosome 3p12-13.5, and positional cloning identified the cause of the disease as CAG repeat expansion in this gene. The SCA7 gene product, ataxin-7, is an 897 amino acid protein with an expandable polyglutamine tract close to its N-terminus. No clues to ataxin-7 function have been obtained from sequence database searches. Here we report that ataxin-7 has a motif of ca. 50 amino acids, related to the phosphate-binding site of arrestins. To test the relevance of this sequence similarity, we introduced the putative ataxin-7 phosphate-binding site into visual arrestin and beta-arrestin. Both chimeric arrestins retain receptor-binding affinity and show characteristic high selectivity for phosphorylated activated forms of rhodopsin and beta-adrenergic receptor, respectively. Although the insertion of a Gly residue (absent in arrestins but present in the putative phosphate-binding site of ataxin-7) disrupts the function of visual arrestin-ataxin-7 chimera, it enhances the function of beta-arrestin-ataxin-7 chimera. Taken together, our data suggest that the arrestin-like site in the ataxin-7 sequence is a functional phosphate-binding site. The presence of the phosphate-binding site in ataxin-7 suggests that this protein may be involved in phosphorylation-dependent binding to its protein partner(s) in the cell.

The minimal genome concept.

Complete genome sequences are becoming available for a large number of diverse species. Quantification of gene content, of gene family expansion, of orthologous gene conservation, as well as their displacement, are now possible - laying the ground for the estimation of the minimal set of proteins sufficient for cellular life. The consensus of computational results suggests a set close to 300 genes. These predictions will be evaluated by engineering of small bacterial genomes.

Conserved transactivation domain shared by interferon regulatory factors and Smad morphogens.

Interferon regulatory factors (IRFs) regulate the transcription of both interferon-inducible genes and interferons themselves. Along with the N-terminal, DNA-binding, winged-helix domain, most IRFs contain the C-terminal domains that are shown to be related to the C-terminal domains in the proteins of the Smad family that mediate transcription activation in the transforming growth factor response pathway. Comparison of the IRF-Smad alignment to the known three-dimensional structure of human tumor suppressor Smad4 suggests that a conserved loop, equivalent to Loop 3 in Smad 4, is a determinant of protein-protein interaction in IRFs.

A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice.

A genetic locus of Pseudomonas aeruginosa was identified that is highly and specifically inducible during infection of neutropenic mice. This locus, ppkA, encodes a protein that is highly homologous to eukaryote-type serine/threonine protein kinases. A ppkA null mutant strain shows reduced virulence in neutropenic mice compared to the wild type. Overexpression of the PpkA protein greatly inhibited the growth of Escherichia coli or P. aeruginosa. However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells.

Large-scale taxonomic profiling of eukaryotic model organisms: a comparison of orthologous proteins encoded by the human, fly, nematode, and yeast genomes.

Comparisons of DNA and protein sequences between humans and model organisms, including the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster, are a significant source of information about the function of human genes and proteins in both normal and disease states. Important questions regarding cross-species sequence comparison remain unanswered, including (1) the fraction of the metabolic, signaling, and regulatory pathways that is shared by humans and the various model organisms; and (2) the validity of functional inferences based on sequence homology. We addressed these questions by analyzing the available fractions of human, fly, nematode, and yeast genomes for orthologous protein-coding genes, applying strict criteria to distinguish between candidate orthologous and paralogous proteins. Forty-two quartets of proteins could be identified as candidate orthologs. Twenty-four Drosophila protein sequences were more similar to their human orthologs than the corresponding nematode proteins. Analysis of sequence substitutions and evolutionary distances in this data set revealed that most C. elegans genes are evolving more rapidly than Drosophila genes, suggesting that unequal evolutionary rates may contribute to the differences in similarity to human protein sequences. The available fraction of Drosophila proteins appears to lack representatives of many protein families and domains, reflecting the relative paucity of genomic data from this species.

Characterization of the large (L) RNA of peanut bud necrosis tospovirus.

The nucleocapsids purified from peanut plants systemically infected with peanut bud necrosis virus (PBNV), a member of the genus Tospovirus, contained both viral(v) and viral complementary(vc) sense L RNAs. Defective forms of L RNA containing 'core polymerase region' were observed. The full length L RNA of PBNV was sequenced using overlapping cDNA clones. The 8911 nucleotide L RNA contains a single open reading frame (ORF) in the vc strand, and encodes a protein of 330 kDa. At the 5' and 3' termini of the v sense RNA there were 247 and 32 nt untranslated regions, respectively, containing an 18 nt complementary sequence with one mismatch. Comparisons of the predicted amino acid sequence of the L protein of PBNV with other members of Bunyaviridae suggest that the L protein of PBNV is a viral polymerase. The L protein had highest identity in the 'core-polymerase domain' with the corresponding regions of other tospoviruses, tomato spotted wilt virus and impatiens necrotic spot virus.

Comparison of archaeal and bacterial genomes: computer analysis of protein sequences predicts novel functions and suggests a chimeric origin for the archaea.

Protein sequences encoded in three complete bacterial genomes, those of Haemophilus influenzae, Mycoplasma genitalium and Synechocystis sp., and the first available archaeal genome sequence, that of Methanococcus jannaschii, were analysed using the BLAST2 algorithm and methods for amino acid motif detection. Between 75% and 90% of the predicted proteins encoded in each of the bacterial genomes and 73% of the M. jannaschii proteins showed significant sequence similarity to proteins from other species. The fraction of bacterial and archaeal proteins containing regions conserved over long phylogenetic distances is nearly the same and close to 70%. Functions of 70-85% of the bacterial proteins and about 70% of the archaeal proteins were predicted with varying precision. This contrasts with the previous report that more than half of the archaeal proteins have no homologues and shows that, with more sensitive methods and detailed analysis of conserved motifs, archaeal genomes become as amenable to meaningful interpretation by computer as bacterial genomes. The analysis of conserved motifs resulted in the prediction of a number of previously undetected functions of bacterial and archaeal proteins and in the identification of novel protein families. In spite of the generally high conservation of protein sequences, orthologues of 25% or less of the M. jannaschii genes were detected in each individual completely sequenced genome, supporting the uniqueness of archaea as a distinct domain of life. About 53% of the M. jannaschii proteins belong to families of paralogues, a fraction similar to that in bacteria with larger genomes, such as Synechocystis sp. and Escherichia coli, but higher than that in H. influenzae, which has approximately the same number of genes as M. jannaschii. Certain groups of proteins, e.g. molecular chaperones and DNA repair enzymes, thought to be ubiquitous and represented in the minimal gene set derived by bacterial genome comparison, are missing in M. jannaschii, indicating massive non-orthologous displacement of genes responsible for essential functions. An unexpectedly large fraction of the M. jannaschii gene products, 44%, shows significantly higher similarity to bacterial than to eukaryotic proteins, compared with 13% that have eukaryotic proteins as their closest homologues (the rest of the proteins show approximately the same level of similarity to bacterial and eukaryotic homologues or have no homologues). Proteins involved in translation, transcription, replication and protein secretion are most closely related to eukaryotic proteins, whereas metabolic enzymes, metabolite uptake systems, enzymes for cell wall biosynthesis and many uncharacterized proteins appear to be 'bacterial'. A similar prevalence of proteins of apparent bacterial origin was observed among the currently available sequences from the distantly related archaeal genus, Sulfolobus. It is likely that the evolution of archaea included at least one major merger between ancestral cells from the bacterial lineage and the lineage leading to the eukaryotic nucleocytoplasm.

Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs.

Positional cloning has already produced the sequences of more than 70 human genes associated with specific diseases. In addition to their medical importance, these genes are of interest as a set of human genes isolated solely on the basis of the phenotypic effect of the respective mutations. We analyzed the protein sequences encoded by the positionally cloned disease genes using an iterative strategy combining several sensitive computer methods. Comparisons to complete sequence databases and to separate databases of nematode, yeast, and bacterial proteins showed that for most of the disease gene products, statistically significant sequence similarities are detectable in each of the model organisms. Only the nematode genome encodes apparent orthologs with conserved domain architecture for the majority of the disease genes. In yeast and bacterial homologs, domain organization is typically not conserved, and sequence similarity is limited to individual domains. Generally, human genes complement mutations only in orthologous yeast genes. Most of the positionally cloned genes encode large proteins with several globular and nonglobular domains, the functions of some or all of which are not known. We detected conserved domains and motifs not described previously in a number of proteins encoded by disease genes and predicted functions for some of them. These predictions include an ATP-binding domain in the product of hereditary nonpolyposis colon cancer gene (a MutL homolog), which is conserved in the HS90 family of chaperone proteins, type II DNA topoisomerases, and histidine kinases, and a nuclease domain homologous to bacterial RNase D and the 3'-5' exonuclease domain of DNA polymerase I in the Werner syndrome gene product.