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Results for “coevolution”

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PubMed1998

Phylogenetic analysis of the Chlamydia trachomatis major outer membrane protein and examination of potential pathogenic determinants.

Phylogenetic analysis was utilized to investigate biological relationships (tissue tropism, disease presentation, and epidemiologic success), as evidenced by coevolution, among human strains of Chlamydia trachomatis. Nucleotide sequences of omp1, the gene encoding the major outer membrane protein (MOMP) of C. trachomatis, were determined for 40 strains representing 11 serovars. These data were combined with available omp1 sequences from GenBank for an analysis encompassing a total of 69 strains representing 17 serovars infecting humans. Phylogenetic analysis of the nucleotide and inferred amino acid sequences showed no evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiologic success). In addition, no specific residues that may have evolved to play a role in determining biologically relevant characteristics of chlamydia, such as tissue specificity, disease presentation, and epidemiologic success, were apparent in the MOMP. These results suggest that variation in MOMP may have arisen from a need to be diverse in the presence of immune pressure rather than as a function of pathogenicity. Therefore, the role of MOMP in disease pathogenesis and infection may be passive, and it may not be the major ligand responsible for directing infection of various human cell types.

Bacterial Outer Membrane Proteins↗

PubMed2006

Genomic structure and phylogeny of the plant pathogen Ralstonia solanacearum inferred from gene distribution analysis.

In the present study, we investigated the gene distribution among strains of the highly polymorphic plant pathogenic beta-proteobacterium Ralstonia solanacearum, paying particular attention to the status of known or candidate pathogenicity genes. Based on the use of comparative genomic hybridization on a pangenomic microarray for the GMI1000 reference strain, we have defined the conditions that allowed comparison of the repertoires of genes among a collection of 18 strains that are representative of the biodiversity of the R. solanacearum species. This identified a list of 2,690 core genes present in all tested strains. As a corollary, a list of 2,338 variable genes within the R. solanacearum species has been defined. The hierarchical clustering based on the distribution of variable genes is fully consistent with the phylotype classification that was previously defined from the nucleotide sequence analysis of four genes. The presence of numerous pathogenicity-related genes in the core genome indicates that R. solanacearum is an ancestral pathogen. The results establish the long coevolution of the two replicons that constitute the bacterial genome. We also demonstrate the clustering of variable genes in genomic islands. Most genomic islands are included in regions with an alternative codon usage, suggesting that they originate from acquisition of foreign genes through lateral gene transfers. Other genomic islands correspond to genes that have the same base composition as core genes, suggesting that they either might be ancestral genes lost by deletion in certain strains or might originate from horizontal gene transfers.

Chromosome Mapping↗

PubMed2006

Population structure of plasmid-containing strains of Streptococcus mutans, a member of the human indigenous biota.

There are suggestions that the phylogeny of Streptococcus mutans, a member of the human indigenous biota that is transmitted mostly mother to child, might parallel the evolutionary history of its human host. The relatedness and phylogeny of plasmid-containing strains of S. mutans were examined based on chromosomal DNA fingerprints (CDF), a hypervariable region (HVR) of a 5.6-kb plasmid, the rRNA gene intergenic spacer region (IGSR), serotypes, and the genotypes of mutacin I and II. Plasmid-containing strains were studied because their genetic diversity was twice as great as that of plasmid-free strains. The CDF of S. mutans from unrelated human hosts were unique, except those from Caucasians, which were essentially identical. The evolutionary history of the IGSR, with or without the serotype and mutacin characters, clearly delineated an Asian clade. Also, a continuous association with mutacin II could be reconstructed through an evolutionary lineage with the IGSR, but not for serotype e. DNA sequences from the HVR of the plasmid produced a well-resolved phylogeny that differed from the chromosomal phylogeny, indicating that the horizontal transfer of the plasmid may have occurred multiple times. The plasmid phylogeny was more congruent with serotype e than with mutacin II evolution, suggesting a possible functional correlation. Thus, the history of this three-tiered relationship between human, bacterium, and plasmid supported both coevolution and independent evolution.

Biological Evolution↗

PubMed1997

Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts.

The transconjugant CB100, harboring the Ti plasmid from the Agrobacterium tumefaciens biovar 2 strain D10B/87 in the chromosomal background of the biovar 1 strain C58, was defective in vir gene induction. This defect was corrected in the presence of virA from pTiA6. Based on this complementation result and an analysis of the induction requirements of the transconjugant CB100 and its parent strains, it was hypothesized that the defective vir gene induction in CB100 was related to a dysfunctional interaction between the pTi-encoded D10B/87 VirA and the chromosome-encoded C58 ChvE. To verify this hypothesis, D10B/87 and C58 virA were compared, and conclusions from this first set of analyses were then corroborated by comparing D10B/87 and C58 chvE. Whereas only a few nucleotide differences were identified in the promoters and 5' ends of the coding regions of D10B/87 and C58 virA, analysis of hybrid virA genes showed that these differences collectively accounted for the poor vir gene induction of strain CB100. In contrast with the sequence similarity of the VirA proteins, extensive divergence was seen between the chromosome-encoded D10B/87 and C58 ChvE. Although D10B/87 chvE introduced in trans had little effect on vir gene induction of CB100, it enhanced the induction response of a strain CB100 derivative in which the chromosomal C58 chvE had been inactivated by marker exchange. These results suggest that chromosomal backgrounds provided by different strains of A. tumefaciens are not equivalent for VirA function. Following conjugative transfer of certain Ti plasmids to a new agrobacterial host, evolution of the newly introduced virA, or coevolution of chvE and virA, may lead to optimization of ChvE-VirA interaction and vir gene induction levels.

Agrobacterium tumefaciens↗

PubMed1998

Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA.

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like beta-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.

Amino Acid Sequence↗

PubMed2004

Evolutionary dynamics of insertion sequences in Helicobacter pylori.

Prokaryotic insertion sequence (IS) elements behave like parasites in terms of their ability to invade and proliferate in microbial gene pools and like symbionts when they coevolve with their bacterial hosts. Here we investigated the evolutionary history of IS605 and IS607 of Helicobacter pylori, a genetically diverse gastric pathogen. These elements contain unrelated transposase genes (orfA) and also a homolog of the Salmonella virulence gene gipA (orfB). A total of 488 East Asian, Indian, Peruvian, and Spanish isolates were screened, and 18 and 14% of them harbored IS605 and IS607, respectively. IS605 nucleotide sequence analysis (n = 42) revealed geographic subdivisions similar to those of H. pylori; the geographic subdivision was blurred, however, due in part to homologous recombination, as indicated by split decomposition and homoplasy tests (homoplasy ratio, 0.56). In contrast, the IS607 populations (n = 44) showed strong geographic subdivisions with less homologous recombination (homoplasy ratio, 0.2). Diversifying selection (ratio of nonsynonymous change to synonymous change, >>1) was evident in approximately 15% of the IS605 orfA codons analyzed but not in the IS607 orfA codons. Diversifying selection was also evident in approximately 2% of the IS605 orfB and approximately 10% of the IS607 orfB codons analyzed. We suggest that the evolution of these elements reflects selection for optimal transposition activity in the case of IS605 orfA and for interactions between the OrfB proteins and other cellular constituents that potentially contribute to bacterial fitness. Taken together, similarities in IS elements and H. pylori population genetic structures and evidence of adaptive evolution in IS elements suggest that there is coevolution between these elements and their bacterial hosts.

DNA Transposable Elements↗

PubMed2005

Only one of the five CheY homologs in Vibrio cholerae directly switches flagellar rotation.

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.

Amino Acid Sequence↗

PubMed1998

Phylogenetic placement of rickettsiae from the ticks Amblyomma americanum and Ixodes scapularis.

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsii Hip2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.

Amino Acid Sequence↗

PubMed2006

Comparing phylogenetic codivergence between polyomaviruses and their hosts.

Seventy-two full genomes corresponding to nine mammalian (67 strains) and two avian (5 strains) polyomavirus species were analyzed using maximum likelihood and Bayesian methods of phylogenetic inference. Our fully resolved and well-supported (bootstrap proportions > 90%; posterior probabilities = 1.0) trees separate the bird polyomaviruses (avian polyomavirus and goose hemorrhagic polyomavirus) from the mammalian polyomaviruses, which supports the idea of spitting the genus into two subgenera. Such a split is also consistent with the different viral life strategies of each group. Simian (simian virus 40, simian agent 12 [Sa12], and lymphotropic polyomavirus) and rodent (hamster polyomavirus, mouse polyomavirus, and murine pneumotropic polyomavirus [MPtV]) polyomaviruses did not form monophyletic groups. Using our best hypothesis of polyomavirus evolutionary relationships and established host phylogenies, we performed a cophylogenetic reconciliation analysis of codivergence. Our analyses generated six optimal cophylogenetic scenarios of coevolution, including 12 codivergence events (P < 0.01), suggesting that Polyomaviridae coevolved with their avian and mammal hosts. As individual lineages, our analyses showed evidence of host switching in four terminal branches leading to MPtV, bovine polyomavirus, Sa12, and BK virus, suggesting a combination of vertical and horizontal transfer in the evolutionary history of the polyomaviruses.

Animals↗

PubMed2026

The organization and dynamics of viral factories.

Viral factories (VFs) are dynamic, virus-induced microcompartments that serve as centralized hubs in the host cell for viral genome replication, transcription, and virion assembly. These structures employ unique viral mechanisms for remodeling cellular architecture to create specialized replication organelles and improve the efficiency of viral propagation. VFs exhibit striking structural and functional diversity among RNA and DNA viruses, from reoviruses and poxviruses to the Nucleocytoviricota phylum. Some are enclosed by host-derived membranes, while others exist as biomolecular condensates from liquid-liquid phase separation. VFs recruit host lipids, cytoskeletal elements, and metabolic enzymes, effectively reprogramming the intracellular environment to favor viral replication. This review provides a comprehensive examination of the molecular composition, ultrastructure, and biogenesis of viral factories across a wide range of viral lineages and host systems. We describe membrane-bound and phase-separated VFs and the mechanisms by which they hijack host machinery to create these replication organelles and explore viral strategies to shield replication intermediates from host immune responses. Additional emphasis is placed on the complex VFs formed by giant viruses in the Nucleocytoviricota, whose ability to spatially compartmentalize replication and transcription, exclude ribosomes, and recruit host mitochondria and membranes blurs the line between viral and cellular organization. By integrating findings from cell biology and evolutionary virology, this review proposes that viral factories offer a conceptual framework for understanding virus-host coevolution and provides new insights into how their organization may have shaped the emergence of eukaryotic complexity.

Nucleocytoviricota↗

PubMed1984

Polyomavirus growth and persistence in Friend erythroleukemic cells.

Infection of Friend erythroleukemic (FL) cells by polyomavirus (Py) invariably results in the selection of persistently infected FL-Py cell lines and clones. Anti-Py serum treatment of FL-Py lines and clones leads to the loss of Py genome and consequent cell cure. Conversely, cure has not been obtained in FL-PytsA cell lines (isolated after infection by a Py thermosensitive early mutant) and their derivative clones cultivated for a long time at nonpermissive temperature (39 degrees C), where viral large-T protein is inactive. Rescue of viral particles has always been obtained after shifting cells to 32 degrees C. Integrated viral genomes were detected by blot hybridization in an FL-PytsA clone at 39 degrees C. Long-term observation of FL-Py cell lines and their derivative clones reveals a reciprocal selection mechanism (coevolution) between the viral and the cellular populations, resulting in either a completely virus-free Py-resistant FL cell line (cure) or in a continuously Py-shedding line bearing Py genome variants. Structural analysis of these viral populations has been carried out, and some viral variants have been isolated and characterized. On the basis of the results obtained, the possible mechanisms of Py persistence in FL cells will be discussed.

Animals↗

PubMed1989

Extensive cell heterogeneity during persistent infection with foot-and-mouth disease virus.

Coevolution of viruses and the host cells occurred in BHK-21 cell cultures persistently infected with foot-and-mouth disease virus (FMDV) (J. C. de la Torre, E. Martínez-Salas, J. Diez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). In the present report we provide evidence of an extreme phenotypic heterogeneity of the cells, which was generated in the course of persistence. A total of 248 stable cell clones isolated from FMDV carrier cultures at early or late passages were analyzed. At least six distinct cell phenotypes were distinguished with regard to cell morphology, resistance to FMDV strain C-S8c1, and cell growth characteristics. No infectious FMDV or viral RNA was detected in variant cell clones, suggesting that the altered phenotypes were caused by inheritable cell modifications, selected in the course of persistence. Thus, the FMDV-BHK-21 carrier cell system must be described as a dynamic interaction between an evolving heterogeneous population of virus and multiple cell variants. We suggest that cell heterogeneity confers a selective advantage for long-term virus and cell survival by providing the cell population with a range of responses toward FMDV.

Animals↗

PubMed1990

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture.

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.

Amino Acid Sequence↗

PubMed1992

Phylogenetic analysis of 48 papillomavirus types and 28 subtypes and variants: a showcase for the molecular evolution of DNA viruses.

Papillomaviruses are attractive models for studying the molecular evolution of DNA viruses because of the large number of isolates that exhibit genomic diversity and host species and tissue specificity. To examine their relationship, we selected two amino acid sequences, one of 52 residues within the early gene E1 and the other of 44 residues within the late gene L1, which allowed insertion- and deletion-free alignment of all accessible papillomavirus sequences. We constructed phylogenetic trees from the amino acid and corresponding nucleotide sequences from 28 published and 20 newly determined animal and human papillomavirus (HPV) genomic sequences by using distance matrix, maximum-likelihood, and parsimony methods. The trees agreed in all important topological aspects. One major branch with two clearly separated clusters contained 11 HPV types associated with epidermodysplasia verruciformis. A second major branch had all the papillomaviruses involved in genital neoplasia and, in distant relationship, the cutaneous papillomaviruses HPV type 2a (HPV-2a), HPV-3, and HPV-10 as well as the "butcher's" papillomavirus HPV-7 and two simian papillomaviruses. Four artiodactyl (even-toed hoofed mammal) papillomaviruses, the cottontail rabbit papillomavirus, and avian (chaffinch) papillomavirus type 1 formed a third major branch. Last, four papillomaviruses exhibited little affinity to any of these three branches; these were the cutaneous types HPV-1a, HPV-4, and HPV-41 and B-group bovine papillomavirus type 4. The phylogeny suggests that some branches of papillomavirus evolution are restricted to particular target tissues and that a general process of long-term papillomavirus-host coevolution has occurred. This latter hypothesis is still conjectural because of bias in the current data base for human types and the paucity of animal papillomavirus sequences. The comparison of evolutionary distances for the most closely related types with those of 28 subtypes and variants of HPV-2, HPV-5, HPV-6, HPV-16, and HPV-18 supports the type as a natural taxonomic unit, with subtypes and variants being expressions of minor intratype genomic diversity similar to that found in the natural populations of all biological species. An exception to this seems to be HPV-2c, which has an evolutionary distance from HPV-2a of the intertype magnitude and may eventually have to be regarded as a distinct type. We describe an experimental approach that estimates the taxonomic and phylogenetic positions of newly identified papillomaviruses without viral isolation and complete genomic sequencing.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

The genetic drift of human papillomavirus type 16 is a means of reconstructing prehistoric viral spread and the movement of ancient human populations.

We have investigated the diversity of a hypervariable segment of the human papillomavirus type 16 (HPV-16) genome among 301 virus isolates that were collected from 25 different ethnic groups and geographic locations. Altogether, we distinguished 48 different variants that had diversified from one another along five phylogenetic branches. Variants from two of these branches were nearly completely confined to Africa. Variants from a third branch were the only variants identified in Europeans but occurred at lower frequency in all other ethnic groups. A fourth branch was specific for Japanese and Chinese isolates. A small fraction of all isolates from Asia and from indigenous as well as immigrant populations in the Americas formed a fifth branch. Important patterns of HPV-16 phylogeny suggested coevolution of the virus with people of the three major human races, namely, Africans, Caucasians, and East Asians. But several minor patterns are indicative of smaller bottlenecks of viral evolution and spread, which may correlate with the migration of ethnic groups in prehistoric times. The colonization of the Americas by Europeans and Africans is reflected in the composition of their HPV-16 variants. We discuss arguments that today's HPV-16 genomes represent a degree of diversity that evolved over a large time span, probably exceeding 200,000 years, from a precursor genome that may have originated in Africa. The identification of molecular variants is a powerful epidemiological and phylogenetic tool for revealing the ancient spread of papillomaviruses, whose trace through the world has not yet been completely lost.

Africa↗

PubMed1993

Simian immunodeficiency viruses from central and western Africa: evidence for a new species-specific lentivirus in tantalus monkeys.

Although up to 50% of African green monkeys (AGMs) are infected by simian immunodeficiency viruses (SIV) in their natural habitat, they remain asymptomatic carriers of these lentiviruses. They provide an attractive model to study not only the origin but also the link among genetic variation, host-virus adaptation, and pathogenicity of primate lentiviruses. SIVagm have been isolated from three species of AGM: the vervet (Cercopithecus pygerythrus), the grivet (Cercopithecus aethiops), and the sabaeus (Cercopithecus sabaeus) monkey. We studied four new SIVagm isolates from a fourth AGM species, the tantalus monkey (Cercopithecus tantalus), caught in the Central African Republic, and four new isolates from feral sabaeus monkeys from Senegal. Antigenic properties and partial env sequences were used to evaluate the diversity among these isolates. Alignment of env sequences in SIVagm isolated from tantalus and sabaeus monkeys permitted detailed mapping of the variable and conserved domains in the external glycoprotein. Genetic distances indicated that SIVagm isolates from tantalus monkeys are the most divergent among SIVagm in feral AGMs in Africa. The fact that AGMs are infected by four distinct lentiviruses, each specific for a single AGM species, supports the hypothesis of a coevolution of these viruses and their natural hosts and suggests that SIV transmission is a rare event among separated AGM species in the wild.

Amino Acid Sequence↗

PubMed1996

Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter.

The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.

Amino Acid Sequence↗

PubMed1997

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.

Ammonium Chloride↗