RESUMO
UNLABELLED: Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies. IMPORTANCE: Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.
Assuntos
Proteínas de Fluorescência Verde/análise , Vírus Sincicial Respiratório Humano/fisiologia , Replicação Viral , Animais , Células Cultivadas , Modelos Animais de Doenças , Feminino , Genótipo , Proteínas de Fluorescência Verde/genética , Humanos , Lactente , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Sistema Respiratório/virologia , Sigmodontinae , Coloração e Rotulagem , VirulênciaRESUMO
The stability and conservation of the sequences of RNA viruses in the field and the high error rates measured in vitro are paradoxical. The field stability indicates that there are very strong selective constraints on sequence diversity. The nature of these constraints is discussed. Apart from constraints on variation in cis-acting RNA and the amino acid sequences of viral proteins, there are other ones relating to the presence of specific dinucleotides such CpG and UpA as well as the importance of RNA secondary structures and RNA degradation rates. Recent other constraints identified in other RNA viruses, such as effects of secondary RNA structure on protein folding or modification of cellular tRNA complements, are also discussed. Using the family Paramyxoviridae, I show that the codon usage pattern (CUP) is (i) specific for each virus species and (ii) that it is markedly different from the host - it does not vary even in vaccine viruses that have been derived by passage in a number of inappropriate host cells. The CUP might thus be an additional constraint on variation, and I propose the concept of codon constellation to indicate the informational content of the sequences of RNA molecules relating not only to stability and structure but also to the efficiency of translation of a viral mRNA resulting from the CUP and the numbers and position of rare codons.
Assuntos
Códon , Sequência Conservada , Mutação , Paramyxoviridae/genética , Sequência de Bases , Regulação Viral da Expressão Gênica , Conformação de Ácido Nucleico , Biossíntese de ProteínasRESUMO
UNLABELLED: The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity. IMPORTANCE: Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.
Assuntos
Variação Genética , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Parainfluenza 5/genética , Vírus da Parainfluenza 5/isolamento & purificação , Infecções por Rubulavirus/veterinária , Infecções por Rubulavirus/virologia , Animais , Humanos , Dados de Sequência Molecular , Vírus da Parainfluenza 5/fisiologia , Inoculações Seriadas , Cultura de VírusRESUMO
There is a paradox between the remarkable genetic stability of measles virus (MV) in the field and the high mutation rates implied by the frequency of the appearance of monoclonal antibody escape mutants generated when the virus is pressured to revert in vitro (S. J. Schrag, P. A. Rota, and W. J. Bellini, J. Virol. 73:51-54, 1999). We established a highly sensitive assay to determine frequencies of various categories of mutations in large populations of wild-type and laboratory-adapted MVs using recombinant viruses containing an additional transcription unit (ATU) encoding enhanced green fluorescent protein (EGFP). Single and double mutations were made in the fluorophore of EGFP to ablate fluorescence. The frequencies of reversion mutants in the population were determined by measuring the appearance of fluorescence indicating a revertant virus. This allows mutation rates to be measured under nonselective conditions, as phenotypic reversion to fluorescence requires only either a single- or a double-nucleotide change and amino acid substitution, which does not affect the length of the nonessential reporter protein expressed from the ATU. Mutation rates in MV are the same for wild-type and laboratory-adapted viruses, and they are an order of magnitude lower than the previous measurement assessed under selective conditions. The actual mutation rate for MV is approximately 1.8 × 10(-6) per base per replication event.
Assuntos
Variação Genética , Vírus do Sarampo/genética , Taxa de Mutação , Substituição de Aminoácidos , Animais , Antígenos CD/metabolismo , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Mutação , Receptores de Superfície Celular/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Replicação ViralRESUMO
Recently, numerous large-scale mumps outbreaks have occurred in vaccinated populations. Clinical isolates sequenced from these outbreaks have invariably been of genotypes distinct from those of vaccine viruses, raising concern that certain mumps virus strains may escape vaccine-induced immunity. To investigate this concern, sera obtained from children 6 weeks after receipt of measles, mumps, and rubella (MMR) vaccine were tested for the ability to neutralize a carefully selected group of genetically diverse mumps virus strains. Although the geometric mean neutralizing antibody titer of the sera was lower against some virus strains than others, all viruses were readily neutralized, arguing against immune escape.
Assuntos
Surtos de Doenças , Vacina contra Sarampo-Caxumba-Rubéola/imunologia , Vírus da Caxumba/imunologia , Caxumba/epidemiologia , Caxumba/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Vacina contra Sarampo-Caxumba-Rubéola/administração & dosagem , Dados de Sequência Molecular , Caxumba/virologia , Vírus da Caxumba/classificação , Vírus da Caxumba/genética , Filogenia , Estados Unidos/epidemiologia , VacinaçãoRESUMO
The Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) has recently decided to modify the current definition of virus species (Code of Virus Classification and Nomenclature Rule 3.21) and will soon ask the full ICTV membership (189 voting members) to ratify the proposed controversial change. In this discussion paper, 14 senior virologists, including six Life members of the ICTV, compare the present and proposed new definition and recommend that the existing definition of virus species should be retained. Since the pros and cons of the proposal posted on the ICTV website are not widely consulted, the arguments are summarized here in order to reach a wider audience.
Assuntos
Classificação/métodos , Virologia/métodos , Vírus/classificação , Terminologia como AssuntoRESUMO
The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99â%) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70â%, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (CâU) or guanine to adenine (GâA) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, AâG and UâC transitions, with preference for next neighbour-nucleotides Uâ=âA>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.
Assuntos
Citidina Desaminase/metabolismo , Vírus do Sarampo/patogenicidade , Vírus da Caxumba/patogenicidade , Vírus Sinciciais Respiratórios/patogenicidade , Replicação Viral , Desaminase APOBEC-3G , Animais , Antivirais/metabolismo , Linhagem Celular , Citidina Desaminase/imunologia , Humanos , Vírus do Sarampo/crescimento & desenvolvimento , Vírus do Sarampo/imunologia , Vírus da Caxumba/crescimento & desenvolvimento , Vírus da Caxumba/imunologia , Mutação Puntual , RNA Viral/genética , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Vírus Sinciciais Respiratórios/imunologiaRESUMO
The global increase in measles vaccination has resulted in a significant reduction of measles mortality. The standard route of administration for the live-attenuated measles virus (MV) vaccine is subcutaneous injection, although alternative needle-free routes, including aerosol delivery, are under investigation. In vitro, attenuated MV has a much wider tropism than clinical isolates, as it can use both CD46 and CD150 as cellular receptors. To compare the in vivo tropism of attenuated and pathogenic MV, we infected cynomolgus macaques with pathogenic or attenuated recombinant MV expressing enhanced green fluorescent protein (GFP) (strains IC323 and Edmonston, respectively) via the intratracheal or aerosol route. Surprisingly, viral loads and cellular tropism in the lungs were similar for the two viruses regardless of the route of administration, and CD11c-positive cells were identified as the major target population. However, only the pathogenic MV caused significant viremia, which resulted in massive virus replication in B and T lymphocytes in lymphoid tissues and viral dissemination to the skin and the submucosa of respiratory epithelia. Attenuated MV was rarely detected in lymphoid tissues, and when it was, only in isolated infected cells. Following aerosol inhalation, attenuated MV was detected at early time points in the upper respiratory tract, suggesting local virus replication. This contrasts with pathogenic MV, which invaded the upper respiratory tract only after the onset of viremia. This study shows that despite in vitro differences, attenuated and pathogenic MV show highly similar in vivo tropism in the lungs. However, systemic spread of attenuated MV is restricted.
Assuntos
Vírus do Sarampo/fisiologia , Tropismo Viral , Animais , Linfócitos B/virologia , Antígeno CD11c/análise , Células Dendríticas/química , Células Dendríticas/virologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Pulmão/virologia , Macaca fascicularis , Vírus do Sarampo/genética , Sistema Respiratório/virologia , Coloração e Rotulagem/métodos , Linfócitos T/virologia , Carga Viral , ViremiaRESUMO
The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.
Assuntos
Hospedeiro Imunocomprometido , Pneumopatias/virologia , Pulmão/virologia , Vírus da Parainfluenza 3 Humana/metabolismo , Infecções por Paramyxoviridae/virologia , Adulto , Sítios de Ligação , Análise Mutacional de DNA , Feminino , Frequência do Gene , Doença Enxerto-Hospedeiro/tratamento farmacológico , Células HEK293 , Humanos , Leucemia Mieloide Aguda , Mutação , Ácido Micofenólico/administração & dosagem , Ácido N-Acetilneuramínico/química , Vírus da Parainfluenza 3 Humana/genética , Infecções por Paramyxoviridae/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/virologia , Receptores Virais/metabolismo , Sirolimo/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Adulto JovemRESUMO
The Jeryl Lynn (JL) vaccine against mumps virus (MuV) contains two components, MuV(JL5) and MuV(JL2), which differ by over 400 nt. Due to the occurrence of bias in the direction of mutation, these differences and those found in nucleotide sequences of different isolates of the minor component in the vaccine (MuV(JL2)) might be due to the effect of ADAR-like deaminases on MuV grown in tissue-cultured cells. A molecular clone of MuV(JL2) (pMuV(JL2)) and MuV(JL2)-specific helper plasmids were constructed in order to investigate molecular interactions between MuV(JL5) and MuV(JL2), to augment the existing molecular clone of MuV(JL5) (pMuV(JL5)) and MuV(JL5)-specific helper plasmids. Genome and mRNA termini of MuV(JL2) were characterized, and an unusual oligo-G insertion transcriptional editing event was detected near the F mRNA polyadenylation site of MuV(JL2), but not of MuV(JL5). Genes encoding glycoproteins of rMuV(JL2) and rMuV(JL5) have been exchanged to characterize the oligo-G insertion, which associated with the specific sequence of the F gene of MuV(JL2) and not with any other genes or the RNA-dependent RNA polymerase of strain MuV(JL2). The results indicate that a single G-to-A sequence change obliterates the co-transcriptional editing of the F mRNA and that this oligo-G insertion does not affect the growth of the virus.
Assuntos
Vacina contra Caxumba/genética , Vírus da Caxumba/classificação , Vírus da Caxumba/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , DNA Viral/análise , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da Espécie , Células VeroRESUMO
UNLABELLED: Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness-mediated by the receptor binding protein and the fusion protein-can be drastically different from the requirements for infection in vivo. Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo. Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection. IMPORTANCE: Mechanistic information about viral infection-information that impacts antiviral and vaccine development-is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3-a globally important respiratory paramyxovirus-in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.
Assuntos
Sistema Respiratório/virologia , Respirovirus/fisiologia , Internalização do Vírus , Animais , Genoma Viral , Humanos , Respirovirus/isolamento & purificação , Respirovirus/patogenicidade , Respirovirus/ultraestrutura , Infecções por Respirovirus/virologia , Análise de Sequência de DNA , Sigmodontinae , VirulênciaRESUMO
This review focuses on new concepts important for the understanding of the pathogenesis of measles virus. First the requirement for specific entry receptors restricts the cell types that measles can enter during the initial stages of infection in the human host. Recently, the paradigm for measles has shifted from an epithelial infection similar to that caused in the respiratory tract by other members of the paramyxoviruses to one which displays more similarity to the infection of the immune system by HIV-1, though the route of infection is different. Secondly we review the role of host proteins that support viral replication as well as those that modify the cellular environment in order to promote measles virus replication. The role of specific virus proteins in the anti-antiviral response is also reviewed. Measles virus counteracts all pathways known to induce interferon synthesis as well as signalling by interferons, exemplifying the importance of these in the virulence/attenuation of the virus. We conclude that only studies in relevant animal model systems or humans or in vitro or ex vivo studies of relevant cell types and tissues will bring us closer to an understanding of the pathogenesis of the virus, factors that have often been overlooked in past studies.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Evasão da Resposta Imune , Imunidade Inata , Interferons/imunologia , Vírus do Sarampo/imunologia , Sarampo , Proteínas Virais , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Humanos , Interferons/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sarampo/imunologia , Sarampo/virologia , Vírus do Sarampo/metabolismo , Vírus do Sarampo/patogenicidade , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas de Ligação a RNA , Receptores Virais/genética , Receptores Virais/imunologia , Receptores Virais/metabolismo , Transdução de Sinais/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Replicação Viral/genética , Replicação Viral/imunologiaAssuntos
Proteínas Luminescentes , Vírus do Sarampo/fisiologia , Sarampo/transmissão , Animais , Astrocitoma , Chlorocebus aethiops , Proteínas de Fluorescência Verde , Sarampo/virologia , Vírus do Sarampo/isolamento & purificação , Microscopia Confocal/métodos , Células Tumorais Cultivadas , Células Vero , Replicação ViralRESUMO
Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.
Assuntos
Genoma Viral , Vírus do Sarampo/genética , Transcrição Gênica , Proteínas Virais/genética , Animais , Chlorocebus aethiops , DNA Viral/genética , Fluorescência , Regulação Viral da Expressão Gênica , Genes Reporter , Variação Genética , Haplorrinos , Humanos , Fases de Leitura Aberta , Plasmídeos , RNA Mensageiro/genética , RNA Viral/genética , Mapeamento por Restrição , Transfecção , Células VeroRESUMO
A recombinant measles virus (MV) expressing red fluorescent protein (MVDsRed1) was used to produce a persistently infected cell line (piNT2-MVDsRed1) from human neural precursor (NT2) cells. A similar cell line (piNT2-MVeGFP) was generated using a virus that expresses enhanced green fluorescent protein. Intracytoplasmic inclusions containing the viral nucleocapsid protein were evident in all cells and viral glycoproteins were present at the cell surface. Nevertheless, the cells did not release infectious virus nor did they fuse to generate syncytia. Uninfected NT2 cells express the MV receptor CD46 uniformly over their surface, whereas CD46 was present in cell surface aggregates in the piNT2 cells. There was no decrease in the overall amount of CD46 in piNT2 compared to NT2 cells. Cell-to-cell fusion was observed when piNT2 cells were overlaid onto confluent monolayers of MV receptor-positive cells, indicating that the viral glycoproteins were correctly folded and processed. Infectious virus was released from the underlying cells, indicating that persistence was not due to gross mutations in the virus genome. Persistently infected cells were superinfected with MV or canine distemper virus and cytopathic effects were not observed. However, mumps virus could readily infect the cells, indicating that superinfection immunity is not caused by general soluble antiviral factors. As MVeGFP and MVDsRed1 are antigenically indistinguishable but phenotypically distinct it was possible to use them to measure the degree of superinfection immunity in the absence of any cytopathic effect. Only small numbers of non-fusing green fluorescent piNT2-MVDsRed1 cells (1 : 300 000) were identified in which superinfecting MVeGFP entered, replicated and expressed its genes.
Assuntos
Antígenos CD/metabolismo , Vírus do Sarampo/fisiologia , Sarampo/virologia , Glicoproteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Fusão Celular , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/virologia , Humanos , Sarampo/metabolismo , Vírus do Sarampo/genética , Vírus do Sarampo/imunologia , Proteína Cofatora de Membrana , Agregação de Receptores , Recombinação Genética , Replicação ViralRESUMO
Measles virus (MV) is the type species of the Morbillivirus genus and its RNA-dependent RNA polymerase complex is comprised of two viral polypeptides, the large (L) and the phospho- (P) proteins. Sequence alignments of morbillivirus L polymerases have demonstrated the existence of three well-conserved domains (D1, D2, and D3) which are linked by two variable hinges (H1 and H2). Epitope tags (c-Myc) were introduced into H1 and H2 to investigate the tolerance of the variable regions to insertions and to probe the flexibility of the proposed domain structures to spatial reorientation. Insertion into H1 abolished polymerase activity whereas introduction into H2 had no effect. The open reading frame of enhanced green fluorescent protein was also inserted into the H2 region of the MV L gene to extend these observations. This resulted in a recombinant protein that was both functional and autofluorescent, although the overall polymerase activity was reduced by over 40%. Two recombinant viruses which contained the chimeric L genes EdtagL(MMc-mycM) and EdtagL(MMEGFPM) were generated. Tagged L proteins were detectable, by indirect immunofluorescence in the case of EdtagL(MMc-mycM) and by autofluorescence in the case of EdtagL(MMEGFPM). We suggest that D3 enjoys a limited conformational independence from the other domains, indicating that the L polymerases of the Mononegavirales may function as multidomain proteins.
Assuntos
Proteínas Luminescentes/genética , Vírus do Sarampo/enzimologia , Fases de Leitura Aberta/genética , RNA Polimerase Dependente de RNA/metabolismo , Animais , Chlorocebus aethiops , Imunofluorescência , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Plasmídeos , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Transfecção , Células VeroRESUMO
In order to investigate which measles virus (MV)-strains use CD46 and/or CD150 (signalling lymphocytic activation molecule, SLAM) as receptors, CHO cells expressing either recombinant CD46 or SLAM were infected with a panel of 28 MV-strains including vaccine strains, wild-type strains with various passage histories and recombinant viruses. We found that SLAM served as a common receptor conferring virus uptake and syncytium formation for all MV-strains tested. Predominantly vaccine and laboratory adapted strains, but also a minor fraction of the wild-type strains tested, could utilize both CD46 and SLAM. Using recombinant viruses, we demonstrate that the single amino acid exchange in the haemagglutinin (H) protein at position 481 Asn/Tyr (H481NY) determines whether the virus can utilize CD46. This amino acid alteration has no affect on the usage of SLAM as receptor, and as such demonstrates that the binding sites for SLAM and CD46 are distinct.