Células persistentemente infectadas por el virus del sarampión son más sensibles a la inducción de apoptósis por TRAIL: Base de una posible terapía para PES y otras encefalitis sarampionosas / Measles virus persistently infected cells are more sensitive to apoptosis induction by TRAIL: Basis for a potential therapy in SSPE and other measles virus encephalitis

Introducción y objetivos: El virus del sarampión (VS)aparte de producir complicaciones graves respiratorias,gastrointestinales, encefalitis, ceguera y sordera queacompañan al sarampión y hacen que mate anualmentecerca de un millón de niños en el mundo, es el causantede encefalitis lentas como la Panencefalitis esclerosantesubaguda (PES) o la Encefalitis sarampionosa de cuerposde inclusión (MIBE). Para estas encefalopatías subagudasprogresivas con desmielinización y atrofia cerebral, de curso mortal tras meses a décadas de evolución no se dispone actualmente de tratamiento efectivo. El objetivo de este estudio es la búsqueda de posibles tratamientos para eliminar selectivamente las células infectadas por el virus y así frenar el progreso de la infección y en último término la eliminación del virus del paciente.Métodos: Estudio de sensibilidad a la inducción de apoptosis por la proteína TRAIL (TNF-related-apoptosis inducing ligand) de células humanas en cultivo sin infectar y persistentemente infectadas por VS. Estudio de los mecanismos de inducción de la apoptosis en la línea celular MOLT3, en la que nuestro laboratorio mostró por primera vez que VS puede inducir apoptosis.Resultados: La infección persistente por VS hace que células humanas se hagan marcadamente más sensibles al suicidio celular inducido por TRAIL exógeno. El mecanismo de esta sensibilización podría explicarse por un aumento de la expresión en la superficie celular de las células infectadas de los receptores funcionales para TRAIL, junto con una reducción de la expresión de la proteína antiapoptótica Bcl2 y una estimulación de la quinasa Akt y del factor de transcripción NFkB, lo que provocaría el suicidio de las células infectadas.Conclusiones: Células persistentemente infectadas porVS son más sensibles a la inducción de suicidio por apoptosis mediado por TRAIL que la células no infectadas. El tratamiento con TRAIL al eliminar las células infectadas podría frenar la progresión del virus o incluso eliminarlo en pacientes con encefalitis subagudas causadas por VS como SSPE o MIBE

Introduction: Measles virus (MV) causes acute infectionswith severe respiratory, neurological, deafness, blindness and gastrointestinal complications that today still kill near one million children annually worldwide. The late measles complications Subacute Sclerosing Panencephalitis (SSPE) or Measles Inclusion Bodies Encephalitis (MIBE) show a protracted progressive and fatal course and for them no efficient treatment is currently available. This work aims to find a way to selectively destroy the infected cells, and doing so limit the progression of VS through the infectedbrain and eventually to eliminate the virus from the patient with SSPE or MIBE.Materials and Methods: Sensitivity to apoptosis induced by exogenous recombinant TRAIL was determined in steady-state MV persistently infected human cell line MOLT3 versus uninfected cells, by chromatin condensation, TUNEL, and caspases activation assays. Expression of TRAIL functional receptors (TRAIL-R1 y TRAIL-R2) and decoy receptors (TRAIL-R3 and TRAIL-R4) was determined by flow cytometry and western- blots. mRNAs of pro- and antiapopoptotic factors were quantitated by RT-PCR and RNAse protection assays. Activation of NFkB by EMSA and supershift electrophoretic assays.Results: MV persistently infected MOLT3 cells becamehighly sensitive to apoptosis induced by exogenous TRAIL.This sensitization could be explained by the observed MV infection up-regulation in the infected cells of the expression of TRAIL functional receptors TRAIL-R1 and TRAIL-R2 and down-regulation of the expression of antiapoptotic factor Bcl2 and activation of the protein-kinase Akt and NF-kB that would provoke the suicide of the infected cells.Conclusions: The high sensitization to induced apoptosisby exogenous TRAIL observed in MV persistently infectedcells suggest that treatment with recombinant TRAIL couldselectively eliminate MV infected cells in the CNS of patients and it could be possible to exploit this agent for the treatment of SSPE or MIBE encephalopaties (AU)

Measles virus fusion protein is palmitoylated on transmembrane-intracytoplasmic cysteine residues which participate in cell fusion.

[3H]palmitic acid was metabolically incorporated into the viral fusion protein (F) of Edmonston or freshly isolated measles virus (MV) during infection of human lymphoid or Vero cells. The uncleaved precursor F0 and the F1 subunit from infected cells and extracellular virus were both labeled, indicating that palmitoylation can take place prior to F0 cleavage and that palmitoylated F protein was incorporated into virus particles. [3H]palmitic acid was released from F protein upon hydroxylamine or dithiothreitol treatment, indicating a thioester linkage. In cells transfected with the cloned MV F gene, in which the cysteines located in the intracytoplasmic and transmembrane domains (Cys 506, 518, 519, 520, and 524) were replaced by serine, a major reduction of [3H]palmitic acid incorporation was observed for F mutated at Cys 506 and, to a lesser extent, at Cys 518 and Cys 524. We also observed incorporation of [3H]palmitic acid in the F1 subunit of canine distemper virus F protein. Cell fusion induced by cotransfection of cells with MV F and H (hemagglutinin) genes was significantly reduced after replacement of Cys 506 or Cys 519 with serine in the MV F gene. Transfection with the F gene with a mutation for Cys 518 abolished cell fusion, although less mutant protein was detected on the cell surface. These results suggest that the F protein transmembrane domain cysteines 506 and 518 participate in structures involved in cell fusion, possibly mediated by palmitoylation.

Sequence divergence of measles virus haemagglutinin during natural evolution and adaptation to cell culture.

Phylogenetic analysis of the sequence of the H gene of 75 measles virus (MV) strains (32 published and 43 new sequences) was carried out. The lineage groups described from comparison of the nucleotide sequences encoding the C-terminal regions of the N protein of MV were the same as those derived from the H gene sequences in almost all cases. The databases document a number of distinct genotype switches that have occurred in Madrid (Spain). Well-documented is the complete replacement of lineage group C2, the common European genotype at that time, with that of group D3 around the autumn of 1993. No further isolations of group C2 took place in Madrid after this time. The rate of mutation of the H gene sequences of MV genotype D3 circulating in Madrid from 1993 to 1996 was very low (5 x 10(-4) per annum for a given nucleotide position). This is an order of magnitude lower than the rates of mutation observed in the HN genes of human influenza A viruses. The ratio of expressed over silent mutations indicated that the divergence was not driven by immune selection in this gene. Variations in amino acid 117 of the H protein (F or L) may be related to the ability of some strains to haemagglutinate only in the presence of salt. Adaptation of MV to different primate cell types was associated with very small numbers of mutations in the H gene. The changes could not be predicted when virus previously grown in human B cell lines was adapted to monkey Vero cells. In contrast, rodent brain-adapted viruses displayed a lot of amino acid sequence variation from normal MV strains. There was no convincing evidence for recombination between MV genotypes.

Identification of two amino acids in the hemagglutinin glycoprotein of measles virus (MV) that govern hemadsorption, HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains.

We have used site-directed mutagenesis of the hemagglutinin (H) glycoprotein of measles virus (MV) to investigate the molecular basis for the phenotypic differences observed between MV vaccine strains and recently isolated wild-type MV strains. The former downregulate CD46, the putative cellular receptor of MV, are positive for hemadsorption, and are fusogenic in HeLa cells, whereas the latter are negative for these phenotypic markers. CD46 downregulation in particular, could have profound consequences for the immunopathology of MV infection, as this molecule protects the cell from complement lysis. Mutagenesis of two amino acids, valine and tyrosine at positions 451 and 481, respectively, in the H protein from the vaccine-like Hallé MV strain to their counterparts, glutamate and asparagine, in the H protein from the wild-type Ma93F MV strain (creating the V451E/Y481N double mutation) abrogated CD46 downregulation, HeLa cell fusion, and hemadsorption. The converse double mutagenesis of the Ma93F H protein (E451V/N481Y) transferred the CD46-downregulating, fusogenic, and hemadsorption functions to this protein. The data provide the first mapping study of the functional domains of MV H. The consequences of these results for MV vaccine design and the role of CD46 in MV infection are discussed.

Molecular epidemiology of measles virus: identification of pathways of transmission and implications for measles elimination.

The nucleotide sequences of either the hemagglutinin or nucleoprotein genes from wild type measles viruses isolated in the United States between 1989 and 1992 differed by < 0.5%. This suggests that the majority of viruses associated with resurgence of measles in the United States belonged to a single indigenous genotype. In contrast, wild type viruses isolated from sporadic outbreaks of measles in the United States during 1994 were genetically heterogeneous. These viruses were more closely related to wild type viruses previously circulating in Europe, Africa, or Japan and were epidemiologically linked to importations or no known source. In addition to demonstrating the utility of genetic analysis in understanding the epidemiology of measles, these data suggest that the transmission of the indigenous virus was interrupted after the 1989-1992 epidemic. Measures to further reduce the incidence of measles in the United States should include efforts to control importation and subsequent spread of measles.

Temporal and geographical distribution of measles virus genotypes.

The nucleotide sequence encoding the C terminus of the nucleocapsid protein of measles virus (MV) is the most variable in the genome. The sequence of this region is reported for 21 new MV strains and for virus RNA obtained from cases of subacute panencephalitis (SSPE) tissue. The nucleotide sequence of a total of 65 MV strains has been analysed using the CLUSTAL program to determine the relationships between the strains. An unrooted tree shows that eight different genotypes can be discerned amongst the sequences analysed so far. The data show that the C-terminal coding sequence of the nucleocapsid gene, although highly variable between strains, is stable in a given strain and does not appear to diverge in tissue culture. It therefore provides a good 'signature' sequence for specific genotypes. The sequence of this region can be used to discriminate new imported viruses from old 'endemic' strains of MV in a geographical area. The different genotypes are not geographically restricted although some appear to be the mainly 'endemic' types in large areas of the world. In global terms there appears to be at least four cocirculating genotypes of MV. The low level of divergence in the Edmonston lineage group isolated before 1970 indicates that some isolates are probably laboratory contaminants. This applies to some SSPE isolates such as the Hallé, Mantooth and Horta-Barbosa strains as well as some wild-type isolates from that period.

Measles virus from a long-term persistently infected human T lymphoblastoid cell line, in contrast to the cytocidal parental virus, establishes an immediate persistence in the original cell line.

To investigate the mechanisms of measles virus (MV) establishment and maintenance of persistence in lymphoid cells, we have established a long-term persistent infection with MV, Edmonston strain, in the human T lymphoblastoid cell line MOLT4, which has been in continuous culture for over 8 years. In this culture, designated MOMP1, more than 98% of cells display viral antigens. The MOMP1 culture is immune to superinfection with MV and is not cured by anti-MV antibodies. No evidence of defective interfering particles was obtained. The persistently infected culture releases an infectious virus showing a miniplaque and thermoresistant modified phenotype that, unlike the parental virus Edmonston strain which produces a lytic infection with extensive cell fusion, establishes an immediate persistence in MOLT4 cells with neither significant loss of cell viability nor cell fusion. This suggests that the modification in the virus suffices to maintain the state of persistence without requiring a coevolution of the host cell during the infection, as has been reported in other persistent virus infections.

Measles virus gene expression in lytic and persistent infections of a human lymphoblastoid cell line.

MOMP1 is a measles virus (MV) long-term steady-state persistently infected culture of the human T lymphoblastoid cell line MOLT4. The analysis of MV gene expression revealed that in MOMP1 cells, the major MV proteins, haemagglutinin (H), phosphoprotein (P), nucleoprotein, fusion (F) and matrix (M), are present and the fusion precursor (F0) is cleaved into F2 and F1 peptides. H and F2 proteins are glycosylated in both lytic and persistent MOLT4 infections. All major proteins are underexpressed in the persistently infected cultures in comparison to the lytically infected cells. However a relatively greater reduction was observed for H, M and P proteins. Pulse-chase labelling experiments indicated that this underexpression of H, M and P proteins was not due to selective degradation of these proteins in the persistent infection (p.i.). The relative amounts of the major monocistronic and dicistronic mRNAs for MV proteins, with the exception of P mRNA, was not altered in the p.i. with respect to lytically infected MOLT4 cells, suggesting that the defective expression of H and M proteins was not due to a restriction in the transcription of their mRNAs. In contrast, the mRNA for P protein, the most abundant MV mRNA in these lytically infected T lymphoid cells, is markedly underexpressed in the homologous p.i. Thus the underexpression of P protein in p.i. could be due to a decreased availability of P mRNA. This unbalanced underexpression of MV proteins may impair the cell fusion and c.p.e. of MV and facilitate viral persistence in human lymphoid cells.

[Hepatitis C virus infection in patients treated with hemodialysis]. / Infección por el virus de la hepatitis C en enfermos tratados con hemodiálisis.

The prevalence of hepatitis C infection was evaluated (Ortho HCV Antibody ELISA Test) in 64 patients with chronic renal failure treated in a single hemodialysis unit. None of these patients was a carrier of hepatitis B virus nor of antibodies against human immunodeficiency virus. Antibodies against hepatitis C virus were detected in 11 patients (17%). The prevalence was higher in the 13 previously diagnosed of non A, non B hepatitis (77%) than in the 51 without previous hepatitis (2%) (p less than 0.001). A relationship between the infection rate and the number of previous blood transfusions was also observed: 5% in the patients without previous transfusions, 13% in the 30 patients who had received between 1 and 10 blood units and 40% in the 15 who had received more than 10 blood units (p less than 0.05). These data suggest that the hepatitis C virus may be responsible for most episodes previously diagnosed as non A, non B hepatitis, and that blood transfusions are the major risk factor.

Visceral leishmaniasis as an opportunistic infection in the acquired immunodeficiency syndrome.

We describe the case of a woman aged 34 years infected with the human immunodeficiency virus and whose illness was complicated by visceral leishmaniasis that ultimately led to her death.

The genome of simian virus 40.

The nucleotide sequence of SV40 DNA was determined, and the sequence was correlated with known genes of the virus and with the structure of viral messenger RNA's. There is a limited overlap of the coding regions for structural proteins and a complex pattern of leader sequences at the 5' end of late messenger RNA. The sequence of the early region is consistent with recent proposals that the large early polypeptide of SV40 is encoded in noncontinguous segments of DNA.

Studies of low molecular weight RNA from cells infected with adenovirus 2. III. The sequence of the promoter for VA-RNA I.

VA-RNA I is one of the very few RNA species produced in animal cells whose transcriptional initiation site is known precisely. We have analyzed the nucleotide sequence of the DNA preceding the 5' end of VA-RNA I and compared it with known prokaryotic promoters and presumptive eukaryotic promoters.

Studies of low molecular weight RNA from cells infected with adenovirus 2. I. The sequences at the 3' end of VA-RNA I.

VA-RNA I is a low molecular weight RNA produced in large amounts in cells infected with adenoviruses. The 3' terminus of this RNA may represent a transcription termination site. We have demonstrated that this RNA occurs in infected cells in several forms which differ in the number of uridylic acid residues at the 3' ends. The nucleotide sequence of a DNA fragment overlapping the 3' end of VA-RNA I has been determined. The DNA could encode up to 4 uridylic acid residues at the 3' end of the RNA. The DNA sequence shows some similarity to known transcription termination sequences in prokaryotic systems.

Studies of low molecular weight RNA from cells infected with adenovirus 2. II. Heterogeneity at the 5' end of VA-RNA I.

The 5'-terminal sequences of VA-RNA I produced in cells infected with adenovirus 2 were analyzed. The RNA contained a mixture of mono-, di-, and triphosphate ends. The largest part of the RNA has 5'-terminal mono-, di-, or triphosphoguanylic acid, but a portion of the RNA has a 5'-terminal mono-, di-, or triphosphoadenylic acid. An RNA with 5'-terminal adenylic acid was purified and gave the same oligonucleotide map as the major form of VA-RNA I, except for the 5'-terminal products. We therefore conclude that transcription of VA-RNA I can initiate at two different nearby sites.

Comparison of the nucleotide sequence of the messenger RNA for the major structural protein of SV40 with the DNA sequence encoding the amino acids of the protein.

The 16S late mRNA from SV40 directs the synthesis of the major viral structural protein, VP1. We have compared the oligonucleotides in the 16S mRNA with those that would be present in a transcript of the portion of SV40 DNA coding for VP1. The results indicate that a segment of about 200 nucleotides of RNA transcribed from a distant part of SV40 DNA have become linked to the transcript of VP1 codons by a bond resistant to phenol extraction and denaturation in formamide.

Effect of poliovirus double-stranded RNA on viral and host-cell protein synthesis.

Cell-free protein-synthesizing systems that initiate on endogenous messenger RNA have been developed from uninfected and poliovirus-infected HeLa cells. Poliovirus double-stranded RNA is an effective inhibitor of protein synthesis in these extracts, and both cell-directed and virus-specific protein synthesis are equally sensitive to the inhibitory action of double-stranded RNA. The concentrations of double-stranded RNA required for inhibition are not achieved in the infected cell at early times after infection when host-cell shut-off occurs, but rather are achieved only late in infection when virus-specific protein synthesis begins to decline. This indicates that double-stranded RNA does not act as a direct agent to inhibit host cell protein synthesis following infection by poliovirus. The possible significance of inhibition by double-stranded RNA of poliovirus-specific protein synthesis is discussed.