RESUMEN
Recent evidence has accumulated which definitively shows that chemokine receptors CCR5 and CXCR4 play an essential role as coreceptors for human immunodeficiency virus type 1 (HIV-1) infection. Flow cytometric analysis permitted us to detect CD38, a surface marker of early differentiation, as well as activation of T cells, on about half of healthy donor-derived CD4(+) T cells. In this study, we focused on the susceptibility of CD38(+) and CD38(-) subsets of CD4(+) T cells to HIV-1 infection with different coreceptor tropisms. About 20% of peripheral blood mononuclear cell-derived resting CD4(+) T cells were recovered into the CD38(+) subset fraction by panning with a monoclonal antibody to CD38. Most of the cells in this CD38(high) fraction also expressed CD45RA and CD62L at higher intensities compared with those of CD38(low) fraction. CCR5(+) T cells predominated in the CD38(-) subset, although cell surface expression of CD4 and CXCR4 was almost similar between both subsets. This difference was consistent with a significantly higher susceptibility of the CD38(-) subset to a macrophage (M)-tropic HIV-1 strain. In contrast, it was shown that a T-tropic strain of HIV-1 could replicate more efficiently in the CD38(+) subset, although viral adsorption rates were similar between both subsets. Thus, the differential susceptibility of CD4(+) T cells to M(-) and T-tropic HIV-1 was associated with their surface expression of CD38.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación/análisis , Linfocitos T CD4-Positivos/inmunología , VIH-1/patogenicidad , NAD+ Nucleosidasa/análisis , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Linfocitos T CD4-Positivos/virología , Células Cultivadas , Citometría de Flujo , Humanos , Selectina L/análisis , Antígenos Comunes de Leucocito/análisis , Glicoproteínas de Membrana , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Receptores CCR5/análisis , Receptores CXCR4/análisis , Subgrupos de Linfocitos T/inmunología , Replicación ViralRESUMEN
In this review we summarize multiple aspects of the human immunodeficiency virus (HIV) protease from both structural and functional viewpoints. After an introductory overview, we provide an up-to-date status report on protease inhibitors (PI). This proceeds from a discussion of PI structural design, to how PI are optimally utilized in highly active antiretroviral triple therapy (one PI along with two reverse transcriptase inhibitors), the emergence of PI resistance, and the natural role of secretory leukocyte PI. Then we switch to another focus: the interaction of HIV protease with other genes in acute and persistent infection, which in turn may have an effect on AIDS pathogenesis. We conclude with a discussion on future directions in HIV treatment, involving multiple-target anti-HIV therapy, vaccine development, and novel reactivation-inhibitory reagents.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/etiología , Inhibidores de la Proteasa del VIH/uso terapéutico , Proteasa del VIH/efectos de los fármacos , Vacunas contra el SIDA , Ensayos Clínicos como Asunto , Diseño de Fármacos , Quimioterapia/tendencias , Genes Virales , VIH-1/patogenicidad , Humanos , Latencia del VirusRESUMEN
Protease inhibitors are widely used in the treatment of human immunodeficiency virus type 1 (HIV-1)-infected individuals and show a drastic effect on the reduction of virus load. We previously reported that doughnut-shaped, protease-defective gp120-containing HIV-1 particles from an L-2 cell clone, carrying a provirus with mutations at the pol (protease), env (gp41) and nef genes, rapidly and more effectively induces virus particle-mediated syncytia formation of uninfected T-cells, than a parental wild-type laboratory strain of HIV-1 (LAI). In this study, we examined the possibility of whether enhanced syncytia formation is mediated by morphologically similar doughnut-shaped particles obtained after treatment of LAI-infected cells with the protease inhibitors L-689, 502, DMP-323, RO-31-8959, and KNI-272. Utilizing such protease inhibitor-induced particles and a clone of MOLT-4 cells, we could not detect any enhancement of syncytia formation, over that seen with wild-type LAI particles. This result should alleviate concerns of patients on highly active antiretroviral therapy (HAART), that protease inhibitors might accelerate progression of the disease through enhanced production of defective, 'immature'-appearing particles.
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Fármacos Anti-VIH/farmacología , Proteasa del VIH/genética , VIH-1/patogenicidad , Inhibidores de Proteasas/farmacología , Linfocitos T/virología , Western Blotting , Línea Celular , Quimioterapia Combinada , VIH-1/efectos de los fármacos , Humanos , Microscopía Electrónica , Linfocitos T/efectos de los fármacosRESUMEN
Screening and confirmatory low cost reagent tests have been developed for detection of anti-hepatitis C virus (HCV). Assays are based on the use of specific synthetic peptides from several structural and non-structural viral proteins. The efficacy of the screening anti-HCV EIA-Spep assay was compared with both Abbott EIA 2.0 (Abbott Laboratories, North Chicago, IL) and Ortho EIA 2.0 (Ortho Diagnostic Systems, Raritan, NJ) anti-HCV detection kits and the confirmatory EIA-Cpep assay was compared with the Abbott Matrix anti-HCV confirmation test. In the EIA-Spep, a pool of 3 peptides was added to each well of a microtiter plate. In EIA-Cpep, each well was separately coated with 1 of 4 peptides and 1 recombinant protein. A total of 867 blood donor samples from Costa Rica tested simultaneously with the 3 screening assays yielded the same specificity and negative predictive values of > or =99.9% and 100%, respectively. A comparative study on voluntary blood donor samples from Honduras, Nicaragua, and El Salvador using the 2 anti-HCV confirmatory assays revealed different patterns that are 46% positive, 24% indeterminate, and 30% negative with the EIA-Cpep assay vs. 31% positive, 48% indeterminate, and 21% negative with the Matrix assay. A study of 71 patient samples from Costa Rica showed a higher correlation between initially reactive samples when analyzed by the Abbott and Ortho kits, than when the assay results were compared between the Abbott and EIA-Spep kits; the latter detected 7 and 15 non-reactive samples, respectively. These results could reflect the use of a similar antigen source for the 2 commercial assays. The presence of HCV RNA in a group of 29 samples analyzed was related to the simultaneous reactivity in all 3 screening assays. None of the discordant samples had detectable levels of HCV RNA. Economic difficulties for health care services in the developing countries of Central America have prevented implementation of routine anti-HCV blood donor screening tests. This is likely to be the primary reason for uncontrolled dissemination of HCV, and the lack of identification of potential high risk groups. Alternative low cost reagents developed locally as described in this article could be a useful tool in the control of HCV spread throughout the developing world.
Asunto(s)
Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/diagnóstico , Oligopéptidos/inmunología , Secuencia de Aminoácidos , Donantes de Sangre , Costos y Análisis de Costo , Países en Desarrollo , Hepacivirus/inmunología , Hepatitis C/sangre , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Tamizaje Masivo/economía , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , ARN Viral/sangre , ARN Viral/genética , Juego de Reactivos para Diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
Apoptosis is an important mechanism of human immunodeficiency virus type 1 (HIV-1)-induced T-cell depletion. Our recent findings revealed mitogenic stimulation-dependent apoptosis induction in healthy donor-derived peripheral blood T-lymphocytes after adsorption with defective HIV-1 particles through acquirement by a subset of CD4+/CD38- cells of specific killer function. Based on these in vitro observations, we have extended the significance of this killing activity of CD4+/CD38- cells directly derived from HIV-1 carriers. The CD4+/CD38- cells from HIV-1-positive individuals showed significantly higher cell-killing activities than those from HIV-1-negative donors by co-culture with allogeneic resting T-cells after mitogenic stimulation. Furthermore, most of the samples induced apoptosis in a Fas-dependent manner. Thus, it is suggested that HIV-1 infection-related apoptosis is triggered by inappropriate activation of a certain resting T-cell subset, presumably due to adsorption with HIV-1 particles.
Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/inmunología , VIH-1/inmunología , Subgrupos de Linfocitos T/inmunología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Anticuerpos Monoclonales , Biomarcadores , Células Cultivadas , Seronegatividad para VIH , Seropositividad para VIH , Humanos , Activación de Linfocitos , Receptor fas/inmunologíaRESUMEN
Apoptosis is a major mechanism whereby HIV-1 depletes uninfected CD4+ and CD8+ T cells. We previously showed that resting peripheral blood T cells derived from healthy donors were killed by an apoptotic mechanism after adsorption to gp120-containing, protease-defective HIV-1 (L-2) particles, more effectively than parental wild-type LAI adsorption or rgp 120-mediated CD4 cross-linking, followed by mitogenic stimulation. Here, we present evidence that the L-2 particle-based apoptosis was induced both in CD4+ and CD8+ cells by generation of effector cells which were mainly derived from a resting memory CD4+CD38- subset. This subset enhanced the CD25 expression on the surface and secreted IFN-gamma in the culture supernatant after L-2 particle exposure. Significant elevation of Fas ligand mRNA was found in the subset by L-2 particle exposure, while expression of Fas antigen on uninfected T cells was induced by exposure to IFN-gamma. These results indicate that L-2 particles can shift the CD4+CD38- subpopulation from a resting to an activated state, and this activation leads to killing of bystander CD4+ and CD8+ T cells by a Fas-mediated mechanism. In fact, purified CD4+CD38- cells exposed to L-2 particles were converted into effector cells that were able to kill autologous as well as allogenic target T cells pretreated with IFN-gamma. Further, we found that the observation of apoptosis due to L-2 particles was a more general phenomenon, that also occurred with Thai primary HIV-1 isolates. These results suggest that such specific types of HIV-1 particles may play a major role in the induction of apoptosis for both bystander CD4+ and CD8+ T cells, through inappropriate activation of CD4+CD38- cells.
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Apoptosis/inmunología , VIH-1/inmunología , VIH-1/patogenicidad , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Citotoxicidad Inmunológica , Cartilla de ADN/genética , Virus Defectuosos/inmunología , Proteína Ligando Fas , Infecciones por VIH/sangre , Infecciones por VIH/etiología , Infecciones por VIH/inmunología , Humanos , Técnicas In Vitro , Activación de Linfocitos , Glicoproteínas de Membrana/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-2/sangre , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T/patologíaAsunto(s)
Genes env , Genes nef , Genes pol , Genes prv , VIH-1/genética , Linfocitos T/virología , Secuencia de Aminoácidos , Secuencia de Bases , Células Clonales , ADN Viral , Endopeptidasas , Humanos , Datos de Secuencia Molecular , Mutación , Provirus/genética , Células Tumorales Cultivadas , ViriónRESUMEN
Newly released HIV-1 particles exhibit an immature morphology, previously reported to be characterized by a doughnut/ring-shaped structure. In this study we showed that among immature extracellular virus particles not only were particles with doughnut-shaped morphology present, but particles with a crescent morphology were also observed. These particles occurred with different frequencies, depending on whether they were in the cell or in cell-free fractions. The crescent-shaped particles were more abundant in the cell-free fractions, whereas the particles in the cell fraction mainly exhibited doughnut-shaped morphology. The crescent-shaped structure may represent an assembly intermediate.
Asunto(s)
VIH-1/ultraestructura , Línea Celular Transformada , Humanos , Virión/ultraestructuraRESUMEN
Apoptosis and syncytium formation are two mechanisms by which human immunodeficiency virus type 1 (HIV-1) impairs uninfected CD4+ T-cell function and are mainly involved in the progression of the disease to AIDS. Previously, we showed that gp120-containing, protease-deficient HIV-1 (L-2) particles generated syncytia by particle-mediated fusion with uninfected cultured CD4+ T cells. Here, we present evidence that such L-2 particles can induce apoptosis in 40 to 50% of T cells which were enriched from HIV-1-negative healthy donor-derived peripheral blood mononuclear cells (PBMC-Ts). Activation of PBMC-Ts with phytohemagglutinin, concanavalin A, or ionomycin after incubation with L-2 particles resulted in the loss of proliferative capacity and gradual induction of apoptosis over 3 days. Wild-type strain LAI particles or recombinant gp120 were markedly less efficient (< or = 15%) at inducing such apoptosis. Western blot (immunoblot) analysis revealed that L-2 particles contained a larger amount of Env gp120 than LAI particles. Either preincubation of PBMC-Ts with a Fas antagonist or preincubation of L-2 particles with soluble CD4 blocked most of the apoptosis. This suggests that L-2-like particles can play a major role in HIV-1-induced apoptosis of uninfected bystander cells.
Asunto(s)
Apoptosis , Proteína gp120 de Envoltorio del VIH , VIH-1 , Linfocitos T/virología , Células Cultivadas , Citometría de Flujo , Humanos , Activación de Linfocitos , Proteínas Recombinantes , Linfocitos T/patología , ViriónRESUMEN
Human immunodeficiency virus type 1 (HIV-1) infection indirectly induces activation-dependent apoptosis in bystander immune CD4+ T-cells, a hallmark of AIDS pathogenesis. It is well known that this pathogenetic event is significantly correlated with a high virus load. Active viral replication occurs in HIV-1 asymptomatic carriers throughout all stages of clinical disease. Most of the HIV-1 in plasma is derived from short-lived infected cells with a half life of a few days; however, a minor population of virus is derived from long-lived persistently and latently infected cells. Recently, the importance of such latent reservoirs for HIV-1 has come to the forefront because of studies with potent antiretroviral inhibitors that block only new rounds of infection. An initial large drop in viral load occurs within two weeks as noted by a decrease in plasma viremia. This is then followed by a slower second-phase decay, since only a small fraction of latently infected resting CD4+ T-cells carry replication-competent, integrated provirus. This review highlights the mechanisms of apoptosis induction in bystander immune cells by both protease-defective, gp120-containing HIV-1 particles, as well as by wild-type virus that appears to be derived predominantly from long-lived infected cells. A model involving the NH2-terminal Nef domain (p7) in this 'bystander apoptosis' event is also presented.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Apoptosis , Proteínas de la Cápside , VIH-1/fisiología , Mutación , Proteínas Virales , Latencia del Virus , Síndrome de Inmunodeficiencia Adquirida/virología , Cápside/metabolismo , Productos del Gen gag/metabolismo , Productos del Gen nef/metabolismo , VIH-1/genética , Humanos , Factores de Tiempo , Virión , Integración Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) derived from an infectious molecular clone pNL432 was extensively passaged in tissue culture by repeated rounds of acute infection. We previously showed the natural occurrence of a nonsense mutation in the vpr gene during continued passage of this virus. In this report, we show that two forms of large deletions (561 and 518 base pairs containing short direct repeats at the deletion junctions) occur after passage 50 in the region that spans the vif and vpr open reading frames. One model to explain the occurrence of these deletion regions is that such mutations result from misalignment of the growing point at a limited number of nucleotide positions. Infection of CD4+ T-cells with a recombinant HIV-1 construct containing the same vif to vpr deletion showed virtually no cytopathogenic phenotype. Thus, misalignment deletions at non-essential accessory genes of HIV-1 might be induced during replication, which result in the generation of virus with a low cytopathogenic potential.
Asunto(s)
Eliminación de Gen , Genes vif , Genes prv , VIH-1/genética , Secuencia de Bases , Linfocitos T CD4-Positivos/virología , Células Clonales , ADN Viral/genética , ADN Viral/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Pase SeriadoRESUMEN
Several recent reports support the hypothesis that apoptosis occurring in leukocytes of human immunodeficiency virus type 1 (HIV-1)-infected individuals is important in progression to AIDS. Specifically, apoptosis of uninfected bystander cells appears critical in the pathogenesis of disease. Here, we present evidence that protease-defective, gp120-containing HIV-1 (L-2) particle preparations specifically induce apoptosis in cells obtained from a subset of promonocytic U937-derived subclones. The rate of apoptosis induction was inversely correlated with the susceptibility of the U937 subclones to wild-type HIV-1 infection. Three types of apoptosis experiments were performed: DNA content analysis by flow cytometry, apoptotic nuclear degradation by fluorescent microscopy and DNA fragmentation analysis by agarose gel electrophoresis. Kinetic analysis revealed that there was a slower induction of apoptosis by L-2 particle preparations than with tumor necrosis factor (TNF)-alpha or anti-Fas antibody. However, there were no significant differences in the initial binding rates of L-2 particles as well as the binding of TNF-alpha or anti-Fas antibody to the U937 subclones. The basal level of protein kinase C activity was higher in high-type subclones compared with low-type subclones. These results suggest that U937 cells can be divided into at least two subpopulations, one that permits a productive HIV-1 infection but is not subjected to L-2 particle preparation-induced apoptosis, while the other poorly replicates HIV-1 and is subjected to L-2 mediated apoptosis, although at a slower rate than found with TNF-alpha or anti-Fas antibody.
Asunto(s)
Apoptosis/inmunología , Endopeptidasas/deficiencia , VIH-1/enzimología , VIH-1/patogenicidad , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Células Clonales , Humanos , Memoria Inmunológica , Linfoma de Células B Grandes Difuso/metabolismo , Proteína Quinasa C/análisis , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/inmunología , Receptor fas/inmunologíaRESUMEN
A pooling system was developed for use in anti-HCV screening of voluntary blood donors at the local Central American Red Cross blood banks, in Nicaragua, El Salvador and Honduras. The commercially available second generation anti-HCV screening kit from Abbott Laboratories (North Chicago, IL) was used with a modification in the initial serum dilution procedure. Pools of five sera were selected for routine screening, based on comparative studies of individual samples and of pools with different sample sizes. During the years 1993 and 1994 a total of 89, 148 voluntary blood donors were screened and a positive prevalence rate of 0.35% was established. Of the initially positive samples, 54% confirmed positive, 30% were indeterminate and 16% were negative using the Abbott Matrix test. Significant differences of positive screening prevalence rates were found in the three countries, with average values of 0.50%, 0.23% and 0.08%, respectively, in Nicaragua, El Salvador and Honduras. These initially positive samples also showed a different confirmatory pattern with a positive rate of 64% in Nicaragua, in contrast to 20% in El Salvador. Only a few samples were available for RT-PCR amplification of HCV-RNA; however, this highly sensitive method did not appear to be more helpful than serology in confirming the HCV donor status. Overall, the data obtained indicate a fluctuation of HCV prevalence in voluntary blood donors among the three Central American countries. Further, differences were found in the percentages of initially screened positives and confirmation patterns. This information appears useful for establishing criteria in future screening policies. Thus, we suggest that the use of pooling for anti-HCV screening is beneficial in countries under development, since there are potential cost savings, as well as benefits in establishment of initial prevalence rates.
Asunto(s)
Donantes de Sangre , Países en Desarrollo , Hepacivirus/aislamiento & purificación , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C/virología , Bancos de Sangre , Costos y Análisis de Costo , Estudios de Evaluación como Asunto , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/sangre , Hepatitis C/inmunología , Humanos , Tamizaje Masivo/economía , Reacción en Cadena de la Polimerasa , ARN Viral/sangre , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
The complete nucleotide and predicted amino acid sequences for open reading frames (ORFs) of the human adenovirus type 41 (Ad41) early region 3 (E3) gene have been determined. The sequence of the Ad41 E3 gene (map units 74 to 83.9) consists of 3,373 nucleotides and has one TATA box and two polyadenylation signals (AATAAA). Analysis of the nucleotide sequence reveals that the E3 gene can encode six ORFs, designated RL1 to RL6. These are all expressed at the mRNA level, as determined by reverse transcription-PCR analysis of AD41-infected cell RNA. When compared with known E3 sequences of most other human adenoviruses deposited in GenBank, the sequences of RL1 to RL3 were found to be unique to subgroup F adenoviruses (Ad40 and Ad41). They encode putative proteins of 173 amino acids (19.4 kDa) and 276 amino acids (31.6 kDa) in one reading frame as well as a 59- amino-acid (6.7 kDa) protein in an overlapping reading frame. RL4 encodes a 90-amino-acid protein (10.1 kDa) with 40% homology to the Ad2 E3 10.4-kDa protein, which induces degradation of the epidermal growth factor receptor and functions together with the Ad2 E3 14.5-kDa protein to protect mouse cell lines against lysis. RL5 encodes a protein of 107 amino acid residues (12.3 kDa) and is analogous to the Ad E3 14.5-kDa protein. RL6 codes for a protein of 122 amino acids (14.7 kDa) that is analogous to the Ad2 14.7-kDa protein, which functions to protect Ad-infected cells from tumor necrosis factor-induced cytolysis. This finding of three unique (RL1 to RL3) E3 gene ORFs may explain why subgroup F adenoviruses differ substantially from other human adenoviruses in their host range; i.e., they replicate predominantly in the host's gastrointestinal rather than respiratory tract. A recent phylogenetic study that compared subgroup F Ad40 DNA sequences with representatives of subgroups B (Ad3), C (Ad2), and E (Ad4) reached a similar conclusion about the uniqueness of RL1 and RL2.
Asunto(s)
Proteínas E3 de Adenovirus/genética , Adenovirus Humanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Viral , Genes Virales , Humanos , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Proviral DNA from cells surviving severe but transient cytopathic effects, mediated by infection with recombinant human immunodeficiency virus type 1 (HIV-1) carrying a single gene mutation at vif, vpr, or vpu, was characterized by use of HIV-1-specific primer pairs in a two-step PCR. Deletion mutations were detected in a region that spanned the vif and vpr open reading frames. Cloning and sequencing of the amplified DNA from this region revealed frequent large deletions in a limited number of nucleotide positions. Analyses of the deletions suggested that (i) genetic recombination, (ii) template-primer slippage, and (iii) misalignment of the growing point during reverse transcription of the HIV-1 genome might be the mechanisms that generated the mutations. Apart from the large deletions, smaller deletions that gave frameshift mutations in vif and/or vpr prevailed. In addition, cells infected with a triple mutant defective in vif, vpr, and vpu did not show any cytopathic effect. Thus, mutations generating multiple accessory gene defects during HIV-1 replication correlate with viral persistence and loss of cytopathogenicity.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Virus Defectuosos/genética , Genes vif , Genes prv , Genes vpu , VIH-1/genética , Mutación , Replicación Viral/genética , Secuencia de Bases , Línea Celular Transformada , Cartilla de ADN , ADN Viral/genética , Virus Defectuosos/inmunología , Mutación del Sistema de Lectura , Genoma Viral , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Cinética , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Provirus/genética , Provirus/inmunología , Eliminación de Secuencia , Linfocitos T , Factores de TiempoRESUMEN
Nine phage isolates infectious for Vibrio vulnificus and falling into four morphological groups were isolated from estuarine waters collected in Louisiana. Of the 60 V. vulnificus strains tested, 87% were susceptible to one or more of the isolates. With the exception of V. fluvialis, Vibrio species other than vulnificus were resistant to infection. A spectrum of enteric bacterial strains were similarly resistant. Susceptibility differences were seen between opaque (virulent) V. vulnificus strains and those with translucent (nonvirulent) colony types, with the former being more susceptible. Susceptibility patterns to infection by the nine phage isolates among the V. vulnificus test strains suggest that the latter may fall into several groups. Other aspects relating to the phage isolates are presented.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Vibrio/virología , Antibiosis , Bacteriófagos/ultraestructura , Microscopía Electrónica , Especificidad de la EspecieRESUMEN
DNA sequencing of the subgroup F human adenovirus serotype 41 (TAK, Ad41) fiber gene revealed the presence of two adjacent open reading frames encoding information for proteins with molecular weights of 60.6 kDa and 41.4 kDa (Pieniazek, et al; Nucleic Acids Res. 18: p. 1901, 1990). In this paper, various approaches were used to characterize the two proteins and determine whether both fibers were expressed in infected cells as well as on viral particles. We initially used a reverse transcriptase-polymerase chain reaction with primers for the short and long fiber genes to amplify mRNA from Ad41 infected HEp-2 cells at 48 h post-infection. Two distinct DNA bands; one slightly larger than 1.1 kbp and the other at about 1.7 kbp were identified. Second, we used polyclonal anti-Ad41 virion and monoclonal anti-Ad5 fiber antibodies to demonstrate that at both 24 and 36 h post-infection, Ad41 expressed two fiber proteins of the expected size. Specifically, by SDS-PAGE, one fiber (short) had a molecular weight of 40 kDa, while the other (long) had a molecular weight of 60 kDa. Third, by electron microscopy, two sizes of fibers were released from CsCl purified virions, both having a characteristic adenovirus morphology, with a knob at one end. The long fiber measured 315A in length and the short fiber was 250A long. These measurements are consistent with the two Ad41 fibers being encoded by the above open reading frames. We also performed a computer search to compare fiber sequences from other human adenovirus serotypes with that of the Ad41 short and long fiber proteins. The primary structure of both Ad41 fibers were found to be similar in that they contained tail, shaft and knob regions. Further, the tail region of both fibers (amino acids 1-42) showed a 74% overall homology to each other and contained the Ad conserved sequence NH2-F-N-P-V-Y-P-Y-COOH. An interesting difference, however, was observed in the shaft region where the long fiber (amino acids 43-389) had twenty-two 16-amino acid repeat motifs, while the short fiber (amino acids 43-233) had only twelve. Finally, we noted that the long fiber knob region was about 15% longer than that of the short fiber, and showed little overall homology. In conclusion, human adenovirus subgroup F (type 41) virions appear to differ from those of all other human adenoviruses (subgenera A-E) in that they contain two fiber genes and correspondingly, two different sized fibers.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Adenovirus Humanos/genética , Proteínas de la Cápside , Cápside/genética , Adenovirus Humanos/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Cápside/química , Cápside/ultraestructura , Línea Celular , Cartilla de ADN/genética , ADN Viral/genética , Humanos , Microscopía Electrónica , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , SerotipificaciónRESUMEN
A surprisingly large number of animal viruses interact with cytoskeletal elements inside infected cells at different stages of replication. For example, a viral protease is activated during assembly of murine leukemia viruses at the cell surface, which causes both the morphological conversion of 'immature' to 'mature' particles and a drastic reduction of the actin stress fiber network.