RÉSUMÉ
We previously generated a prototype monkey-tropic human immunodeficiency virus type 1 (HIV-1) designated NL-DT5R. This viral clone has a small region of simian immunodeficiency virus (SIV) within Gag capsid (CA) protein and also SIV Vif protein, but displays a poor growth phenotype in simian cells. To improve the growth potential of NL-DT5R, we have constructed a series of its gag variant viruses. Out of fourteen viral clones generated, five were infectious for simian HSC-F cells, and two of the infectious variants grew similarly with NL-DT5R. Taking their genome structures into consideration, our data here clearly show that a narrow CA region within the Gag protein, i.e., the domain around cyclophilin A (CypA)-binding loop, is critical for the growth ability of HIV-1 in simian cells.
Sujet(s)
Acides aminés/analyse , Cyclophiline A/analyse , Produits du gène gag/analyse , Produits du gène gag/physiologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/physiologie , Virus de l'immunodéficience simienne/physiologie , Lymphocytes T/virologie , Séquence d'acides aminés , Animaux , Lignée cellulaire , Prolifération cellulaire , Cyclophiline A/physiologie , Modèles animaux de maladie humaine , Cellules épithéliales/virologie , Produits du gène vif/analyse , Produits du gène vif/physiologie , Humains , Macaca fascicularis , Données de séquences moléculaires , MutationRÉSUMÉ
Neuropathologically, human immunodeficiency virus (HIV) is associated with a range of inflammatory disorders, extensive cortical neuronal loss, and dendritic and synaptic damage. Although the mechanisms resulting in these abnormalities are still unclear, the neurotoxic effects are thought to be due in part to viral products including the tat gene product. We have previously shown that Tat when presented to neurons extracellularly interacts with neuronal cell membranes to cause neuronal excitation and toxicity in fmole amounts. To determine the role of Tat in mediating HIV encephalitis (HIVE), we detected tat mRNA and protein in tissue extracts of nine patients with HIVE and seven patients without HIVE. Despite long autopsy times and significant degradation, tat mRNA was detected in 4/9 patients with HIVE but not in any of the seven patients without dementia. Similarly, the env mRNA was also detected in 5/9 patients with HIVE but not in the patients without HIVE. However, vif mRNA was detected in both groups of patients with (5/9) or without (2/7) HIVE. Using protein extracts from the brains of the same groups of patients we were unable to detect Tat by enzyme linked immunosorbant assay (ELISA) (sensitivity of 2 ng Tat/ml of brain tissue). However, Tat could be detected immunohistochemically and in protein extracts from the brains of rhesus macaques with encephalitis due to a chimeric strain of HIV and simian immunodeficiency virus (SHIV). Our observations support the role of Tat in the neuropathogenesis of HIV and SHIV encephalitis.
Sujet(s)
Démence associée au SIDA/virologie , Encéphalite virale/virologie , Lobe frontal/composition chimique , Protéines du gène tat/analyse , Gènes tat , Infections à VIH/virologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , ARN messager/analyse , ARN viral/analyse , Démence associée au SIDA/métabolisme , Démence associée au SIDA/anatomopathologie , Adulte , Sujet âgé , Animaux , Technique de Western , Encéphalite virale/métabolisme , Encéphalite virale/anatomopathologie , Test ELISA , Femelle , Lobe frontal/anatomopathologie , Lobe frontal/virologie , Produits du gène env/analyse , Produits du gène vif/analyse , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/isolement et purification , Humains , Poumon/composition chimique , Poumon/anatomopathologie , Poumon/virologie , Macaca mulatta , Mâle , Adulte d'âge moyen , Modifications postmortem , RT-PCR , Sensibilité et spécificité , Virus de l'immunodéficience simienne/génétique , Produits du gène tat du virus de l'immunodéficience humaine , Produits du gène vif du virus de l'immunodéficience humaineRÉSUMÉ
Consecutive enzymatic reactions on analytes affinity-bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been used for detecting protein synthesis errors occuring at the N-terminus. The usefulness of this method was demonstrated by analyzing two commercially available recombinant HIV proteins with affinity tags at the N-terminus, and histatin-5, a peptide with multiple histidine residues. The high specificity, sensitivity, and speed of analysis make this method especially useful in obtaining N-terminal sequencing information of histidine-tagged recombinant proteins.
Sujet(s)
Chromatographie d'affinité/méthodes , Métaux , Protéines recombinantes/analyse , Spectrométrie de masse MALDI/méthodes , Protéines virales/analyse , Séquence d'acides aminés , Produits du gène vif/analyse , Protéine de capside p24 du VIH/analyse , VIH-1 (Virus de l'Immunodéficience Humaine de type 1) , Données de séquences moléculaires , Protéines/analyse , Produits du gène vif du virus de l'immunodéficience humaineRÉSUMÉ
The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.
Sujet(s)
Produits du gène vif/biosynthèse , Gènes vif , VIH (Virus de l'Immunodéficience Humaine)/métabolisme , Virus de l'immunodéficience simienne/métabolisme , Virion/métabolisme , Séquence d'acides aminés , Anticorps , Séquence nucléotidique , Amorces ADN , Réplication de l'ADN , ADN viral/biosynthèse , Délétion de gène , Produits du gène vif/analyse , VIH (Virus de l'Immunodéficience Humaine)/génétique , VIH-2 (Virus de l'Immunodéficience Humaine de type 2)/génétique , VIH-2 (Virus de l'Immunodéficience Humaine de type 2)/métabolisme , Cellules HeLa , Humains , Données de séquences moléculaires , Mutagenèse , Fragments peptidiques/composition chimique , Fragments peptidiques/immunologie , Réaction de polymérisation en chaîne , Provirus/génétique , Provirus/métabolisme , Protéines recombinantes/analyse , Protéines recombinantes/biosynthèse , Virus de l'immunodéficience simienne/génétique , Transfection , Virion/génétique , Produits du gène vif du virus de l'immunodéficience humaineRÉSUMÉ
A fragment of the genome of human immunodeficiency virus type 1 (HIV-1) coding for p23 protein, the product of vif gene, was cloned in a plasmid vector pUR291. The resulting recombinant plasmid pLacVif1 was conducive in E. coli cells to the synthesis of a hybrid polypeptide with molecular weight of 136 kDa containing antigenic determinants of p23 protein of HIV-1. The employment of this polypeptide for analysis of HIV-1-positive sera by indirect enzyme immunoassay showed that vif-specific antibodies were found in 53% of the cases and their appearance was not related to the stage of the disease.
Sujet(s)
Spécificité des anticorps , Escherichia coli/génétique , Produits du gène vif , Anticorps anti-VIH/sang , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/immunologie , Clonage moléculaire/méthodes , Produits du gène vif/analyse , Produits du gène vif/génétique , Séropositivité VIH/diagnostic , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Humains , Techniques immunoenzymatiques , Masse moléculaire , Plasmides/génétique , Protéines recombinantes/analyse , Protéines recombinantes/génétique , Produits du gène vif du virus de l'immunodéficience humaineRÉSUMÉ
Two molecularly cloned coisolates of human immunodeficiency virus type 1 (HIV-1) have been found to exhibit different phenotypes of viral expression, either rapid and cytopathic (N1T-A virus) or delayed and noncytopathic (N1T-E virus [X. Ma, K. Sakai, F. Sinangil, E. Golub, and D. J. Volsky, Virology 176:184-194, 1990]). To identify the viral genetic elements responsible for these phenotypes, we prepared reciprocal recombinants in different regions of N1T-A and N1T-E viral genomes. Infectivity experiments with the recombinant viruses revealed that the rapid/cytopathic (N1T-A-like) phenotype assorted cleanly with the V1f-coding region and Vif expression. The smallest HIV-1 DNA region that conferred the complete phenotypic switch was a 284-bp NdeI-StuI fragment within the vif open reading frame. Nucleotide sequence analysis revealed a 35-bp deletion starting at nucleotide 218 in the N1T-E vif gene. A 23-kDa Vif protein was detected by immunoblotting using Vif-specific antiserum in extracts of cells infected with N1T-A but not N1T-E virus. No detectable vif protein was found in association with sedimented particles of either virus. Cotransfection of a eucaryotic vif expression plasmid with N1T-E DNA complemented the N1T-E defect; rapid/cytopathic infection similar to that in N1T-A-transfected cells was observed. We conclude that Vif controls the rate, and consequently the cytopathic outcome, of HIV-1 infection.