RESUMO
To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.
Assuntos
Produtos do Gene gag , Vírus da Imunodeficiência Felina , Vírus da Imunodeficiência Símia , Montagem de Vírus , Dedos de Zinco , Animais , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Produtos do Gene gag/química , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Gatos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Linhagem Celular , Nucleocapsídeo/metabolismo , Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , FenótipoRESUMO
CD8+ T-cells play a crucial role in the control of HIV replication. HIV-specific CD8+ T-cell responses rapidly expand since the acute phase of the infection, and it has been observed that HIV controllers harbor CD8+ T-cells with potent anti-HIV capacity. The development of CD8+ T-cell-based vaccine against HIV-1 has focused on searching for immunodominant epitopes. However, the strong immune pressure of CD8+ T-cells causes the selection of viral variants with mutations in immunodominant epitopes. Since HIV-1 mutations are selected under the context of a specific HLA-I, the circulation of viral variants with these mutations is highly predictable based on the most prevalent HLA-I within a population. We previously demonstrated the adaptation of circulating strains of HIV-1 to the HLA-A*02 molecule by identifying mutations under positive selection located in GC9 and SL9 epitopes derived from the Gag protein. Also, we used an in silico prediction approach and evaluated whether the mutations found had a higher or lower affinity to the HLA-A*02. Although this strategy allowed predicting the interaction between mutated peptides and HLA-I, the functional response of CD8+ T-cells that these peptides induce is unknown. In the present work, peripheral blood mononuclear cells from 12 HIV-1+ HLA-A*02:01+ individuals were stimulated with the mutated and wild-type peptides derived from the GC9 and SL9 epitopes. The functional profile of CD8+ T-cells was evaluated using flow cytometry, and the frequency of subpopulations was determined according to their number of functions and the polyfunctionality index. The results suggest that the quality of the response (polyfunctionality) could be associated with the binding affinity of the peptide to the HLA molecule, and the functional profile of specific CD8+ T-cells to mutated epitopes in individuals under cART is maintained.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Linfócitos T CD8-Positivos , Colômbia , Epitopos , Produtos do Gene gag , Antígenos HLA-A , Humanos , Epitopos Imunodominantes , Leucócitos Mononucleares , PeptídeosRESUMO
During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.
Assuntos
HIV-1 , Regiões 5' não Traduzidas , Adenosina/genética , Adenosina/metabolismo , Produtos do Gene gag/genética , HIV-1/metabolismo , Metilação , RNA Viral/genética , RNA Viral/metabolismo , Vírion/metabolismoRESUMO
Murine leukemia virus (MLV) requires the infected cell to divide to access the nucleus to integrate into the host genome. It has been determined that MLV uses the microtubule and actin network to reach the nucleus at the early stages of infection. Several studies have shown that viruses use the dynein motor protein associated with microtubules for their displacement. We have previously reported that dynein light-chain roadblock type 2 (Dynlrb2) knockdown significantly decreases MLV infection compared to nonsilenced cells, suggesting a functional association between this dynein light chain and MLV preintegration complex (PIC). In this study, we aimed to determine if the dynein complex Dynlrb2 subunit plays an essential role in the retrograde transport of MLV. For this, an MLV mutant containing the green fluorescent protein (GFP) fused to the viral protein p12 was used to assay the PIC localization and speed in cells in which the expression of Dynlrb2 was modulated. We found a significant decrease in the arrival of MLV PIC to the nucleus and a reduced net speed of MLV PICs when Dynlrb2 was knocked down. In contrast, an increase in nuclear localization was observed when Dynlrb2 was overexpressed. Our results suggest that Dynlrb2 plays an essential role in MLV retrograde transport. IMPORTANCE Different viruses use different components of cytoplasmic dynein complex to traffic to their replication site. We have found that murine leukemia virus (MLV) depends on dynein light-chain Dynlrb2 for infection, retrograde traffic, and nuclear entry. Our study provides new information regarding the molecular requirements for retrograde transport of MLV preintegration complex and demonstrates the essential role of Dynlrb2 in MLV infection.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Dineínas do Citoplasma/genética , Dineínas/metabolismo , Vírus da Leucemia Murina/crescimento & desenvolvimento , Replicação Viral/genética , Células 3T3 , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular , Núcleo Celular/virologia , Dineínas/genética , Produtos do Gene gag/genética , Células HEK293 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos , Microtúbulos/metabolismoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) can cause life-threatening diseases for which there are no effective treatments. Prevention of HTLV-1 infection requires massive testing of pregnant women, blood for transfusion, and organs for transplantation, as well as safe sex. In this context, serological assays are widely used for monitoring HTLV-1 infections. Despite the necessity for recombinant antigens to compose serological tests, there is little information available on procedures to produce recombinant HTLV-1/2 antigens for serological diagnostic purposes. In this work, we tested a series of genetic constructions to select those more amenable for production in bacterial systems. To overcome the constraints in expressing sections of viral envelope proteins in bacteria, we have used the p24 segment of the gag protein as a scaffold to display the immunogenic regions of gp46 and gp21. Nine recombinant antigenic proteins derived from HTLV-1 and five derived from HTLV-2 were successfully purified. The HTLV-1 antigens showed high efficiency in discriminating HTLV-positive samples from HTLV-negative samples using enzyme-linked immunosorbent assays. Interestingly, HTLV-1-positive samples showed a high level of cross-reaction with HTLV-2 antigens. This finding is explained by the high sequence conservation between the structural proteins of these two highly related viruses. In summary, the results presented in this work provide a detailed description of the methods used to produce recombinant HTLV-1 and HTLV-2 antigens, and they demonstrate that the HTLV-1 antigens show strong potential for serological diagnosis of HTLV-1 infections.
Assuntos
Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Ensaio de Imunoadsorção Enzimática , Feminino , Produtos do Gene gag/genética , Infecções por HTLV-I/diagnóstico , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 2 Humano/genética , Humanos , GravidezRESUMO
The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag-Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.
Assuntos
Proteínas do Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Montagem de Vírus , Motivos de Aminoácidos , Animais , Células COS , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Produtos do Gene gag/genética , HIV-1/genética , Vírus da Imunodeficiência Felina/química , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Anemia Infecciosa Equina/genética , Mutação , Conformação Proteica em alfa-Hélice , Vírion/genética , Vírion/metabolismoRESUMO
Feline immunodeficiency virus (FIV), genus Lentivirus, is responsible for feline immunodeficiency syndrome in domestic cats. FIV has been classified into six subtypes: A, B, C, D, E and F, based on regions of the env gene as well as the gag gene. In Argentina, the circulation of subtypes B and E was reported more than two decades ago. The objective of this work was to study the FIV variants circulating presently in the city of Buenos Aires in naturally infected cats utilizing a nested PCR targeting the gag gene. A phylogenetic comparison with representative sequences of five previously published subtypes shows a clustering with subtypes A and B. This is the first report of FIV subtype A in Argentina.
Assuntos
Síndrome de Imunodeficiência Adquirida Felina/epidemiologia , Produtos do Gene env/genética , Produtos do Gene gag/genética , Vírus da Imunodeficiência Felina/classificação , Vírus da Imunodeficiência Felina/genética , Animais , Argentina/epidemiologia , Gatos , Síndrome de Imunodeficiência Adquirida Felina/virologia , Genes env/genética , Vírus da Imunodeficiência Felina/isolamento & purificação , Filogenia , Reação em Cadeia da PolimeraseRESUMO
Feline immunodeficiency virus (FIV) is an important cat pathogen worldwide whose biological and pathophysiological properties resemble those of human immunodeficiency virus type 1 (HIV-1). Therefore, the study of FIV not only benefits its natural host but is also useful for the development of antiviral strategies directed against HIV-1 infections in humans. FIV assembly results from the multimerization of a single but complex viral polypeptide, the Gag precursor. In this review, we will first give an overview of the current knowledge of the proteins encoded by the FIV pol, env, rev, vif, and orf-A genes, and then we will describe and discuss in detail the critical roles that each of the FIV Gag domains plays in virion morphogenesis. Since retroviral assembly is an attractive target for therapeutic interventions, gaining a better understanding of this process is highly desirable.
Assuntos
Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Vírion/fisiologia , Montagem de Vírus , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/fisiologia , Vírus da Imunodeficiência Felina/química , Vírus da Imunodeficiência Felina/genética , Modelos Moleculares , Conformação Proteica , Vírion/metabolismoRESUMO
The formation of immature lentiviral particles is dependent on the multimerization of the Gag polyprotein at the plasma membrane of the infected cells. One key player in the virus assembly process is the capsid (CA) domain of Gag, which establishes the protein-protein interactions that give rise to the hexagonal lattice of Gag molecules in the immature virion. To gain a better understanding of the functional equivalence between the CA proteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively), we generated a series of chimeric FIV Gag proteins in which the CA-coding region was partially or totally replaced by its SIV counterpart. All the FIV Gag chimeras were found to be assembly-defective; however, all of them are able to interact with wild-type SIV Gag and be recruited into extracellular virus-like particles, regardless of the SIV CA sequences present in the chimeric FIV Gag. The results presented here markedly contrast with our previous findings showing that chimeric SIVs carrying FIV CA-derived sequences are assembly-competent. Overall, our data support the notion that although the SIV and FIV CA proteins share 51% amino acid sequence similarity and exhibit a similar organization, i.e., an N-terminal domain joined by a flexible linker to a C-terminal domain, their functional exchange between these different lentiviruses is strictly dependent on the context of the recipient Gag precursor.
Assuntos
Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Animais , Células COS , Proteínas do Capsídeo/metabolismo , Chlorocebus aethiopsRESUMO
The serine incorporator 5 (SERINC5) is a recently discovered restriction factor that inhibits viral infectivity by preventing fusion. Retroviruses have developed strategies to counteract the action of SERINC5, such as the expression of proteins like negative regulatory factor (Nef), S2, and glycosylated Gag (glycoGag). These accessory proteins downregulate SERINC5 from the plasma membrane for subsequent degradation in the lysosomes. The observed variability in the action of SERINC5 suggests the participation of other elements like the envelope glycoprotein (Env) that modulates susceptibility of the virus towards SERINC5. The exact mechanism by which SERINC5 inhibits viral fusion has not yet been determined, although it has been proposed that it increases the sensitivity of the Env by exposing regions which are recognized by neutralizing antibodies. More studies are needed to understand the role of SERINC5 and to assess its utility as a therapeutic strategy.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Proteínas de Membrana/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Produtos do Gene gag/metabolismo , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Terapia de Alvo Molecular , Virulência , Internalização do Vírus , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity.
Assuntos
Produtos do Gene nef/metabolismo , HIV-1/fisiologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Animais , Linhagem Celular , Mutação da Fase de Leitura , Técnicas de Inativação de Genes , Produtos do Gene gag/metabolismo , Produtos do Gene nef/genética , Produtos do Gene pol/metabolismo , Humanos , Pan troglodytes , Ligação Proteica , Vírus da Imunodeficiência Símia/genéticaRESUMO
Endogenous retroviruses are regarded as ideal genetic markers for evolutionary analyses. Birds were some of the initial vertebrates found to contain endogenous retroviruses. However, few studies have investigated the presence and distribution of endogenous retroviruses in goose. In this study, we detected the avian sarcoma and leukosis virus gag gene in the genomic DNA of 8 Chinese native breeds using polymerase chain reaction method. The results indicated that a 1.2-kb avian sarcoma and leukosis virus gag sequence was integrated into all 8 goose breeds. The mean genetic pairwise distance was 0.918% among the investigated geese. To the best of our knowledge, this is the first report demonstrating the presence of the endogenous retroviruses in the domestic goose genome. The genetic structure should be further examined in the domestic goose.
Assuntos
Alpharetrovirus/genética , Anseriformes/genética , Evolução Molecular , Produtos do Gene gag/genética , Animais , Anseriformes/virologia , Cruzamento , DNA Mitocondrial/genética , Produtos do Gene gag/isolamento & purificação , GenomaRESUMO
BACKGROUND: Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV-IE) is a baculovirus recently identified in our laboratory, with high pathogenicity to the soybean looper, Chrysodeixis includens (Lepidoptera: Noctuidae) (Walker, 1858). In Brazil, the C. includens caterpillar is an emerging pest and has caused significant losses in soybean and cotton crops. The PsinSNPV genome was determined and the phylogeny of the p26 gene within the family Baculoviridae was investigated. RESULTS: The complete genome of PsinSNPV was sequenced (Roche 454 GS FLX - Titanium platform), annotated and compared with other Alphabaculoviruses, displaying a genome apparently different from other baculoviruses so far sequenced. The circular double-stranded DNA genome is 139,132 bp in length, with a GC content of 39.3 % and contains 141 open reading frames (ORFs). PsinSNPV possesses the 37 conserved baculovirus core genes, 102 genes found in other baculoviruses and 2 unique ORFs. Two baculovirus repeat ORFs (bro) homologs, bro-a (Psin33) and bro-b (Psin69), were identified and compared with Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV) and Trichoplusia ni single nucleopolyhedrovirus (TnSNPV) bro genes and showed high similarity, suggesting that these genes may be derived from an ancestor common to these viruses. The homologous repeats (hrs) are absent from the PsinSNPV genome, which is also the case in ChchNPV and TnSNPV. Two p26 gene homologs (p26a and p26b) were found in the PsinSNPV genome. P26 is thought to be required for optimal virion occlusion in the occlusion bodies (OBs), but its function is not well characterized. The P26 phylogenetic tree suggests that this gene was obtained from three independent acquisition events within the Baculoviridae family. The presence of a signal peptide only in the PsinSNPV p26a/ORF-20 homolog indicates distinct function between the two P26 proteins. CONCLUSIONS: PsinSNPV has a genomic sequence apparently different from other baculoviruses sequenced so far. The complete genome sequence of PsinSNPV will provide a valuable resource, contributing to studies on its molecular biology and functional genomics, and will promote the development of this virus as an effective bioinsecticide.
Assuntos
Evolução Molecular , Produtos do Gene gag/genética , Lepidópteros/genética , Nucleopoliedrovírus/genética , Animais , Lepidópteros/virologiaRESUMO
Lichen planus (LP) is a common inflammatory skin disease of unknown etiology. Reports of a common transactivation of quiescent human endogenous retroviruses (HERVs) support the connection of viruses to the disease. HERVs are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancer and autoimmune diseases. We explored the transcriptional activity of HERV sequences as well as the antiviral restriction factor and interferon-inducible genes in the skin from LP patients and healthy control (HC) donors. The study included 13 skin biopsies from patients with LP and 12 controls. Real-time PCR assay identified significant decrease in the HERV-K gag and env mRNA expression levels in LP subjects, when compared to control group. The expressions of HERV-K18 and HERV-W env were also inhibited in the skin of LP patients. We observed a strong correlation between HERV-K gag with other HERV sequences, regardless the down-modulation of transcripts levels in LP group. In contrast, a significant up-regulation of the cytidine deaminase APOBEC 3G (apolipoprotein B mRNA-editing), and the GTPase MxA (Myxovirus resistance A) mRNA expression level was identified in the LP skin specimens. Other transcript expressions, such as the master regulator of type I interferon-dependent immune responses, STING (stimulator of interferon genes) and IRF-7 (interferon regulatory factor 7), IFN-ß and the inflammassome NALP3, had increased levels in LP, when compared to HC group. Our study suggests that interferon-inducible factors, in addition to their role in innate immunity against exogenous pathogens, contribute to the immune control of HERVs. Evaluation of the balance between HERV and interferon-inducible factor expression could possibly contribute to surveillance of inflammatory/malignant status of skin diseases.
Assuntos
Retrovirus Endógenos/metabolismo , Produtos do Gene env/metabolismo , Produtos do Gene gag/metabolismo , Interferon beta/metabolismo , Líquen Plano/imunologia , Proteínas de Membrana/metabolismo , Proteínas da Gravidez/metabolismo , Pele/metabolismo , Superantígenos/metabolismo , Desaminase APOBEC-3G , Adulto , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Retrovirus Endógenos/genética , Feminino , Regulação Viral da Expressão Gênica , Produtos do Gene env/genética , Produtos do Gene gag/genética , Humanos , Vigilância Imunológica , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/genética , Líquen Plano/genética , Líquen Plano/virologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas da Gravidez/genética , Pele/virologia , Superantígenos/genética , Ativação Transcricional , Regulação para Cima , Adulto JovemRESUMO
To gain insight into the functional relationship between the capsid (CA) domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively), we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD) of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD) of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.
Assuntos
Proteínas do Capsídeo/genética , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Símia/genética , Células HEK293 , HumanosRESUMO
The Gag polyprotein of feline immunodeficiency virus (FIV) assembles at the plasma membrane of the infected cells. We aimed to identify the FIV Gag domains that interact and promote Gag multimerization. To do this we generated a series of Gag subdomains and tested their ability to associate with full-length Gag and be recruited into extracellular virus-like particles (VLPs). Removal of 37 residues from the C-terminus of FIV Gag and deletion of the N-terminal and central regions of the nucleocapsid (NC) domain attenuated but did not abrogate association with wild-type Gag, whereas a Gag mutant protein encompassing the matrix (MA) and capsid (CA) domains interacted poorly with full-length Gag. Association with wild-type Gag was abolished by deleting most of the NC together with the N-terminal 40 residues of the MA, which most likely reflects the inability of this Gag mutant to bind RNA. Notably, the CA-NC Gag subdomain both associated with wild-type Gag and was recruited into particles in a proportion close to 50â% of the total Gag-related protein mass of VLPs. Moreover, both a Gag protein lacking the C-terminal p2 peptide and a nonmyristoylated version of the polyprotein exhibited a transdominant-negative effect on the assembly of wild-type Gag. Analysis of Gag mutants carrying internal deletions within the CA revealed that the N-terminal and the C-terminal domains of the CA are necessary for Gag assembly. Our results demonstrate that the FIV CA-NC region constitutes the principal self-interaction domain of Gag and that the RNA-binding capacity of Gag is necessary for its multimerization.
Assuntos
Produtos do Gene gag/genética , Vírus da Imunodeficiência Felina/genética , Multimerização Proteica/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Membrana Celular/virologia , Chlorocebus aethiops , Produtos do Gene gag/biossíntese , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/patogenicidade , Dados de Sequência Molecular , Nucleocapsídeo/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas de Ligação a RNA/genética , Ratos , Alinhamento de Sequência , Vaccinia virus/genética , Vaccinia virus/patogenicidade , Proteínas da Matriz Viral/genética , Montagem de Vírus/genéticaRESUMO
Yellow Fever vaccine is one of the most efficacious human vaccines ever made. The vaccine (YF 17D) virus induces polyvalent immune responses, with a mixed TH1/TH2 CD4(+) cell profile, which results in robust T CD8(+) responses and high titers of neutralizing antibody. In recent years, it has been suggested that early events after yellow fever vaccination are crucial to the development of adequate acquired immunity. We have previously shown that primary immunization of humans and monkeys with YF 17D virus vaccine resulted in the early synthesis of IFN-γ. Herein we have demonstrated, for the first time that early IFN-γ production after yellow fever vaccination is a feature also of murine infection and is much more pronounced in the C57BL/6 strain compared to the BALB/c strain. Likewise, in C57BL/6 strain, we have observed the highest CD8(+) T cells responses as well as higher titers of neutralizing antibodies and total anti-YF IgG. Regardless of this intense IFN-γ response in mice, it was not possible to see higher titers of IgG2a in relation to IgG1 in both mice lineages. However, IgG2a titers were positively correlated to neutralizing antibodies levels, pointing to an important role of IFN-γ in eliciting high quality responses against YF 17D, therefore influencing the immunogenicity of this vaccine.
Assuntos
Imunidade Adaptativa/imunologia , Imunização , Interferon gama/biossíntese , Vacina contra Febre Amarela/imunologia , Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Chlorocebus aethiops , Feminino , Produtos do Gene gag/imunologia , Humanos , Imunoglobulina G/sangue , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Recombinação Genética/genética , Baço/imunologia , Células Vero , Febre Amarela/sangue , Febre Amarela/virologiaRESUMO
Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.
Assuntos
Anticorpos Antivirais , Western Blotting/normas , Sistema Nervoso Central/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Paraparesia Espástica Tropical/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Sistema Nervoso Central/metabolismo , Ensaio de Imunoadsorção Enzimática , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/líquido cefalorraquidiano , Paraparesia Espástica Tropical/sangue , Paraparesia Espástica Tropical/líquido cefalorraquidiano , Sensibilidade e EspecificidadeRESUMO
Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.
Assuntos
Humanos , Anticorpos Antivirais , Western Blotting/normas , Sistema Nervoso Central/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Paraparesia Espástica Tropical/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/líquido cefalorraquidiano , Sistema Nervoso Central/metabolismo , Ensaio de Imunoadsorção Enzimática , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/líquido cefalorraquidiano , Paraparesia Espástica Tropical/sangue , Paraparesia Espástica Tropical/líquido cefalorraquidiano , Sensibilidade e EspecificidadeRESUMO
The genome of the Caprine Arthritis-Encephalitis Virus (CAEV) encodes the polycistronic precursor protein p55(gag). Proteolytic cleavage of p55(gag) generates the viral core proteins. Some studies suggest that the CAEV p55(gag) protein contains epitopes or antigenic determinants for these core proteins. This work reinforces this hypothesis and demonstrates that monoclonal antibodies (MAbs) that are directed against the capsid protein (p28) of CAEV are also reactive against the precursor p55(gag) protein and the intermediate cleavage products, p44, p36 and p22. The major activity of the MAbs was directed against p28. The MAbF12 binding site in p28 was found to be a linear epitope with a structure that is stable after SDS treatment and remains unaltered after ß-mercaptoethanol (ß-ME) treatment. The MAbF12 binding site in the p55(gag), p36 and p22 proteins was found to be a linear epitope with cross-linked sulphide bonds. In conclusion, these findings suggest that the p28 epitope is presented differently from the epitope in the polycistronic precursor protein p55(gag). The highly immunogenic p28 contains a linear epitope that is detergent-stable and is not altered by ß-ME treatment, whereas the p55(gag) epitope contains a linear epitope susceptible to denaturing agents.