RESUMO
An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Processamento de Proteína Pós-Traducional , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Motivos de Aminoácidos , Anticorpos Neutralizantes/biossíntese , Glicosilação , Proteína gp120 do Envelope de HIV/administração & dosagem , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Imunidade Humoral/efeitos dos fármacos , Imunogenicidade da Vacina , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de Partículas Semelhantes a VírusRESUMO
The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.
Assuntos
Citoplasma/química , HIV-1/fisiologia , Receptores de HIV/fisiologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Tropismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/fisiologiaRESUMO
Killed and live attenuated influenza virus vaccines are effective in preventing and curbing the spread of influenza epidemics when the strains present in the vaccines are closely matched with the predicted epidemic strains. These vaccines are primarily targeted to induce immunity to the variable major target antigen, hemagglutinin (HA) of influenza virus. However, current vaccines are not effective in preventing the emergence of new pandemic or highly virulent viruses. New approaches are being investigated to develop universal influenza virus vaccines as well as to apply more effective vaccine delivery methods. Conserved vaccine targets including the influenza M2 ion channel protein and HA stalk domains are being developed using recombinant technologies to improve the level of cross protection. In addition, recent studies provide evidence that vaccine supplements can provide avenues to further improve current vaccies.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Pandemias/prevenção & controle , Vacinação , Proteínas da Matriz Viral/imunologia , Animais , Sequência Conservada/imunologia , Proteção Cruzada/imunologia , Reações Cruzadas/imunologia , Vias de Administração de Medicamentos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vacinas contra Influenza/biossíntese , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Estrutura Terciária de Proteína , Estados Unidos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/biossíntese , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Proteínas da Matriz Viral/genéticaRESUMO
There is an urgent need to develop novel approaches for vaccination against emerging pathogenic avian influenza viruses as a priority for pandemic preparedness. Influenza virus-like particles (VLPs) have been suggested and developed as a new generation of non-egg-based cell culture-derived vaccine candidates against influenza infection. Influenza VLPs are formed by a self-assembly process incorporating structural proteins into budding particles composed of the hemagglutinin (HA), neuraminidase (NA) and M1 proteins, and may include additional influenza proteins such as M2. Animals vaccinated with VLPs were protected from morbidity and mortality resulting from lethal influenza infections. The protective mechanism of influenza VLP vaccines was similar to that of the currently licensed influenza vaccines inducing neutralizing antibodies and hemagglutination inhibition activities. Current studies demonstrate that influenza VLP approaches can be a promising alternative approach to developing a vaccine for pandemic influenza viruses. The first human clinical trial of a recombinant pandemic-like H5N1 influenza VLP vaccine was initiated in July 2007 (Bright et al., unpublished).
Assuntos
Surtos de Doenças/prevenção & controle , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Vírion/imunologia , Animais , Vetores Genéticos , Humanos , Virus da Influenza A Subtipo H5N1/imunologia , Spodoptera , Vírion/isolamento & purificaçãoRESUMO
To identify and explore the activity of compounds which may act as anti-HIV virucidal agents, we have investigated platinum compounds, especially those containing N-donor aromatic ligands. After screening over 70 related agents, including N-donor aromatic ligands and metal precursors, we have identified a novel class of platinum(II) complexes with 2-pyridyl-1,2,4-triazine derivatives and Pt(II) formulations with these derivatives (ptt compounds) as having the highest anti-HIV activity. The maximum activity was observed when the agents were added immediately post-infection. The ptt agents did not block cell fusion activity of HIV-1 Env proteins in cells bearing CD4X4 or CD4R5 receptors, indicating a lack of interaction with the Env protein. The ptt compounds exhibit low toxicity for human epithelial cells, and are thus promising candidates for use as microbicides or antiviral agents against HIV.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/prevenção & controle , Compostos de Platina/farmacologia , Triazinas/farmacologia , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/toxicidade , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1 , Cinética , Fusão de Membrana/efeitos dos fármacos , Camundongos , Compostos de Platina/química , Compostos de Platina/toxicidade , Triazinas/química , Triazinas/toxicidadeRESUMO
SER virus, a paramyxovirus closely related to simian virus 5, induces no syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT), and truncation or mutations of the CT result in enhanced syncytium formation (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77:167-178, 2003; S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). We hypothesized that the presence of the long CT serves to stabilize the metastable conformation of the F protein. We observed that the hemifusion, cytoplasmic content mixing, and syncytium formation ability of the wild-type SER virus F coexpressed with the SER virus hemagglutinin-neuraminidase (HN) protein was enhanced, both qualitatively and quantitatively, at elevated temperatures. We also observed enhanced hemifusion, content mixing, and syncytium formation in SER virus F- and HN-expressing cells at reduced pH conditions ranging between 4.8 and 6.2. We have obtained evidence that in contrast to other paramyxoviruses, entry of SER virus into cells occurs by a low-pH-dependent process, indicating that the conversion to the fusion-active state for SER virus F is triggered by exposure to reduced pH.
Assuntos
Fusão de Membrana , Respirovirus/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Linhagem Celular , Cricetinae , Regulação Viral da Expressão Gênica , Células Gigantes/fisiologia , Cobaias , Concentração de Íons de Hidrogênio , Respirovirus/genética , Respirovirus/fisiologia , Temperatura , Proteínas Virais de Fusão/genéticaRESUMO
Sin Nombre virus (SNV) is a major representative of the New World hantaviruses and the most common cause of hantavirus pulmonary syndrome (HPS) with high mortality in North America. Unlike other members of the family Bunyaviridae which mature in the Golgi complex, New World hantaviruses have been previously reported to mature at the cell surface. For family Bunyaviridae viruses, retention of the viral glycoproteins at the Golgi complex is thought to be responsible for their Golgi maturation. In our studies, the majority of SNV glycoproteins, G1 and G2, was localized in the Golgi complex when expressed from a full-length GPC clone or in SNV-infected cells, in agreement with data for other members of the family Bunyaviridae, including the Old World hantaviruses. However, the SNV glycoproteins could also be detected at the cell surface at advanced posttransfection or postinfection time points. G1 expressed in the absence of G2 did not accumulate in the Golgi, but remained predominantly associated with the endoplasmic reticulum (ER). Overexpressed amounts of apparently misfolded G1 were aggregated in a subcellular compartment likely to represent the aggresome. Unexpectedly, an additional major pool of G1 was detected intracellularly in SNV-infected and GPC-expressing transfected cells, by using a SNV G1-specific Fab antibody. This pool of G1 is predominantly localized in late endosomes-lysosomes.
Assuntos
Vírus Sin Nombre/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Cricetinae , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Microscopia Imunoeletrônica , Transporte Proteico , Ratos , Frações Subcelulares/metabolismo , Transfecção , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/genéticaRESUMO
SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.
Assuntos
Proteína HN/fisiologia , Fusão de Membrana , Paramyxoviridae/fisiologia , Proteínas Virais de Fusão/química , Sequência de Aminoácidos , Citoplasma/química , Dados de Sequência Molecular , Mutação , Relação Estrutura-Atividade , Proteínas Virais de Fusão/fisiologiaRESUMO
SER virus is a member of the family Paramyxoviridae, genus Rubulavirus, which has been isolated from pigs. It is very closely related to SV5 virus serologically, in protein profile, and in nucleotide sequence. However, unlike SV5, SER induces minimal syncytium formation in infected CV-1 or BHK cells. Fluorescence transfer experiments between labeled erythrocytes and infected MDBK cells revealed that SER also induces hemifusion and pore formation with reduced efficiency. The virion polypeptide profiles of SER and SV5 are very similar, except that the SER F1 subunit shows an apparent molecular weight that is about 2 kDa higher than that of SV5. Comparison of the deduced amino acid sequences revealed the SER F (551 aa) to be longer than SV5 F (529 aa) by 22 residues in the cytoplasmic tail (CT) domain. The HN and M gene sequences of the viruses were found to be very similar. The SER F showed minimal fusion activity when coexpressed with either SV5 or SER HN. In contrast, SV5 F was highly fusogenic when coexpressed with either HN protein, indicating that the restricted fusion capacity of SER virus is a property of its F protein. Truncation in the CT of SER F by 22 residues completely rescued its ability to cause syncytium formation, whereas other truncations rescued syncytium formation partially. These results demonstrate that an elongated CT of a paramyxovirus F protein suppresses its membrane fusion activity.
Assuntos
Fusão de Membrana/fisiologia , Rubulavirus/fisiologia , Animais , Sequência de Bases , Bovinos , Células Cultivadas , Chlorocebus aethiops , Cricetinae , Citoplasma/metabolismo , DNA Viral , Cães , Expressão Gênica , Células HeLa , Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Humanos , Dados de Sequência Molecular , Fenótipo , Rubulavirus/genética , Rubulavirus/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/genéticaRESUMO
The involvement of CXCR4 and CCR5 coreceptors in apoptosis induced by the HIV envelope (Env) proteins has not been well defined. We found that simian human immunodeficiency virus (SHIV) virus-like particles (VLPs) containing HIV Env proteins preferentially induce apoptosis of cells corresponding to their coreceptor usage in a CD4+ T cell line. We also demonstrated that induction of apoptosis by SHIV VLPs is correlated with coreceptor usage in a non-T cell line. We examined the effects of SHIV VLPs containing Env proteins derived from either a T-cell-tropic HIV (BH10) strain or a dual-tropic HIV (89.6) strain on induction of apoptosis in recombinant CD4+ human osteosarcoma (HOS) cells expressing either CXCR4 (HOS-CD4.CXCR4) or CCR5 coreceptors (HOS-CD4.CCR5). HOS-CD4.CXCR4 or HOS-CD4.CCR5 cells were activated with concanavalin A and cocultured with VLPs. By TUNEL (TdT-mediated dUTP-X nick end labeling) fluorescence staining and flow cytometry assays, SHIV BH10 VLPs were found to preferentially induce apoptosis in HOS-CD4.CXCR4 cells but not in HOS-CD4 or HOS-CD4.CCR5 cells. On the other hand, SHIV 89.6 VLPs induced an elevated level of apoptosis in both HOS-CD4.CXCR4 and HOS-CD4.CCR5 cells in a dose-dependent fashion. These data demonstrate that T-cell-tropic BH10 Env preferentially utilizes CXCR4, but not CCR5, for induction of apoptosis, whereas dual-tropic 89.6 Env induces apoptosis in both CXCR4- and CCR5-containing cell lines.
Assuntos
Apoptose , HIV-1 , Receptores CCR5/fisiologia , Receptores CXCR4/fisiologia , Proteínas do Envelope Viral/metabolismo , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Concanavalina A/farmacologia , Produtos do Gene gag/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/metabolismo , Infecções por HIV/etiologia , Humanos , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Células Tumorais Cultivadas , Proteínas do Envelope Viral/imunologiaRESUMO
West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5'- and 3'-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3'-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5'-end completely abrogated virus replication. Replacement of the conserved 5'-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5'- and 3'-end genome sequences. However, the presence of extensive nonrelated sequences at both 5'- and 3'-ends could not be repaired.
Assuntos
Genoma Viral , Vírus do Nilo Ocidental/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Replicon , Transcrição Gênica , Transfecção , Replicação Viral/genética , Vírus do Nilo Ocidental/patogenicidadeRESUMO
Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with alpha-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of the Bunyaviridae family.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Complexo de Golgi/metabolismo , Orthohantavírus/metabolismo , Proteínas do Core Viral/metabolismo , Animais , Capsídeo/química , Capsídeo/genética , Linhagem Celular , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Imunofluorescência , Orthohantavírus/genética , Infecções por Hantavirus/virologia , Vírus La Crosse/metabolismo , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
During viral maturation, the cytoplasmic tail of the murine leukemia virus (MuLV) envelope (Env) protein undergoes proteolytic cleavage by the viral protease to release the 16-amino-acid R peptide, and this cleavage event activates the Env protein's fusion activity. We introduced Gly and/or Ser residues at different positions upstream of the R peptide in the cytoplasmic tail of the Friend MuLV Env protein and investigated their effects on fusion activity. Expression in HeLa T4 cells of a mutant Env protein with a single Gly insertion after I619, five amino acids upstream from the R peptide, induced syncytium formation with overlaid XC cells. Env proteins containing single or double Gly-Ser insertions after F614, 10 amino acids upstream from the R peptide, induced syncytium formation, and mutant proteins with multiple Gly insertions induced various levels of syncytium formation between HeLa T4 and XC cells. Immunoprecipitation and surface biotinylation assays showed that most of the mutants had surface expression levels comparable to those of the wild-type or R peptide-truncated Env proteins. Fluorescence dye redistribution assays also showed no hemifusion in the Env proteins which did not induce fusion. Our results indicate that insertion mutations in the cytoplasmic tail of the MuLV Env protein can suppress the inhibitory effect of the R peptide on membrane fusion and that there are differences in the effects of insertions in two regions in the cytoplasmic tail upstream of the R peptide.
Assuntos
Vírus da Leucemia Murina/metabolismo , Fusão de Membrana , Fragmentos de Peptídeos/fisiologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células HeLa , Humanos , Vírus da Leucemia Murina/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Envelope Viral/químicaRESUMO
In this study, we investigated the role of the membrane-proximal region of the human parainfluenza virus type 2 (HPIV2) F protein by mutational analysis, including deletion, insertion, and substitution. Deletion or replacement of the entire 12 amino acid region (aa 474-485) of the HPIV2 F protein completely abolished its fusion activity when coexpressed with the HPIV2 HN protein. Deletion of groups of four of aa 478-485, single alanine, or other amino acid substitutions among aa 478-485 had minimal or limited effects on HPIV2 F/HN-induced cell fusion. However, a significant reduction in, or complete inhibition of, fusion activity was observed when aa 474-477 were deleted, or the N475, F476, or F477 residues were singly substituted with alanine. In addition, insertions of four amino acids at this region or deletion of eight or more amino acids significantly reduced F protein fusion activity. The oligomerization patterns and levels of cell surface expression of the mutant F proteins were compared to those of the wild-type HPIV2 F protein. The mutant HPIV2 F proteins defective in fusion were also found to be unable to initiate hemifusion, indicating that there is a specific requirement for three specific amino acids as well as the spacing in this region for initiating lipid mixing.
Assuntos
Fusão de Membrana/fisiologia , Vírus da Parainfluenza 2 Humana/fisiologia , Proteínas Virais de Fusão/fisiologia , Aminoácidos , Sítios de Ligação , Membrana Celular/metabolismo , Expressão Gênica , Engenharia Genética , Células HeLa , Humanos , Mutagênese , Vírus da Parainfluenza 2 Humana/genética , Vírus da Parainfluenza 2 Humana/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
By the use of a mouse model, the enteropathic effects of the simian immunodeficiency virus (SIV) surface unit (SU) envelope glycoprotein were explored. Purified SU (0.01-0.45 nmol) was administered intraperitoneally to 6- to 8-day-old mouse pups and induced a dose-dependent diarrheal response. Surgical introduction of SU into adult mouse intestinal loops revealed fluid accumulation without histological alterations and SU-treated unstripped intestinal mucosa induced chloride (Cl(-)) secretory currents in Ussing chambers. Similarly to rotavirus NSP4, the first described viral enterotoxin, SU induced a transient increase in intracellular calcium levels and increased inositol 1,4,5-triphosphate (IP(3)) levels in HT-29 cells. These data indicate the calcium response is mediated by IP(3). The presence of diarrhea and fluid accumulation within intestinal loops in the absence of histological alterations and induction of Cl(-) secretory currents demonstrate that SIV contains an enterotoxic domain localized within SU and is the second viral enterotoxin described.
Assuntos
Enterotoxinas/toxicidade , Glicoproteínas/toxicidade , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas do Envelope Viral/toxicidade , Animais , Animais Lactentes , Cálcio/metabolismo , Linhagem Celular , Cloretos/metabolismo , Diarreia/induzido quimicamente , Relação Dose-Resposta a Droga , Enterotoxinas/isolamento & purificação , Enterotoxinas/farmacocinética , Glicoproteínas/isolamento & purificação , Células HT29 , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Imunodeficiência Símia/química , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
In addition to the viral envelope (Env) proteins, host cell-derived proteins have been reported to be present in human immunodeficiency virus and simian immunodeficiency virus (SIV) envelopes, and it has been postulated that they may play a role in infection. We investigated whether the incorporation of host cell proteins is affected by the structure and level of incorporation of viral Env proteins. To compare the cellular components incorporated into SIV particles and how this is influenced by the structure of the cytoplasmic domain, we compared SIV virions with full-length and truncated Env proteins. The levels of HLA-I and HLA-II molecules were found to be significantly (15- to 25-fold) higher in virions with full-length Env than in those with a truncated Env. Virions with a truncated Env were also found to be less susceptible to neutralization by specific antibodies against HLA-I or HLA-II proteins. We also compared the level of incorporation into SIV virions of a coexpressed heterologous viral glycoprotein, the influenza virus hemagglutinin (HA) protein. We found that SIV infection of cells expressing influenza virus HA resulted in the production of phenotypically mixed SIV virions containing influenza virus HA as well as SIV envelope proteins. The HA proteins were more effectively incorporated into virions with full-length Env than in virions with truncated Env. The phenotypically mixed particles with full-length Env, containing higher levels of HA, were sensitive to neutralization with anti-HA antibody, whereas virions with truncated Env proteins and containing lower levels of HA were more resistant to neutralization by anti-HA antibody. In contrast, SIV virions with truncated Env proteins were found to be highly sensitive to neutralization by antisera to SIV, whereas virions with full-length Env proteins were relatively resistant to neutralization. These results indicate that the cytoplasmic domain of SIV Env affects the incorporation of cellular as well as heterologous viral membrane proteins into the SIV envelope and may be an important determinant of the sensitivity of the virus to neutralizing antibodies.
Assuntos
Citoplasma/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas do Envelope Viral/metabolismo , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Testes de Neutralização , Testes de Precipitina , Estrutura Terciária de Proteína , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Transfecção , Proteínas do Envelope Viral/imunologia , Montagem de VírusRESUMO
The cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors.
Assuntos
Antígenos CD4/metabolismo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Peptídeos/metabolismo , Receptores Virais/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Fusão Celular , HIV/genética , HIV/metabolismo , Células HeLa , Humanos , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
Through cognate interaction between antigen-specific B-cell and CD4(+) alphabeta T cells, the CD4(+) alphabeta T cells secrete cytokines that initiate immunoglobulin (Ig) class switching from IgM to IgG. In this study, we show that formalin-inactivated influenza PR8 virus induces virus-specific IgM and IgG responses in the absence of CD4(+) T cells and that all four subclasses of IgG are produced. The immunized CD4-deficient mice were also found to be completely protected against lethal infection with live, pathogenic influenza virus. The ability of CD4(+) T-cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8(+) T cells with an anti-CD8 monoclonal antibody. In contrast, alphabeta T-cell-deficient mice (TCRbeta(-/-)) were not found to produce significant amounts of IgG upon immunization with formalin-inactivated PR8 virus. These results suggest that CD4(-) CD8(-) double-negative alphabeta T cells are playing a role in regulating Ig class switching in the absence of CD4(+) T cells.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Fatores Etários , Animais , Antígenos CD4/genética , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Embrião de Galinha , Cães , Formaldeído/farmacologia , Humanos , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Neutralização , Baço/citologiaRESUMO
A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.
Assuntos
Vacinas contra a AIDS/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas contra a AIDS/efeitos adversos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/genética , Humanos , Esquemas de Imunização , Camundongos , Dados de Sequência Molecular , Testes de Neutralização , Pan troglodytes , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Proteínas do Envelope Viral/genéticaRESUMO
A number of studies indicate that the transmembrane domain, the cytoplasmic domain, or both regions of viral surface glycoproteins are involved in quaternary structure formation. In this report, the transmembrane domain and cytoplasmic tail coding sequence of the fusion (F) glycoprotein gene from parainfluenza type 2 virus was truncated by PCR and the resulting gene (PI2F') was expressed in HeLa-T4 cells by using the vaccinia virus-T7 transient expression system. Pulse-chase experiments indicated that the anchor-free PI2F' was expressed and processed into F(1) and F(2) subunits. Both the processed and the unprocessed anchor-free PI2F' proteins were found to be efficiently secreted into the culture medium. Examination of the oligomeric form of the anchor-free PI2F' by chemical cross-linking demonstrated that it assembles posttranslationally into dimers and trimers with a pattern similar to that of the wild-type PI2F protein. In an effort to better understand the biological properties of the truncated form of PI2F', we anchored PI2F' by a glycosyl-phosphatidylinositol (GPI) linkage. The GPI-anchored PI2F' protein, when coexpressed with PI2HN, did not induce cell fusion seen as syncytium formation, but was found to initiate lipid mixing (hemifusion) as observed by transfer of R-18 rhodamine from red blood cells to the GPI-PI2F'/PI2HN cotransfected cells. The results therefore indicate that the extracellular domain of the PI2 fusion protein contains not only the structural information sufficient to direct assembly into higher oligomers, but also is competent to initiate membrane fusion, suggesting that the anchor-free PI2F' may be useful for further structural studies.