RESUMEN
The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.
Asunto(s)
Productos del Gen env , VIH-1 , Productos del Gen env del Virus de la Inmunodeficiencia Humana , Antígenos Virales/genética , Línea Celular , Productos del Gen env/química , Productos del Gen env/genética , VIH-1/fisiología , Mutación , Dominios Proteicos , Proteínas de la Matriz Viral/metabolismo , Replicación Viral/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Abstract Dengue is a major worldwide public health problem, especially in the tropical and subtropical regions of the world. Primary infection with a single Dengue virus serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients who experience secondary infection with a different serotype can progress to a more severe form of the disease, called dengue hemorrhagic fever. The four Dengue virus serotypes (1–4) are antigenically and genetically distinct and each serotype is composed of multiple genotypes. In this study we isolated one Dengue virus 1 serotype, named BR/Alfenas/2012, from a patient with dengue hemorrhagic fever in Alfenas, South Minas Gerais, Brazil and molecular identification was performed based on the analysis of NS5 gene. Swiss mice were infected with this isolate to verify its potential to induce histopathological alterations characteristic of dengue. Liver histopathological analysis of infected animals showed the presence of inflammatory infiltrates, hepatic steatosis, as well as edema, hemorrhage and necrosis focal points. Phylogenetic and evolutionary analyses based on the envelope gene provided evidence that the isolate BR/Alfenas/2012 belongs to genotype V, lineage I and it is probably derived from isolates of Rio de Janeiro, Brazil. The isolate BR/Alfenas/2012 showed two unique amino acids substitutions (SER222THRE and PHE306SER) when compared to other Brazilian isolates from the same genotype/lineage. Molecular models were generated for the envelope protein indicating that the amino acid alteration PHE 306 SER could contribute to a different folding in this region located within the domain III. Further genetic and animal model studies using BR/Alfenas/2012 and other isolates belonging to the same lineage/genotype could help determine the relation of these genetic alterations and dengue hemorrhagic fever in a susceptible population.
Asunto(s)
Animales , Humanos , Ratones , Virus del Dengue/clasificación , Virus del Dengue/genética , Dengue/virología , Variación Genética , Genotipo , Filogenia , Sustitución de Aminoácidos , Estructuras Animales/patología , Brasil , Modelos Animales de Enfermedad , Virus del Dengue/aislamiento & purificación , Productos del Gen env/química , Productos del Gen env/genética , Histocitoquímica , Microscopía , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Proteínas no Estructurales Virales/genéticaRESUMEN
O HTLV-1 é o vírus causador da leucemia/linfoma de célula T no adulto e de uma desordem neurológica conhecida por mielopatia associada ao HTLV ou paraparesia espástica tropical. Um dos modos de transmissão é pelo sangue contaminado e seus subprodutos e, devido ao risco de infecções associadas ao HTLV sua pesquisa na triagem de doadores de sangue foi introduzida no Brasil a partir de 1993. Os kits diagnósticos utilizados nos bancos de sangue nacionais são na sua maioria comprados de empresas estrangeiras. O Brasil não detém a tecnologia para produção deste material e há a necessidade de produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, mostramos a expressão da gp21/HTLV-1 em Escherichia coli e sua reatividade frente a anticorpos monoclonais e de pacientes infectados. Expressar tais proteínas é o primeiro passo para obtenção de conjuntos diagnósticos com tecnologia brasileira.
HTLV-1 is the virus that causes T cell lymphoma/leukemia in adults and a neurological disorder known as HTLV-associated myelopathy or tropical spastic paraparesis. One of the transmission means is through contaminated blood and its byproducts. Because of the risk of HTLV-associated infections, screening for HTLV was introduced for Brazilian blood donors in 1993. Most of the diagnostic kits used in the national blood banks are bought from foreign companies. Brazil does not have the technology to produce this material and there is a need to produce diagnostic systems with national technology. In this study, we show the expression of gp21/HTLV-1 in Escherichia coli and its reactivity towards monoclonal antibodies and the antibodies of infected patients. Expressing these proteins is the first step towards obtaining diagnostic kits with Brazilian biotechnology.