RESUMO
Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.
Assuntos
Adenosina Trifosfatases , Proteínas de Ligação a DNA/metabolismo , HIV-1/fisiologia , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Vacúolos/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Genes Reporter/genética , HIV-1/ultraestrutura , Humanos , Ligação Proteica , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismoRESUMO
A P157S mutation in the reverse transcriptase (RT) of human immunodeficiency virus type 1 conferred fivefold resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine in cell culture. Interestingly, the P157S mutation resulted in increased sensitivity (two- to threefold) to 3'-azido-3'-deoxythymidine (AZT) and to (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). A similar increase in susceptibility to AZT and to PMPA was also conferred by the M184V mutation in RT.
Assuntos
Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/genética , Lamivudina/farmacologia , Mutação Puntual , Inibidores da Transcriptase Reversa/farmacologia , Resistência Microbiana a Medicamentos/genética , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/isolamento & purificação , HIV-1/genética , HIV-1/patogenicidade , Células HeLa , Humanos , Prolina/genética , Serina/genéticaRESUMO
After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.
Assuntos
Capsídeo/química , HIV-1/fisiologia , Dobramento de Proteína , Montagem de Vírus/fisiologia , Sequência de Aminoácidos , Capsídeo/genética , Linhagem Celular , Sequência Conservada/genética , Produtos do Gene gag/química , HIV-1/química , HIV-1/crescimento & desenvolvimento , HIV-1/ultraestrutura , Humanos , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/fisiologia , Ressonância Magnética Nuclear Biomolecular , Mutação Puntual , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão , Linfócitos T , Proteínas da Matriz Viral/química , Vírion/ultraestruturaRESUMO
The carboxyl-terminal domain, residues 146 to 231, of the human immunodeficiency virus-1 (HIV-1) capsid protein [CA(146-231)] is required for capsid dimerization and viral assembly. This domain contains a stretch of 20 residues, called the major homology region (MHR), which is conserved across retroviruses and is essential for viral assembly, maturation, and infectivity. The crystal structures of CA(146-231) and CA(151-231) reveal that the globular domain is composed of four helices and an extended amino-terminal strand. CA(146-231) dimerizes through parallel packing of helix 2 across a dyad. The MHR is distinct from the dimer interface and instead forms an intricate hydrogen-bonding network that interconnects strand 1 and helices 1 and 2. Alignment of the CA(146-231) dimer with the crystal structure of the capsid amino-terminal domain provides a model for the intact protein and extends models for assembly of the central conical core of HIV-1.
