RESUMO
Retroviral reverse transcription takes place within the virion core, where nucleocapsid (NC) protein (NCp) molecules cover the dimeric RNA genome. NCp thus has structural roles in the virion core but is also extensively involved in viral DNA synthesis and virion assembly. To further characterize the role of human immunodeficiency virus type 1 NCp7 during replication of the viral genome, we investigated the relationship between NCp7 and reverse transcriptase (RT) either directly or within nucleoprotein complexes in vitro. We show that NCp7 interacts directly with RT and enhances synthesis of full-length cDNA by increasing RT processivity. Using NCp7 mutants, we show that the complete amino acid sequence of NCp7 is required for functional interactions with RT. Our results suggest that NCp7 plays a role in recruitment of RT into stable and functional nucleoprotein complexes during viral DNA synthesis.
Assuntos
Proteínas do Capsídeo , Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Transcriptase Reversa do HIV/metabolismo , Nucleoproteínas/metabolismo , Proteínas Virais , Sequência de Aminoácidos , Capsídeo/genética , Produtos do Gene gag/genética , Dados de Sequência Molecular , Mutação , Nucleoproteínas/genética , Ligação Proteica , Transcrição Gênica , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4CX4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.
Assuntos
Proteínas do Capsídeo , Capsídeo/fisiologia , Produtos do Gene gag/fisiologia , HIV-1/fisiologia , HIV-1/ultraestrutura , Proteínas Virais , Replicação Viral , Dedos de Zinco/fisiologia , Capsídeo/genética , DNA Viral , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Células HeLa , Humanos , Mutagênese , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Vírion/ultraestrutura , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
It has been shown that peptide libraries are powerful tools for the identification of peptides showing new binding specificity. This technology was applied to the isolation of peptides binding to HIV-1 nucleocapsid protein (NCp7). Three different prolin reach peptide sequences, interacting with NCp7, were isolated, from a constrained phage displayed-peptide library of 10(8) independent clones. The three peptide sequences, isolated from the peptide library, were shown to bind NCp7 in the region 30-52. Moreover, two of them share the PP-(D/E)R consensus sequence.
