RESUMO
Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is a major virion component, essential for various steps of virus replication in cells, such as entry and maturation, and cell fusion. In addition, gB is a strong inducer of the immune response in humans and has been involved in neuropathogenesis. To analyze gB during infection, a recombinant HSV-1 was generated containing gB fused to the green fluorescent protein (GFP). The GFP-gB fusion protein was incorporated into fully infectious viral particles. In cells infected with the recombinant KGFP-gB, the spontaneous fluorescence emitted by the fusion protein was observed as early as 5 h post infection, and its transport through cell compartments was followed during an entire viral replication cycle. The results show that GFP can be inserted into an essential viral envelope component of HSV-1 such as gB while preserving the infectivity of the resulting recombinant. This virus allows the investigation of several events of the viral life cycle involving gB, and provides the basis for the development of new diagnostic assays.
Assuntos
Herpesvirus Humano 1/fisiologia , Proteínas Luminescentes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Proteínas de Fluorescência Verde , Herpesvirus Humano 1/genética , Humanos , Proteínas Luminescentes/genética , Mutação , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/metabolismo , Células Vero , Proteínas do Envelope Viral/genética , Montagem de VírusRESUMO
Herpes simplex virus type 1 (HSV-1) is a human pathogen of the alphaherpesvirus family which infects and spreads in the nervous system. Glycoproteins play a key role in the process of assembly and maturation of herpesviruses, which is essential for neuroinvasion and transneuronal spread. Glycoprotein B (gB) is a main component of the HSV-1 envelope and is necessary for the production of infectious particles. The cytoplasmic domain of gB, the longest one among HSV-1 glycoproteins, contains several highly conserved peptide sequences homologous to motifs involved in intracellular sorting. To determine the specific roles of these motifs in processing, subcellular localization, and the capacity of HSV-1 gB to complement a gB-null virus, we generated truncated or point mutated forms of a green fluorescent protein (GFP)-tagged gB. GFP-gB with a deletion in the acidic cluster DGDADEDDL (amino acids [aa] 896 to 904) behaved the same as the parental form. Deletion or disruption of the YTQV motif (aa 889 to 892) abolished internalization and reduced complementation by 60%. Disruption of the LL motif (aa 871 to 872) impaired the return of the protein to the trans-Golgi network (TGN) while enhancing its recycling to the plasma membrane. Truncations from residue E 857 abolished transport and processing of the truncated proteins, which had null complementation activity, through the Golgi complex. Altogether, our results favor a model in which HSV-1 gets its final envelope in the TGN, and they suggest that endocytosis, albeit not necessary, might play a role in infectivity.
Assuntos
Citoplasma/metabolismo , Herpesvirus Humano 1/patogenicidade , Mutação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Transporte Biológico , Células COS , Linhagem Celular , Chlorocebus aethiops , Endocitose , Teste de Complementação Genética , Proteínas de Fluorescência Verde , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Vero , Proteínas do Envelope Viral/genéticaRESUMO
Herpes simplex virus type 1 (HSV-1) is a neuroinvasive human pathogen that spreads in the nervous system in functionally connected neurons. Determining how HSV-1 components are sorted in neurons is critical to elucidate the mechanisms of virus neuroinvasion. By using recombinant viruses expressing glycoprotein B (gB) tagged with green fluorescent protein (GFP), the subcellular localization of this envelope protein was visualized in infected hippocampal neurons in culture. Results obtained using a fully infectious recombinant virus containing GFP inserted into the ectodomain of gB support the view that capsids and gB are transported separately in neuron processes. Moreover, they show that during infection gB is sorted to the dendritic tree and the axons of polarized hippocampal neurons. However, GFP insertion into the cytoplasmic tail of gB impaired the maturation of the resulting fusion protein and caused its retention in the endoplasmic reticulum. The defective protein did not gain access to axons of infected neurons. These results suggest that the cytoplasmic tail of gB plays a role in maturation and transport and subsequently in axonal sorting in differentiated hippocampal neurons.