RESUMO
The intracellular fate of internalized virus-receptor complexes is suspected of influencing the efficiency of virus infection. However, direct evidence of a link between infection and the fate of internalized virus has been difficult to obtain. To directly address this question, we generated human 293 cell lines stably expressing comparable cell surface levels of three different members of the somatostatin receptor family (SSTR) which have natural differences in intracellular trafficking. Utilizing a glycoprotein that recognizes SSTR, we found that distinctive receptor subtype-specific destinations correlated with observable differences in the level of infection. Infection via SSTR-2 and -3 is restricted at a point after receptor binding and endocytosis but prior to penetration into the host cytoplasm. In contrast, entry via SSTR-5 featured a slower internalization with greater dependence on cholesterol. Quantitative real-time PCR showed that virus bound to SSTR-5 was directed to an intracellular environment that allowed near-wild-type (WT) levels of penetration, possibly due to a more favorable complement of host cell proteases, whereas SSTR-2 and -3 directed virions to a degradative compartment in which cytosol penetration was less efficient. Taken together, the results support that the superior receptor capacity of SSTR-5 results from its internalization into a cellular compartment that is more favorable to the cytoplasmic penetration of viral cores and reverse transcription. They suggest that the intracellular destination of internalized complexes is an important characteristic of a virus receptor and may have exerted a selective pressure on the choice of an entry receptor during evolution of viral glycoproteins.
Assuntos
Vírus da Leucemia Murina/fisiologia , Receptores de Somatostatina/biossíntese , Receptores Virais/biossíntese , Internalização do Vírus , Linhagem Celular , Humanos , Vírus da Leucemia Murina/genéticaRESUMO
Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.
Assuntos
Técnicas de Transferência de Genes/instrumentação , Vírus da Leucemia Murina de Moloney/genética , Receptores de Somatostatina/metabolismo , Somatostatina/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus , Animais , Sítios de Ligação , Linhagem Celular , Marcação de Genes/instrumentação , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/química , Vírus da Leucemia Murina de Moloney/fisiologia , Ligação Proteica , Engenharia de Proteínas , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Receptores Virais/genética , Receptores Virais/metabolismo , Somatostatina/química , Somatostatina/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
We examined 299 methicillin-resistant, community-associated Staphylococcus aureus isolates from Florida and Washington State for the presence of the USA300 epidemic clone. Pulsed-field gel electrophoresis demonstrated the epidemic clone in 43% of our S. aureus strains and in isolates from both states. The majority of the USA300 isolates (88%) were from wound infections.
