ABSTRACT
We recently reported an unconventional mechanism by which miRNAs inhibit HIV-1 viral production. This occurs when miRNAs bind nonspecifically to the viral structural protein Gag, interfering with viral RNA-mediated Gag assembly at the plasma membrane. Consequently, misassembled viral complexes are redirected into the endocytic pathway where they are delivered to lysosomes for degradation. In this study, we demonstrate that autophagy is a critical mediator of the viral degradation pathway and that this pathway is not HIV-1 specific. Misassembled viral complexes were found to colocalize extensively with LC3 and p62 in late endosomes/lysosomes, demonstrating a convergence of autophagy with functional degradative compartments. Knocking down autophagosome formation machineries reduced this convergence, while treatment with autophagy-inducer rapamycin enhanced the convergence. Furthermore, similar autophagy-dependent nonspecific miRNA inhibition of murine leukemia virus (MLV) assembly was shown. Overall, these results reveal autophagy as a crucial regulator of the retroviral degradation pathway in host cells initiated by nonspecific miRNA-Gag interactions. These findings could have significant implications for understanding how cells may regulate retroviral complex assembly by miRNA expression and autophagy, and raise the possibility that similar regulations can occur in other biological contexts.
Subject(s)
Humans , Autophagy , Cell Membrane , Metabolism , Gene Products, gag , Genetics , Metabolism , HEK293 Cells , HIV-1 , Metabolism , Lysosomes , Metabolism , MicroRNAs , Genetics , Metabolism , Virus AssemblyABSTRACT
BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.
Subject(s)
Humans , Antiretroviral Therapy, Highly Active , CD4 Lymphocyte Count , Enzyme-Linked Immunospot Assay , Gene Products, gag , Haplotypes , HIV , HIV Infections , HIV-1 , Immunity, Cellular , Interferon-gamma , RNA , T-Lymphocytes, CytotoxicABSTRACT
BACKGROUND: HIV-specific immunity, such as strong CD4+ helper and CD8+ cytotoxic T lymphocytes (CTL) responses, develops soon after HIV infection, but usually it can not control HIV replication which ultimately results in severe immune deficiency. HIV-specific CTLs, which are induced by HIV-specific CD4+ helper responses, are the key to cellular immune control of HIV. Measurement of HIV-1-specific CTLs using recombinant Gag protein may be very useful, because it is not restricted by HLA haplotype of the infected individual. MATERIALS AND METHODS: Enzyme-linked immunospot (ELISPOT) assays by using recombinant Gag protein were performed to evaluate HIV-1-specific gamma-interferon cellular responses of 25 HIV-1 infected Korean patients, who had been treated at least for the prior 12 months with highly active antiretroviral therapy at Catholic University Kangnam St. Mary's Hospital. RESULTS: The study group consisted of 25 chronically HIV-infected individuals with a median age of 51 years. The median CD4 counts were 556/mm3 (range:369-994/mm3) and HIV RNA titers were < 25 copies/mL (range: <25-180copies/mL). HIV-1-specific ELISPOT assay results range from 0 to 49 SFCs/2 x 10(5) PBMCs (median, 23.5 SFCs/2 x 10(5) PBMCs). CMV pp65-specific ELISPOT assay results range from 5 to 591 SFCs/2 x 10(5) PBMCs (median, 34 SFCs/2 x 10(5) PBMCs). There was no correlation between CD4 counts and HIV-1-specific SFCs measured by ELISPOT using recombinant protein. CONCLUSION: ELISPOT assays by using recombinant Gag protein may be considerable value in the assessment of cell-mediated immunity of HIV-1 infected patients.