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1.
Sci Adv ; 9(44): eadh3642, 2023 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-37922361

RESUMEN

Unintegrated retroviral DNA is transcriptionally silenced by host chromatin silencing factors. Here, we used the proteomics of isolated chromatin segments method to reveal viral and host factors associated with unintegrated HIV-1DNA involved in its silencing. By gene silencing using siRNAs, 46 factors were identified as potential repressors of unintegrated HIV-1DNA. Knockdown and knockout experiments revealed POLE3 as a transcriptional repressor of unintegrated HIV-1DNA. POLE3 maintains unintegrated HIV-1DNA in a repressive chromatin state, preventing RNAPII recruitment to the viral promoter. POLE3 and the recently identified host factors mediating unintegrated HIV-1 DNA silencing, CAF1 and SMC5/SMC6/SLF2, show specificity toward different forms of unintegrated HIV-1DNA. Loss of POLE3 impaired HIV-1 replication, suggesting that repression of unintegrated HIV-1DNA is important for optimal viral replication. POLE3 depletion reduces the integration efficiency of HIV-1. POLE3, by maintaining a repressive chromatin structure of unintegrated HIV-1DNA, ensures HIV-1 escape from innate immune sensing in primary CD4+ T cells.


Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , VIH-1/genética , ADN Viral/genética , Cromatina/genética , Integración Viral , Infecciones por VIH/genética , Inmunidad Innata
2.
Sci Adv ; 9(15): eadg6265, 2023 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-37043562

RESUMEN

Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.


Asunto(s)
Hepatitis B Crónica , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B/genética , Antivirales/farmacología , Antivirales/uso terapéutico , ARN , Hepatitis B Crónica/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Replicación Viral
3.
Cell Host Microbe ; 28(2): 335-349.e6, 2020 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-32504577

RESUMEN

Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.


Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Preescolar , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Células HEK293 , Células Hep G2 , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/inmunología , Humanos , Lactante , Ratones , Ratones Noqueados , Conformación Proteica
4.
Proc Natl Acad Sci U S A ; 117(3): 1678-1688, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31915293

RESUMEN

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.


Asunto(s)
Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatopatías/genética , Alcaloides de Pirrolicidina/farmacología , Animales , Trasplante de Células , Quimera , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Hepatitis B , Virus de la Hepatitis B , Hepatocitos/trasplante , Proteínas de Homeodominio/genética , Humanos , Hidrolasas/genética , Subunidad gamma Común de Receptores de Interleucina/genética , Hígado/patología , Hepatopatías/patología , Malaria , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Plasmodium falciparum
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