RESUMEN
Our functional genomic RNAi screens have identified the protein components of the FACT (facilitates chromatin transcription) complex, SUPT16H and SSRP1, as top host factors that negatively regulate HIV-1 replication. FACT interacts specifically with histones H2A/H2B to affect assembly and disassembly of nucleosomes, as well as transcription elongation. We further investigated the suppressive role of FACT proteins in HIV-1 transcription. First, depletion of SUPT16H or SSRP1 protein enhances Tat-mediated HIV-1 LTR (long terminal repeat) promoter activity. Second, HIV-1 Tat interacts with SUPT16H but not SSRP1 protein. However, both SUPT16H and SSRP1 are recruited to LTR promoter. Third, the presence of SUPT16H interferes with the association of Cyclin T1 (CCNT1), a subunit of P-TEFb, with the Tat-LTR axis. Removing inhibitory mechanisms to permit HIV-1 transcription is an initial and key regulatory step to reverse post-integrated latent HIV-1 proviruses for purging of reservoir cells. We therefore evaluated the role of FACT proteins in HIV-1 latency and reactivation. Depletion of SUPT16H or SSRP1 protein affects both HIV-1 transcriptional initiation and elongation and spontaneously reverses latent HIV-1 in U1/HIV and J-LAT cells. Similar effects were observed with a primary CD4+ T cell model of HIV-1 latency. FACT proteins also interfere with HTLV-1 Tax-LTR-mediated transcription and viral latency, indicating that they may act as general transcriptional suppressors for retroviruses. We conclude that FACT proteins SUPT16H and SSRP1 play a key role in suppressing HIV-1 transcription and promoting viral latency, which may serve as promising gene targets for developing novel HIV-1 latency-reversing agents.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas de Unión al ADN/fisiología , VIH-1/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Virus Linfotrópico T Tipo 1 Humano/fisiología , Factores de Transcripción/fisiología , Factores de Elongación Transcripcional/fisiología , Latencia del Virus/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/virología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Ciclina T/fisiología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Células HEK293 , Duplicado del Terminal Largo de VIH , VIH-1/genética , Proteínas del Grupo de Alta Movilidad/antagonistas & inhibidores , Proteínas del Grupo de Alta Movilidad/genética , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Modelos Biológicos , Factor B de Elongación Transcripcional Positiva/fisiología , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Elongación Transcripcional/antagonistas & inhibidores , Factores de Elongación Transcripcional/genética , Latencia del Virus/genéticaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman's disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60's role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV+/EBV- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.
RESUMEN
Lentiviral vectors are gene delivery vehicles that integrate into the host genome of dividing and non-dividing mammalian cells facilitating long-term transgene expression. Lentiviral vector versatility is greatly increased by incorporating heterologous viral envelope proteins onto the vector particles instead of the native envelope, conferring on these pseudotyped vectors a modified tropism and host range specificity. We investigated the pseudotyping efficiency of HIV-1 based lentiviral vectors with alphaviral envelope proteins from the Chikungunya Virus (CHIKV-G) and Sindbis Virus (SINV-G). Following vector production optimisation, titres for the CHIKV-G pseudotype were comparable to the VSV-G pseudotype but those for the SINV-G pseudotype were significantly lower. High titre CHIKV-G pseudotyped vector efficiently transduced various human and mouse neural cell lines and normal human astrocytes (NHA) in vitro. Although transduction was broad, tropism for NHAs was observed. In vivo stereotaxic delivery in striatum, thalamus and hippocampus respectively in the adult rat brain revealed localised transduction restricted to striatal astrocytes and hippocampal dentate granule neurons. Transduction of different subtypes of granule neurons from precursor to post-mitotic stages of differentiation was evident in the sub-granular zone and dentate granule cell layer. No significant inflammatory response was observed, but comparable to that of VSV-G pseudotyped lentiviral vectors. Robust long-term expression followed for three months post-transduction along with absence of neuroinflammation, coupled to the selective and unique neuron/glial tropism indicates that these vectors could be useful for modelling and gene therapy studies in the CNS.
Asunto(s)
Astrocitos/fisiología , Virus Chikungunya/genética , Vectores Genéticos/genética , Lentivirus/genética , Neuronas/fisiología , Transducción Genética/métodos , Proteínas del Envoltorio Viral/genética , Línea Celular , Células HEK293 , HumanosRESUMEN
OBJECTIVE: We have previously described the generation of coxsackievirus and adenovirus receptor (α CAR)-targeted vector, and shown that intramuscular delivery in mouse leg muscles resulted in specific retrograde transduction of lumbar-motor neurons (MNs). Here, we utilized the α CAR-targeted vector to investigate the in vivo neuroprotective effects of lentivirally expressed IGF-1 for inducing neuronal survival and ameliorating the neuropathology and behavioral phenotypes of the SOD1G93A mouse model of ALS. METHODS: We produced cell factories of IGF-1 expressing lentiviral vectors (LVs) bearing α CAR or Vesicular Stomatitis Virus glycoprotein (VSV-G) on their surface so as to compare neuroprotection from MN transduced versus muscle transduced cells. We performed intramuscular delivery of either α CAR IGF-1 or VSVG IGF-1 LVs into key muscles of SOD1G93A mice prior to disease onset at day 28. Motor performance, coordination and gait analysis were assessed weekly. RESULTS: We observed substantial therapeutic efficacy only with the α CAR IGF-1 LV pretreatment with up to 50% extension of survival compared to controls. α CAR IGF-1 LV-treated animals retained muscle tone and had better motor performance during their prolonged survival. Histological analysis of spinal cord samples at end-stage further confirmed that α CAR IGF-1 LV treatment delays disease onset by increasing MN survival compared with age-matched controls. Intrastriatal injection of α CAR eGFP LV in rats leads to transduction of neurons and glia locally and neurons in olfactory bulb distally. INTERPRETATION: Our data are indicative of the efficacy of the α CAR IGF-1 LV in this model and support its candidacy for early noninvasive neuroprotective therapy in ALS.
RESUMEN
IFI44 is an interferon-alfa inducible protein, and is associated with infection of several viruses. However, IFI44 elicits minimal antiviral effects on these viruses, and its exact role is still unknown. Here we show that IFI44 inhibits HIV-1 replication in vitro. Through depletion of endogenous IFI44 or overexpression of IFI44 we confirm that IFI44 suppresses HIV-1 LTR promoter activity and affects viral transcription. Furthermore, we find that IFI44 localizes to nuclei and binds to the HIV-1 LTR promoter in HIV-1 infected cells. Removing suppression of HIV-1 transcription benefits reactivation of HIV-1 proviruses for purging latent reservoirs. We demonstrate that depletion of endogenous IFI44 in J-LAT cells induces reactivation of latent HIV-1. Based on these results, we propose a model in which IFI44 is recruited to the HIV-1 LTR, which may suppress viral transcription and prevent reactivation of latent HIV-1. Our study suggests a previously unrecognized anti-HIV phenomenon for interferon-stimulated proteins.