RESUMEN
Primate lentiviruses have evolved sophisticated strategies to suppress the immune response of their host species. For example, HIV-2 and most simian immunodeficiency viruses (SIVs) use their accessory protein Nef to prevent T cell activation and antiviral gene expression by downmodulating the T cell receptor CD3. This Nef function was lost in HIV-1 and other vpu-encoding viruses suggesting that the acquisition of Vpu-mediated NF-κB inhibition reduced the selection pressure for inhibition of T cell activation by Nef. To obtain further insights into the modulation of NF-κB activity by primate lentiviral accessory factors, we analyzed 32 Vpr proteins from a large panel of divergent primate lentiviruses. We found that those of SIVcol and SIVolc infecting Colobinae monkeys showed the highest efficacy in suppressing NF-κB activation. Vpr-mediated inhibition of NF-κB resulted in decreased IFNß promoter activity and suppressed type I IFN induction in virally infected primary cells. Interestingly, SIVcol and SIVolc differ from all other primate lentiviruses investigated by the lack of both, a vpu gene and efficient Nef-mediated downmodulation of CD3. Thus, primate lentiviruses have evolved at least three alternative strategies to inhibit NF-κB-dependent immune activation. Functional analyses showed that the inhibitory activity of SIVolc and SIVcol Vprs is independent of DCAF1 and the induction of cell cycle arrest. While both Vprs target the IKK complex or a factor further downstream in the NF-κB signaling cascade, only SIVolc Vpr stabilizes IκBα and inhibits p65 phosphorylation. Notably, only de-novo synthesized but not virion-associated Vpr suppressed the activation of NF-κB, thus enabling NF-κB-dependent initiation of viral gene transcription during early stages of the replication cycle, while minimizing antiviral gene expression at later stages. Our findings highlight the key role of NF-κB in antiviral immunity and demonstrate that primate lentiviruses follow distinct evolutionary paths to modulate NF-κB-dependent expression of viral and antiviral genes.
Asunto(s)
Infecciones por VIH/inmunología , Evasión Inmune/inmunología , FN-kappa B/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Animales , Apoptosis/fisiología , Western Blotting , Línea Celular , Colobus , Citometría de Flujo , VIH/inmunología , Humanos , Activación de Linfocitos/inmunología , Reacción en Cadena de la Polimerasa , Virus de la Inmunodeficiencia de los Simios/inmunologíaRESUMEN
BACKGROUND: Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. RESULTS: In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. CONCLUSIONS: Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process.
Asunto(s)
VIH-1/fisiología , Microscopía Confocal/métodos , Virología/métodos , Desencapsidación Viral , Línea Celular , Humanos , ARN Viral/análisis , Coloración y Etiquetado/métodos , Uridina/análogos & derivados , Uridina/metabolismo , Proteínas Virales/análisisRESUMEN
Lentiviruses, the genus of retrovirus that includes HIV-1, rarely endogenize. Some lemurs uniquely possess an endogenous lentivirus called PSIV ("prosimian immunodeficiency virus"). Thus, lemurs provide the opportunity to study the activity of host defense factors, such as TRIM5α, in the setting of germ line invasion. We characterized the activities of TRIM5α proteins from two distant lemurs against exogenous retroviruses and a chimeric PSIV. TRIM5α from gray mouse lemur, which carries PSIV in its genome, exhibited the narrowest restriction activity. One allelic variant of gray mouse lemur TRIM5α restricted only N-tropic murine leukemia virus (N-MLV), while a second variant restricted N-MLV and, uniquely, B-tropic MLV (B-MLV); both variants poorly blocked PSIV. In contrast, TRIM5α from ring-tailed lemur, which does not contain PSIV in its genome, revealed one of the broadest antiviral activities reported to date against lentiviruses, including PSIV. Investigation into the antiviral specificity of ring-tailed lemur TRIM5α demonstrated a major contribution of a 32-amino-acid expansion in variable region 2 (v2) of the B30.2/SPRY domain to the breadth of restriction. Data on lemur TRIM5α and the prediction of ancestral simian sequences hint at an evolutionary scenario where antiretroviral specificity is prominently defined by the lineage-specific expansion of the variable loops of B30.2/SPRY.
Asunto(s)
Proteínas Portadoras/metabolismo , Lemur/inmunología , Retroviridae/inmunología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Análisis por Conglomerados , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Numerous in vitro studies attribute to human TRIM5α some modest anti-HIV-1 activity and human population studies suggest some differential effect of TRIM5α polymorphisms on disease progression. If the activity of TRIM5α were relevant in vivo, it could result in positive selection on the viral capsid. To address this issue, we identified 10 positively selected sites in HIV-1 capsid from multiple viral strains and generated 17 clade B viruses carrying a minor (i.e. low frequency) residue or an alanine at those positions. All recombinant viruses were susceptible to the modest effect of common human TRIM5α and allelic variants R136Q, and H419Y; H43Y and G249D TRIM5α were generally inactive. Increased sensitivity to TRIM5α was observed for some capsid variants, suggesting that minor residues are selected against in human populations. On the other hand, the modest potency of human TRIM5α does not translate in escape mutations in the viral capsid.
Asunto(s)
Adaptación Biológica , Proteínas Portadoras/inmunología , Proteína p24 del Núcleo del VIH/inmunología , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/inmunología , VIH-1/patogenicidad , Factores de Restricción Antivirales , Proteína p24 del Núcleo del VIH/genética , VIH-1/genética , Humanos , Selección Genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
PURPOSE OF REVIEW: This review highlights new roles of the large family of tripartite motif (TRIM) proteins in antiviral defense. RECENT FINDINGS: Recent research explores the participation of several TRIM family members in regulating the innate immune response. A large number of TRIM genes are upregulated upon treatment by interferon and are directly involved in signaling (TRIM5, 13, 16, 20, 21, 22, 23, 25, 27, 30, 32 and 38). Notably, TRIM5α has been identified as a 'pattern recognition receptor' triggering a cascade of signals upon viral recognition, and contributing to the establishment of the antiviral state. SUMMARY: The identification of new roles for TRIM5α and other family members contributes to an emerging paradigm of host antiretroviral factors as mediators of the innate immune response and of the antiviral state. This leads both to direct therapeutic applications, such as gene therapy, and to the possibility of immune modulation.
Asunto(s)
Proteínas Portadoras/inmunología , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , Inmunidad Innata , Receptores de Reconocimiento de Patrones/fisiología , Animales , Factores de Restricción Antivirales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Humanos , Receptores de Reconocimiento de Patrones/química , Receptores de Reconocimiento de Patrones/inmunología , Transducción de Señal , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína LigasasRESUMEN
SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.
Asunto(s)
Evolución Molecular , VIH-2/patogenicidad , Interacciones Huésped-Patógeno , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Sitios de Unión , Análisis por Conglomerados , VIH-1/inmunología , VIH-1/patogenicidad , VIH-2/inmunología , Humanos , Filogenia , Primates , Unión Proteica , Mapeo de Interacción de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5' but not 3' region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation.