RESUMEN
Ghrelin is a peptide hormone with various important physiological functions. The unique feature of ghrelin is its serine 3 acyl-modification, which is essential for ghrelin activity. The major form of ghrelin is modified with n-octanoic acid (C8:0) by ghrelin O-acyltransferase. Various acyl modifications have been reported in different species. However, the underlying mechanism by which ghrelin is modified with various fatty acids remains to be elucidated. Herein, we report the purification of bovine, porcine, and equine ghrelins. The major active form of bovine ghrelin was a 27-amino acid peptide with an n-octanoyl (C8:0) modification at Ser3. The major active form of porcine and equine ghrelin was a 28-amino acid peptide. However, porcine ghrelin was modified with n-octanol (C8:0), whereas equine ghrelin was modified with n-butanol (C4:0) at Ser3. This study indicates the existence of structural divergence in ghrelin and suggests that it is necessary to measure the minor and major forms of ghrelin to fully understand its physiology.
Asunto(s)
Ghrelina , Animales , Ghrelina/metabolismo , Ghrelina/química , Caballos , Bovinos , Porcinos , Secuencia de Aminoácidos , Acilación , Caprilatos/metabolismoRESUMEN
HIV-1 drug resistance genotypic tests have primarily been performed by Sanger sequencing of gene segments encoding different drug target proteins. Since the number of targets has increased with the addition of a new class of antiretroviral drugs, a simple high-throughput system for assessing nucleotide sequences throughout the HIV-1 genome is required. Here, we developed a new solution using nanopore sequencing of viral pangenomes amplified by PCR. Benchmark tests using HIV-1 molecular clones demonstrated an accuracy of up to 99.9%. In addition, validation tests of our protocol in 106 clinical samples demonstrated high concordance of drug resistance and tropism genotypes (92.5% and 98.1%, respectively) between the nanopore sequencing-based results and archived clinical determinations made based on Sanger sequencing data. These results suggest that our new approach will be a powerful solution for the comprehensive survey of HIV-1 drug resistance mutations in clinical settings.
Asunto(s)
Farmacorresistencia Viral , Genoma Viral , Infecciones por VIH , VIH-1 , Mutación , Secuenciación de Nanoporos , VIH-1/genética , VIH-1/efectos de los fármacos , Farmacorresistencia Viral/genética , Secuenciación de Nanoporos/métodos , Humanos , Infecciones por VIH/virología , Infecciones por VIH/tratamiento farmacológico , Genotipo , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
VLPCOV-01 is a lipid nanoparticle-encapsulated self-amplifying RNA (saRNA) vaccine that expresses a membrane-anchored receptor-binding domain (RBD) derived from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. A phase 1 study of VLPCOV-01 is conducted (jRCT2051210164). Participants who completed two doses of the BNT162b2 mRNA vaccine previously are randomized to receive one intramuscular vaccination of 0.3, 1.0, or 3.0 µg VLPCOV-01, 30 µg BNT162b2, or placebo. No serious adverse events have been reported. VLPCOV-01 induces robust immunoglobulin G (IgG) titers against the RBD protein that are maintained up to 26 weeks in non-elderly participants, with geometric means ranging from 5,037 (95% confidence interval [CI] 1,272-19,940) at 0.3 µg to 12,873 (95% CI 937-17,686) at 3 µg compared with 3,166 (95% CI 1,619-6,191) with 30 µg BNT162b2. Neutralizing antibody titers against all variants of SARS-CoV-2 tested are induced. VLPCOV-01 is immunogenic following low-dose administration. These findings support the potential for saRNA as a vaccine platform.
Asunto(s)
COVID-19 , Vacunas , Humanos , Persona de Mediana Edad , Vacunas contra la COVID-19/efectos adversos , Vacuna BNT162 , SARS-CoV-2/genética , ARN , COVID-19/prevención & control , Vacunas de ARNmRESUMEN
Several vaccines have been widely used to counteract the global pandemic caused by SARS-CoV-2. However, due to the rapid emergence of SARS-CoV-2 variants of concern (VOCs), further development of vaccines that confer broad and longer-lasting protection against emerging VOCs are needed. Here, we report the immunological characteristics of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 Spike (S) receptor binding domain (RBD), which is membrane-anchored by fusing with an N-terminal signal sequence and a C-terminal transmembrane domain (RBD-TM). Immunization with saRNA RBD-TM delivered in lipid nanoparticles (LNP) efficiently induces T-cell and B-cell responses in non-human primates (NHPs). In addition, immunized hamsters and NHPs are protected against SARS-CoV-2 challenge. Importantly, RBD-specific antibodies against VOCs are maintained for at least 12 months in NHPs. These findings suggest that this saRNA platform expressing RBD-TM will be a useful vaccine candidate inducing durable immunity against emerging SARS-CoV-2 strains.
Asunto(s)
COVID-19 , Vacunas , Animales , Cricetinae , Humanos , SARS-CoV-2/genética , COVID-19/prevención & control , Motivo de Reconocimiento de ARN , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
The number of genetic variations in the SARS-CoV-2 genome has been increasing primarily due to continuous viral mutations. Here, we report that the human APOBEC3A (A3A) cytidine deaminase plays a critical role in the induction of C-to-U substitutions in the SARS-CoV-2 genome. Bioinformatic analysis of the chronological genetic changes in a sequence database indicated that the largest UC-to-UU mutation signature, consistent with APOBEC-recognized nucleotide motifs, was predominant in single-stranded RNA regions of the viral genome. In SARS-CoV-2-infected cells, exogenous expression of A3A but not expression of other APOBEC proteins induced UC-to-UU mutations in viral RNA (vRNA). Additionally, the mutated C bases were often located at the tips in bulge or loop regions in the vRNA secondary structure. Interestingly, A3A mRNA expression was drastically increased by interferons (IFNs) and tumour necrosis factor-α (TNF-α) in epithelial cells derived from the respiratory system, a site of efficient SARS-CoV-2 replication. Moreover, the UC-to-UU mutation rate was increased in SARS-CoV-2 produced from lung epithelial cells treated with IFN-ß and TNF-α, but not from CRISPR/Cas9-based A3A knockout cells. Collectively, these findings demonstrate that A3A is a primary host factor that drives mutations in the SARS-CoV-2 RNA genome via RNA editing.
Asunto(s)
Citidina Desaminasa , Mutación , SARS-CoV-2 , Humanos , COVID-19/metabolismo , COVID-19/virología , Citidina Desaminasa/metabolismo , Genoma Viral , ARN Viral/genética , SARS-CoV-2/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Macaque-tropic HIV-1 (HIV-1mt) variants have been developed to establish preferable primate models that are advantageous in understanding HIV-1 infection pathogenesis and in assessing the preclinical efficacy of novel prevention/treatment strategies. We previously reported that a CXCR4-tropic HIV-1mt, MN4Rh-3, efficiently replicates in peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques homozygous for TRIMCyp (CMsTC). However, the CMsTC challenged with MN4Rh-3 displayed low viral loads during the acute infection phase and subsequently exhibited short-term viremia. These virological phenotypes in vivo differed from those observed in most HIV-1-infected people. Therefore, further development of the HIV-1mt variant was needed. In this study, we first reconstructed the MN4Rh-3 clone to produce a CCR5-tropic HIV-1mt, AS38. In addition, serial in vivo passages allowed us to produce a highly adapted AS38-derived virus that exhibits high viral loads (up to approximately 106 copies ml-1) during the acute infection phase and prolonged periods of persistent viremia (lasting approximately 16 weeks postinfection) upon infection of CMsTC. Whole-genome sequencing of the viral genomes demonstrated that the emergence of a unique 15-nt deletion within the vif gene was associated with in vivo adaptation. The deletion resulted in a significant increase in Vpr protein expression but did not affect Vif-mediated antagonism of antiretroviral APOBEC3s, suggesting that Vpr is important for HIV-1mt adaptation to CMsTC. In summary, we developed a novel CCR5-tropic HIV-1mt that can induce high peak viral loads and long-term viremia and exhibits increased Vpr expression in CMsTC.
Asunto(s)
Productos del Gen vpr , Infecciones por VIH , Seropositividad para VIH , VIH-1 , Virus de la Inmunodeficiencia de los Simios , Animales , VIH-1/genética , Leucocitos Mononucleares , Macaca fascicularis , Virus de la Inmunodeficiencia de los Simios/genética , Viremia , Replicación ViralRESUMEN
Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity. IMPORTANCE In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sustitución de Aminoácidos , Sueros Inmunes , Aminoácidos/genética , Nucleótidos , MutaciónRESUMEN
High genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1. IMPORTANCE HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases. In this study, we developed a new analytical system for HIV-1 sequence data obtained by nanopore sequencing. The error rate of this method was reduced to ~0.06%. We applied this system for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection, which were extracted from 373 cases of HIV patients based on our retrospective analysis of HIV-1 drug resistance genotyping test results. We found that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Our protocol will be useful for epidemiological surveillance to examine HIV-1 transmission as well as for genotypic tests of HIV-1 drug resistance in clinical settings.
Asunto(s)
Infecciones por VIH , VIH-1 , Secuenciación de Nanoporos , Genoma Viral , Infecciones por VIH/diagnóstico , Infecciones por VIH/epidemiología , VIH-1/genética , Humanos , Filogenia , Recombinación Genética , Estudios Retrospectivos , Análisis de Secuencia de ADNRESUMEN
There were five epidemic waves of coronavirus disease 2019 in Japan between 2020 and 2021. It remains unclear how the domestic waves arose and abated. To better understand this, we analyzed the pangenomic sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and characterized the molecular epidemiological features of the five epidemic waves in Japan. In this study, we performed deep sequencing to determine the pangenomic SARS-CoV-2 sequences of 1,286 samples collected in two cities far from each other, Tokyo Metropolis and Nagoya. Then, the spatiotemporal genetic changes of the obtained sequences were compared with the sequences available in the Global Initiative on Sharing All Influenza Data (GISAID) database. A total of 873 genotypes carrying different sets of mutations were identified in the five epidemic waves. Phylogenetic analysis demonstrated that sharp displacements of lineages and genotypes occurred between consecutive waves over the 2 years. In addition, a wide variety of genotypes were observed in the early half of each wave, whereas a few genotypes were detected across Japan during an entire wave. Phylogenetically, putative descendant genotypes observed late in each wave displayed regional clustering and evolution in Japan. The genetic diversity of SARS-CoV-2 displayed uneven dynamics during each epidemic wave in Japan. Our findings provide an important molecular epidemiological basis to aid in controlling future SARS-CoV-2 epidemics.
RESUMEN
ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.
Asunto(s)
COVID-19 , Glicoproteínas , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Proteínas Virales , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Glicoproteínas/metabolismo , Humanos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: In vitro selection experiments identified viruses resistant to integrase strand transfer inhibitors (INSTIs) carrying mutations in the G-tract (six guanosines) of the 3'-polypurine tract (3'-PPT). A clinical study also reported that mutations in the 3'-PPT were observed in a patient receiving dolutegravir monotherapy. However, recombinant viruses with the 3'-PPT mutations that were found in the clinical study were recently shown to be susceptible to INSTIs. OBJECTIVES: To identify the specific mutation(s) in the G-tract of the 3'-PPT for acquiring INSTI resistance, we constructed infectious clones bearing single or multiple mutations and systematically characterized the susceptibility of these clones to both first- and second-generation INSTIs. METHODS: The infectious clones were tested for their infectivity and susceptibility to INSTIs in a single-cycle assay using TZM-bl cells. RESULTS: A single mutation of thymidine (T) at the fifth position (GGG GTG) in the G-tract of the 3'-PPT had no effect on INSTI resistance. A double mutation, cytidine (C) or 'T' at the second position and 'T' at the fifth position (GCG GTG and GTG GTG), increased resistance to INSTIs, with the appearance of a plateau in the maximal percentage inhibition (MPI) of the dose-response curves, consistent with a non-competitive mechanism of inhibition. CONCLUSIONS: Mutations at the second and fifth positions in the G-tract of the 3'-PPT may result in complex resistance mechanism(s), rather than simply affecting INSTI binding at the IN active site.
Asunto(s)
Infecciones por VIH , Inhibidores de Integrasa VIH , Integrasa de VIH , VIH-1 , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/farmacología , Inhibidores de Integrasa VIH/uso terapéutico , VIH-1/genética , Humanos , MutaciónRESUMEN
Viral proteins interact with different sets of host cell components throughout the viral life cycle and are known to localize to the intracellular membraneless organelles (MLOs) of the host cell, where formation/dissolution is regulated by phase separation of intrinsically disordered proteins and regions (IDPs/IDRs). Viral proteins are rich in IDRs, implying that viruses utilize IDRs to regulate phase separation of the host cell organelles and augment replication by commandeering the functions of the organelles and/or sneaking into the organelles to evade the host immune response. This review aims to integrate current knowledge of the structural properties and intracellular localizations of viral IDPs to understand viral strategies in the host cell. First, the properties of viral IDRs are reviewed and similarities and differences with those of eukaryotes are described. The higher IDR content in viruses with smaller genomes suggests that IDRs are essential characteristics of viral proteins. Then, the interactions of the IDRs of flaviviruses with the MLOs of the host cell are investigated with emphasis on the viral proteins localized in the nucleoli and stress granules. Finally, the possible roles of viral IDRs in regulation of the phase separation of organelles and future possibilities for antiviral drug development are discussed.
Asunto(s)
Infecciones por Flavivirus/virología , Flavivirus/fisiología , Interacciones Huésped-Patógeno , Animales , Flavivirus/genética , Infecciones por Flavivirus/fisiopatología , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Orgánulos/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The causative agent of the COVID-19 pandemic, SARS-CoV-2, is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the ACE2 receptor and is cleaved by TMPRSS2. However, whether S mutations affect SARS-CoV-2 cell entry remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than that of SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural and binding analyses. Nevertheless, the D614G mutation does not affect neutralization by antisera against prototypic viruses. Taken together, we conclude that the D614G mutation increases cell entry by acquiring higher affinity to ACE2 while maintaining neutralization susceptibility. Based on these findings, further worldwide surveillance is required to understand SARS-CoV-2 transmissibility among humans.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/prevención & control , Mutación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Internalización del Virus , Unión Competitiva , COVID-19/epidemiología , COVID-19/virología , Humanos , Modelos Moleculares , Pandemias , Unión Proteica , Dominios Proteicos , Receptores Virales/metabolismo , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
In HIV-1-infected patients, antiretroviral therapy (ART) is a key factor that may impact commensal microbiota and cause the emergence of side effects. However, it is not fully understood how long-term ART regimens have diverse impacts on the microbial compositions over time. Here, we performed 16S ribosomal RNA gene sequencing of the fecal and salivary microbiomes in patients under different long-term ART. We found that ART, especially conventional nucleotide/nucleoside reverse transcriptase inhibitor (NRTI)-based ART, has remarkable impacts on fecal microbial diversity: decreased α-diversity and increased ß-diversity over time. In contrast, dynamic diversity changes in the salivary microbiome were not observed. Comparative analysis of bacterial genus compositions showed a propensity for Prevotella-enriched and Bacteroides-poor gut microbiotas in patients with ART over time. In addition, we observed a gradual reduction in Bacteroides but drastic increases in Succinivibrio and/or Megasphaera under conventional ART. These results suggest that ART, especially NRTI-based ART, has more suppressive impacts on microbiota composition and diversity in the gut than in the mouth, which potentially causes intestinal dysbiosis in patients. Therefore, NRTI-sparing ART, especially integrase strand transfer inhibitor (INSTI)- and/or non-nucleotide reverse transcriptase inhibitor (NNRTI)-containing regimens, might alleviate the burden of intestinal dysbiosis in HIV-1-infected patients under long-term ART.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Microbioma Gastrointestinal/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/microbiología , Boca/microbiología , Adulto , Disbiosis/tratamiento farmacológico , Disbiosis/microbiología , Disbiosis/virología , Femenino , Infecciones por VIH/virología , Seropositividad para VIH/tratamiento farmacológico , Seropositividad para VIH/virología , VIH-1/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
Binding of HIV-1 capsid (CA) to cleavage and polyadenylation specificity factor 6 (CPSF6) is hypothesized to provide a significant fitness advantage to in vivo viral replication, explaining why CA-CPSF6 interactions are strictly conserved in primate lentiviruses. We recently identified a Q4R mutation in CA after propagation of an interferon (IFN)-ß-hypersensitive CA mutant, RGDA/Q112D (H87R, A88G, P90D, P93A and Q112D) virus, in IFN-ß-treated cells. The Q4R substitution conferred significant IFN-ß resistance to the RGDA/Q112D virus by affecting several properties of the virus, including the sensitivity to myxovirus resistance protein B (MxB), the kinetics of reverse transcription, and the initiation of uncoating. Notably, the Q4R substitution restored the CPSF6 interaction of the RGDA/Q112D virus. To better understand how the Q4R substitution modulated the CA-CPSF6 interaction, we generated a series of CA mutants harboring substitutions at the 4th and 112th residues. In contrast to the effect in the RGDA/Q112D background, the Q4R substitution diminished CA-CPSF6 interaction in an otherwise wild-type virus. Our genetic and structural analyses revealed that while either the Q4R or Q112D substitution impaired CA-CPSF6 interaction, the combination of these substitutions restored this interaction. These results suggest that the 4th and 112th residues in HIV-1 CA cooperatively modulate CA-CPSF6 interactions, further highlighting the tremendous levels of plasticity in primate lentivirus CA, which is one of the barriers to antiretroviral therapy in HIV-1-infected individuals.
Asunto(s)
Proteínas de la Cápside/genética , Cápside/química , VIH-1/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Proteínas de la Cápside/química , Línea Celular , Humanos , Mutación , Transcripción Reversa , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/químicaRESUMEN
Type I interferons (IFNs), including alpha IFN (IFN-α) and IFN-ß, potently suppress HIV-1 replication by upregulating IFN-stimulated genes (ISGs). The viral capsid protein (CA) partly determines the sensitivity of HIV-1 to IFNs. However, it remains to be determined whether CA-related functions, including utilization of known host factors, reverse transcription, and uncoating, affect the sensitivity of HIV-1 to IFN-mediated restriction. Recently, we identified an HIV-1 CA variant that is unusually sensitive to IFNs. This variant, called the RGDA/Q112D virus, contains multiple mutations in CA: H87R, A88G, P90D, P93A, and Q112D. To investigate how an IFN-hypersensitive virus can evolve to overcome IFN-ß-mediated blocks targeting the viral capsid, we adapted the RGDA/Q112D virus in IFN-ß-treated cells. We successfully isolated IFN-ß-resistant viruses which contained either a single Q4R substitution or the double amino acid change G94D/G116R. These two IFN-ß resistance mutations variably changed the sensitivity of CA binding to human myxovirus resistance B (MxB), cleavage and polyadenylation specificity factor 6 (CPSF6), and cyclophilin A (CypA), indicating that the observed loss of sensitivity was not due to interactions with these known host CA-interacting factors. In contrast, the two mutations apparently functioned through distinct mechanisms. The Q4R mutation dramatically accelerated the kinetics of reverse transcription and initiation of uncoating of the RGDA/Q112D virus in the presence or absence of IFN-ß, whereas the G94D/G116R mutations affected reverse transcription only in the presence of IFN-ß, most consistent with a mechanism of the disruption of binding to an unknown IFN-ß-regulated host factor. These results suggest that HIV-1 can exploit multiple, known host factor-independent pathways to avoid IFN-ß-mediated restriction by altering capsid sequences and subsequent biological properties.IMPORTANCE HIV-1 infection causes robust innate immune activation in virus-infected patients. This immune activation is characterized by elevated levels of type I interferons (IFNs), which can block HIV-1 replication. Recent studies suggest that the viral capsid protein (CA) is a determinant for the sensitivity of HIV-1 to IFN-mediated restriction. Specifically, it was reported that the loss of CA interactions with CPSF6 or CypA leads to higher IFN sensitivity. However, the molecular mechanism of CA adaptation to IFN sensitivity is largely unknown. Here, we experimentally evolved an IFN-ß-hypersensitive CA mutant which showed decreased binding to CPSF6 and CypA in IFN-ß-treated cells. The CA mutations that emerged from this adaptation indeed conferred IFN-ß resistance. Our genetic assays suggest a limited contribution of known host factors to IFN-ß resistance. Strikingly, one of these mutations accelerated the kinetics of reverse transcription and uncoating. Our findings suggest that HIV-1 selected multiple, known host factor-independent pathways to avoid IFN-ß-mediated restriction.
Asunto(s)
Proteínas de la Cápside/genética , Cápside/efectos de los fármacos , Cápside/metabolismo , VIH-1/efectos de los fármacos , VIH-1/genética , Interferón beta/metabolismo , Interferón beta/farmacología , Ciclofilina A , Células HEK293 , Infecciones por VIH/virología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Mutación , Proteínas de Resistencia a Mixovirus , Transcripción Reversa , Células THP-1 , Replicación Viral/efectos de los fármacos , Factores de Escisión y Poliadenilación de ARNmRESUMEN
The HIV-1 capsid (CA) utilizes CPSF6 for nuclear entry and integration site targeting. Previous studies demonstrated that the HIV-1 CA C-terminal domain (CTD) contains a highly conserved K182 residue involved in interaction with CPSF6. In contrast, certain HIV-2 strains possess a substitution at this residue (K182R). To assess whether CA-CPSF6 interaction via the CA CTD is conserved among primate lentiviruses, we examined resistance of several HIV-1- and HIV-2-lineage viruses to a truncated form of CPSF6, CPSF6-358. The results demonstrated that viruses belonging to the HIV-2-lineage maintain interaction with CPSF6 regardless of the presence of the K182R substitution, in contrast to the case with HIV-1-lineage viruses. Our structure-guided mutagenesis indicated that the differential requirement for CA-CPSF6 interaction is regulated in part by residues near the 182nd amino acid of CA. These results demonstrate a previously unrecognized distinction between HIV-1 and HIV-2, which may reflect differences in their evolutionary histories.
Asunto(s)
Cápside/metabolismo , VIH-1/genética , VIH-2/genética , Factores de Escisión y Poliadenilación de ARNm/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Cápside/ultraestructura , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , VIH-2/crecimiento & desarrollo , VIH-2/metabolismo , Interacciones Huésped-Patógeno , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Paired-end deep sequencing is a powerful tool to investigate integration sites of the HIV-1 genome in infected cells. Integration sites of HIV-1 proviral DNA carrying intact LTR ends have been well documented. In contrast, integration sites of proviral DNA with aberrant ends, which emerge infrequently but can also induce replication-competent viruses, have not been extensively examined, in part, because of the lack of a suitable bioinformatics method for deep sequencing. Here, we report a novel bioinformatics protocol, named the VINSSRM, to search for integration sites of proviral DNA carrying intact and aberrant LTR ends using paired-end deep sequencing data. The protocol incorporates split-read mapping to assign viral and human genome parts within read sequences and overlapping paired-end read merging to construct long error-corrected sequences. The VINSSRM not only consistently detects integration sites similar to the conventional method but also provides information on additional integration sites, including those of proviral DNA with aberrant ends, which were mainly found in non-exonic regions of the human genome. Therefore, the VINSSRM may help us to understand HIV-1 integration, persistence of infected cells, and viral latency.
Asunto(s)
Genoma Viral , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Provirus/genética , Integración Viral/genética , Biología Computacional , ADN Viral/genética , Infecciones por VIH/virología , Humanos , Sensibilidad y EspecificidadRESUMEN
Antiretroviral therapy (ART) against HIV-1 infection offers the promise of controlling disease progression and prolonging the survival of HIV-1-infected patients. However, even the most potent ART regimens available today cannot cure HIV-1. Because patients will be exposed to ART for many years, physicians and researchers must anticipate the emergence of drug-resistant HIV-1, potential adverse effects of the current drugs, and need for future drug development. In this study, we screened a small-molecule compound library using cell-based anti-HIV-1 assays and discovered a series of novel anti-HIV-1 compounds, 4-oxoquinolines. These compounds exhibited potent anti-HIV-1 activity (EC50â¯<â¯0.1⯵M) with high selectivity indexes (CC50/EC50â¯>â¯2500) and favorable pharmacokinetic profiles in mice. Surprisingly, our novel compounds have a chemical backbone similar to the clinically used integrase (IN) strand transfer inhibitor (INSTI) elvitegravir, although they lack the crucial 3-carboxylate moiety needed for the common INSTI diketo motif. Indeed, the new 4-oxoquinoline derivatives have no detectable INSTI activity. In addition, various drug-resistant HIV-1 strains did not display cross resistance to these compounds. Interestingly, time-of-addition experiments indicated that the 4-oxoquinoline derivative remains its anti-HIV-1 activity even after the viral integration stage. Furthermore, the compounds significantly suppressed p24 antigen production in HIV-1 latently infected cells exposed with tumor necrosis factor alpha. These findings suggest that our 4-oxoquinoline derivatives with no 3-carboxylate moiety may become novel lead compounds in the development of anti-HIV-1 drugs.