RESUMO
Standard genotypic antiretroviral resistance testing, performed by bulk sequencing, does not readily detect variants that comprise <20% of the circulating HIV-1 RNA population. Nevertheless, it is valuable in selecting an antiretroviral regimen after antiretroviral failure. In patients with poor adherence, resistant variants may not reach this threshold. Therefore, deep sequencing would be potentially valuable for detecting minority resistant variants. We compared bulk sequencing and deep sequencing to detect HIV-1 drug resistance at the time of a second-line protease inhibitor (PI)-based antiretroviral regimen failure. Eligibility criteria were virologic failure (HIV-1 RNA load of >500 copies/ml) of a first-line nonnucleoside reverse transcriptase inhibitor-based regimen, with at least the M184V mutation (lamivudine resistance), and second-line failure of a lopinavir/ritonavir (LPV/r)-based regimen. An amplicon-sequencing approach on the Roche 454 system was used. Six patients with viral loads of >90,000 copies/ml and one patient with a viral load of 520 copies/ml were included. Mutations not detectable by bulk sequencing during first- and second-line failure were detected by deep sequencing during second-line failure. Low-frequency variants (>0.5% of the sequence population) harboring major protease inhibitor resistance mutations were found in 5 of 7 patients despite poor adherence to the LPV/r-based regimen. In patients with intermittent adherence to a boosted PI regimen, deep sequencing may detect minority PI-resistant variants, which likely represent early events in resistance selection. In patients with poor or intermittent adherence, there may be low evolutionary impetus for such variants to reach fixation, explaining the low prevalence of PI resistance.
Assuntos
Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores da Protease de HIV/administração & dosagem , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação de Sentido Incorreto , Adulto , Feminino , Inibidores da Protease de HIV/farmacologia , HIV-1/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Viral , Sensibilidade e Especificidade , Carga Viral , Adulto JovemRESUMO
Here we present new sequence data from HIV-1 subtype C-infected long-term survivors (LTS) from Karonga District, Malawi. Gag and env sequence data were produced from nine individuals each of whom has been HIV-1 positive for more than 20 years. We show that the three amino acid deletion in gag p17 previously described from these LTS is not real and was a result of an alignment error. We find that the use of dried blood spots for DNA-based studies is limited after storage for 20 years. We also show some unlikely amino acid changes in env C2-V3 in LTS over time and different patterns of genetic divergence among LTS. Although no clear association between mutations and survival could be shown, amino acid changes that are present in more than one LTS may, in the future, be shown to be important.
Assuntos
Genes env/genética , Antígenos HIV/genética , Sobreviventes de Longo Prazo ao HIV/estatística & dados numéricos , Soropositividade para HIV/genética , HIV-1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Adulto , Sequência de Aminoácidos , Feminino , Soropositividade para HIV/epidemiologia , HIV-1/isolamento & purificação , Humanos , Malaui/epidemiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Filogenia , Reação em Cadeia da Polimerase , Deleção de Sequência/genéticaRESUMO
The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here.
Assuntos
Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Genes Mitocondriais/genética , Filogenia , Poríferos/classificação , Poríferos/genética , RNA Ribossômico 28S/genética , Animais , Organismos Aquáticos/enzimologia , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Funções Verossimilhança , Dados de Sequência Molecular , Poríferos/enzimologia , Análise de Sequência de DNARESUMO
In this preliminary study we show that in 2008, 3 years after antiretroviral therapy was introduced into the Karonga District, Malawi, a greater than expected number of drug-naive individuals have been infected with HIV-1 subtype C virus harboring major and minor drug resistance mutations (DRMs). From a sample size of 40 reverse transcriptase (RT) consensus sequences from drug-naive individuals we found five showing NRTI and four showing NNRTI mutations with one individual showing both. From 29 protease consensus sequences, again from drug-naive individuals, we found evidence of minor DRMs in three. Additional major and minor DRMs were found in clonal sequences from a number of individuals that were not present in the original consensus sequences. This clearly illustrates the importance of sequencing multiple HIV-1 variants from individuals to fully assess drug resistance.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação de Sentido Incorreto , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/isolamento & purificação , Humanos , Malaui , Dados de Sequência Molecular , Filogenia , População Rural , Análise de Sequência de DNARESUMO
Binding to a chemokine receptor, either CCR5 or CXCR4, by the gp120 glycoprotein is an essential step in the pathway by which HIV enters host cells. Recently, CCR5 antagonists have been developed that obstruct binding of CCR5 by gp120, thus inhibiting host cell entry. Resistance to such CCR5 antagonists may emerge, however, through the selection of viral strains capable of utilizing CXCR4 receptors. This study explores the evolutionary context of emergence, and in many cases decline, of dominant CXCR4-usage (X4) during disease progression within a number of individuals. Of seven individuals exhibiting a switch to dominant CXCR4 usage, such dominance is transient in five of them with CCR5-usage (R5) re-emerging to dominate the viral population later in disease progression. Three individuals conform to documented X4 transience in that the re-emergence of R5 dominance is an outgrowth from the predominant R5 strain. However, in two individuals we observe a novel pathway for R5 re-emergence in that R5 strains emerge to dominate late in disease progression through continued evolution of the X4 population. This suggests that the molecular mechanism of such switches between R5 and X4-usage is strain specific and that no single mechanism is shared between individuals. These findings have implications for the understanding of the mechanisms of potential emergence of resistance to CCR5 antagonists through use of the CXCR4 receptor and support the importance to have an appropriately optimized background therapy for use with entry inhibitors and, as for all HAART, to monitor drug resistance in a comprehensive manner.
Assuntos
Evolução Molecular , HIV-1/patogenicidade , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Progressão da Doença , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Humanos , Filogenia , Receptores CCR5/genética , Receptores CCR5/metabolismo , Receptores CXCR4/genéticaRESUMO
The env gene of human immunodeficiency virus (HIV) is a functionally important gene responsible for the production of protein products (gp120 and gp41) involved in host cell recognition, binding, and entry. This occurs through a complex and, as yet, not fully understood process of protein-protein interaction and within and between protein functional communication. Exposure on the surface of active HIV virions means the gp120-gp41 complexes are subjected to intense immune system pressure and have, therefore, evolved mechanisms to avoid neutralization. Using protein-coding sequences representing all the HIV type-1 (HIV-1) group M subtypes, we have identified amino acids within the env gene whose evolution is inextricably linked over the entire HIV-1 group M epidemic. We identified 848 pairs of coevolving residues (involving 263 out of 764 amino acid sites), which represent 0.29% of all possible pairs. Of the coevolving pairs, 68% were significantly correlated by hydrophobicity, molecular weight, or both hydrophobicity and molecular weight. Subsequent grouping of coevolving pairs resulted in the identification of 290 groups of amino acid residues, with the size of these groups ranging from 2 to 10 amino acid residues. Many of these dependencies are correlated by function including CD4 binding, coreceptor binding, glycosylation, and protein-protein interaction. This analysis provides important information regarding the functional dependencies observed within all the HIV-1 group M subtypes and may assist in the identification of functional protein domains and therapeutic targets within the HIV-1 env gene.
Assuntos
Evolução Molecular , Genes env/genética , HIV-1/classificação , HIV-1/genética , Aminoácidos , Antígenos CD4/química , Redes Reguladoras de Genes , Glicosilação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Peso Molecular , Nucleotídeos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Currently, the identification of groups of amino acid residues that are important in the function, structure, or interaction of a protein can be both costly and prohibitively complex, involving vast numbers of mutagenesis experiments. Here, we present the application of a novel computational method, which identifies the presence of coevolution in a data set, thereby enabling the a priori identification of amino acid residues that play an important role in protein function. We have applied this method to the heat shock protein (Hsp) protein-folding system, studying the network between Hsp70, Hsp90, and Hop (heat shock-organizing protein). Our analysis has identified functional residues within the tetratricopeptide repeat (TPR) 1 and 2A domains in Hop, previously shown to be interacting with Hsp70 and Hsp90, respectively. Further, we have identified significant residues elsewhere in Hop within domains that have been recently proposed as being important for Hop interaction with Hsp70 and/or Hsp90. In addition, several amino acid sites present in groups of coevolution were identified as 3-dimensionally or linearly proximal to functionally important sites or domains. Based on our results, we also investigate a further functional domain within Hop, between TPR1 and TPR2A, which we suggest as being functionally important in the interaction of Hop with both Hsp70 and Hsp90 whether directly or otherwise. Our method has identified all the previously characterized functionally important regions in this system, thereby indicating the power of this method in the a priori identification of important regions for site-directed mutagenesis studies.
Assuntos
Biologia Computacional/métodos , Evolução Molecular , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico/genética , Aminoácidos/química , Aminoácidos/genética , Animais , Simulação por Computador , Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico/química , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Protein evolution depends on intramolecular coevolutionary networks whose complexity is proportional to the underlying functional and structural interactions among sites. Here we present a novel approach that vastly improves the sensitivity of previous methods for detecting coevolution through a weighted comparison of divergence between amino acid sites. The analysis of the HIV-1 Gag protein detected convergent adaptive coevolutionary events responsible for the selective variability emerging between subtypes. Coevolution analysis and functional data for heat-shock proteins, Hsp90 and GroEL, highlight that almost all detected coevolving sites are functionally or structurally important. The results support previous suggestions pinpointing the complex interdomain functional interactions within these proteins and we propose new amino acid sites as important for interdomain functional communication. Three-dimensional information sheds light on the functional and structural constraints governing the coevolution between sites. Our covariation analyses propose two types of coevolving sites in agreement with previous reports: pairs of sites spatially proximal, where compensatory mutations could maintain the local structure stability, and clusters of distant sites located in functional domains, suggesting a functional dependency between them. All sites detected under adaptive evolution in these proteins belong to coevolution groups, further underlining the importance of testing for coevolution in selective constraints analyses.
Assuntos
Evolução Molecular , Seleção Genética , Sequência de Aminoácidos , Chaperonina 60/genética , Produtos do Gene gag/genética , HIV-1/genética , Proteínas de Choque Térmico HSP90/genética , Modelos Genéticos , Dados de Sequência Molecular , Filogenia , Proteínas/químicaRESUMO
In a population-based study in northern Malawi we investigated HIV-1 subtype C gag and env gene sequences associated with long-term survival. DNA samples were available from 31 individuals surviving between population surveys carried out in the 1980s and 1990s. Most survivors with paired sequences dating from the 1980s and the 1990s had a three codon deletion in the gag p17 region of the sequence retrieved from the sample collected in the 1990s that was not present in the sequence from the same individual dating from the 1980s. This deletion was also not present in any other 1980s sequences from Malawi, but was common in samples collected in Malawi in the 1990s. The deletion is equivalent to the loss of three amino acids in the D helix region of the gag protein, and may be associated with longer survival and onward transmission.
Assuntos
Códon , Produtos do Gene gag/genética , Antígenos HIV/genética , Sobreviventes de Longo Prazo ao HIV , Deleção de Sequência , Proteínas Virais/genética , Sequência de Aminoácidos , Produtos do Gene gag/química , Antígenos HIV/química , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Virais/química , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
Recent studies have demonstrated the emergence of human immunodeficiency virus type 1 (HIV-1) subtypes with various levels of fitness. Using heterogeneous maximum-likelihood models of adaptive evolution implemented in the PAML software package, with env sequences representing each HIV-1 group M subtype, we examined the various intersubtype selective pressures operating across the env gene. We found heterogeneity of evolutionary mechanisms between the different subtypes with a category of amino acid sites observed that had undergone positive selection for subtypes C, F1, and G, while these sites had undergone purifying selection in all other subtypes. Also, amino acid sites within subtypes A and K that had undergone purifying selection were observed, while these sites had undergone positive selection in all other subtypes. The presence of such sites indicates heterogeneity of selective pressures within HIV-1 group M subtype evolution that may account for the various levels of fitness of the subtypes.
Assuntos
Evolução Molecular , Produtos do Gene env/química , Produtos do Gene env/genética , HIV-1/classificação , Seleção Genética , Sequência de Aminoácidos , Genes env , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Humanos , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Human immunodeficiency virus type 1 (HIV-1) subtype C is responsible for more than 55% of HIV-1 infections worldwide. When this subtype first emerged is unknown. We have analyzed all available gag (p17 and p24) and env (C2-V3) subtype C sequences with known sampling dates, which ranged from 1983 to 2000. The majority of these sequences come from the Karonga District in Malawi and include some of the earliest known subtype C sequences. Linear regression analyses of sequence divergence estimates (with four different approaches) were plotted against sample year to estimate the year in which there was zero divergence from the reconstructed ancestral sequence. Here we suggest that the most recent common ancestor of subtype C appeared in the mid- to late 1960s. Sensitivity analyses, by which possible biases due to oversampling from one district were explored, gave very similar estimates.