RESUMO
Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered.
Assuntos
Infecções por HIV , Inibidores de Integrase de HIV , HIV-1 , Humanos , HIV-1/fisiologia , Replicação Viral/genética , DNA Viral , Mutação , Inibidores de Integrase de HIV/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Piridonas/farmacologia , Infecções por HIV/tratamento farmacológico , Farmacorresistência Viral/genéticaRESUMO
IMPORTANCE: Although HIV replication can be effectively inhibited by antiretroviral therapy, this does not result in a cure as the available drugs do not inactivate the integrated HIV-1 DNA in infected cells. Consequently, HIV-infected individuals need lifelong therapy to prevent viral rebound. Several preclinical studies indicate that CRISPR-Cas gene-editing systems can be used to achieve permanent inactivation of the viral DNA. It was previously shown that this inactivation was due to small inactivating mutations at the targeted sites in the HIV genome and to excision or inversion of the viral DNA fragment between two target sites. We, here, demonstrate that CRISPR-Cas treatment also causes large unintended deletions, which can include surrounding chromosomal sequences. As the loss of chromosomal sequences may cause oncogenic transformation of the cell, such unintended large deletions form a potential safety risk in clinical application of this antiviral application and possibly all CRISPR-Cas gene-editing approaches.
Assuntos
Sistemas CRISPR-Cas , DNA Viral , Edição de Genes , Infecções por HIV , HIV-1 , Provírus , Deleção de Sequência , Humanos , Sistemas CRISPR-Cas/genética , DNA Viral/genética , Edição de Genes/métodos , Edição de Genes/normas , Infecções por HIV/genética , Infecções por HIV/terapia , Infecções por HIV/virologia , HIV-1/genética , Provírus/genética , Deleção Cromossômica , Segurança do PacienteRESUMO
The CRISPR-Cas9 system has been used for genome editing of various organisms. We reported inhibition of the human immunodeficiency virus (HIV) in cell culture infections with a single guide RNA (gRNA) and subsequent viral escape, but complete inactivation of infectious HIV with certain combinations of two gRNAs. The new RNA-guided endonuclease system CRISPR-Cas12a (formerly Cpf1) may provide a more promising tool for genome engineering with increased activity and specificity. We compared Cas12a to the original Cas9 system for inactivation of the integrated HIV DNA genome. Superior antiviral activity is reported for Cas12a, which can achieve full HIV inactivation with only a single gRNA (called crRNA). We propose that the different architecture of Cas9 versus Cas12a endonuclease explains this effect. We also disclose that DNA cleavage by the Cas12a endonuclease and subsequent DNA repair causes mutations with a sequence profile that is distinct from that of Cas9. Both CRISPR systems can induce the typical small deletions around the site of DNA cleavage and subsequent repair, but Cas12a does not induce the pure DNA insertions that are routinely observed for Cas9. Although these typical signatures are apparent in many literature studies, this is the first report that documents these striking differences.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleases/metabolismo , HIV/genética , Linhagem Celular , DNA Viral/química , Edição de Genes , Genoma Viral , Células HEK293 , Humanos , Mutação , RNA/química , Linfócitos T/virologiaAssuntos
Farmacorresistência Viral , Infecções por HIV , HIV-1 , Compostos Heterocíclicos com 3 Anéis , Mutação , Oxazinas , Piperazinas , Piridonas , Compostos Heterocíclicos com 3 Anéis/farmacologia , HIV-1/genética , HIV-1/efeitos dos fármacos , Humanos , Piperazinas/farmacologia , Farmacorresistência Viral/genética , Oxazinas/farmacologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Transcrição Reversa , Inibidores de Integrase de HIV/farmacologiaRESUMO
Antiretroviral therapy (ART) can effectively suppress ongoing HIV replication and block disease progression, but the infection is never cured due to the persistence of a small pool of latently infected cells hosting integrated replication-competent HIV proviruses. However, the vast majority of HIV proviruses in ART-treated patients are replication-incompetent due to a variety of genetic defects. Most defective proviruses (around 90%) contain large internal deletions or are G-to-A hypermutated, resulting in destruction of most if not all viral open reading frames, which is consistent with the idea that cytotoxic T cells (CTLs) effectively remove cells that produce viral antigens. An intriguing subclass of defective proviruses (around 10%) that are consistently detected in such patients carry a small deletion or a point mutation in a relatively precise and well conserved region near the 5' end of the HIV genome, in the area that encodes the major splice donor (MSD) site and the packaging signal Ñ° in the viral RNA genome. Why this subclass of proviruses is defective has never been properly understood. We now propose a mechanistic scenario for how these MSD-Ñ° mutations can prevent viral protein expression. Based on ample results in literature, we argue that MSD inactivation triggers the activity of the 5'-polyadenylation site, resulting in the production of ultra-short non-protein-coding HIV transcripts.
Assuntos
Genoma Viral , HIV-1/genética , Mutação , Provírus/genética , Sítios de Splice de RNA , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , Humanos , Poliadenilação , Integração ViralRESUMO
Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5'ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and the trans-acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein.IMPORTANCE The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.
Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene tat/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Splicing de RNA , RNA Viral/genética , Produtos do Gene tat/genética , Células HEK293 , HIV-1/isolamento & purificação , Humanos , Replicação ViralRESUMO
In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity.
Assuntos
Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Replicação Viral/imunologia , Animais , Imuno-Histoquímica , Imunofenotipagem , Macaca mulatta , Reação em Cadeia da Polimerase , Vacinas AtenuadasRESUMO
Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This â¼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome.
Assuntos
Repetição Terminal Longa de HIV/genética , HIV-1/fisiologia , MicroRNAs/genética , RNA Viral/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologia , Proteínas Argonautas/metabolismo , Sequência de Bases , Regulação Viral da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Sequências Repetidas Invertidas , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , RNA Viral/metabolismo , Transcrição Gênica , Replicação ViralRESUMO
Many potent antiviral drugs have been developed against HIV-1, and their combined action is usually successful in achieving durable virus suppression in infected individuals. This success is based on two effects: additive or even synergistic virus inhibition and an increase in the genetic threshold for development of drug resistance. More recently, several genetic approaches have been developed to attack the HIV-1 genome in a gene therapy setting. We set out to test the combinatorial possibilities for a therapy based on the CRISPR-Cas9 and RNA interference (RNAi) mechanisms that attack the viral DNA and RNA, respectively. When two different sites in the HIV-1 genome were targeted, either with dual CRISPR-Cas9 antivirals or with a combination of CRISPR-Cas9 and RNAi antivirals, we observed additive inhibition, much like what was reported for antiviral drugs. However, when the same or overlapping viral sequence was attacked by the antivirals, rapid escape from a CRISPR-Cas9 antiviral, assisted by the error-prone nonhomologous end joining (NHEJ) DNA repair machinery, accelerated the development of cross-resistance to the other CRISPR-Cas9 or RNAi antiviral. Thus, genetic antiviral approaches can be combined, but overlap should be avoided.
Assuntos
Sistemas CRISPR-Cas , DNA Viral/antagonistas & inibidores , Farmacorresistência Viral/genética , Regulação Viral da Expressão Gênica , Genoma Viral , HIV-1/genética , RNA Viral/antagonistas & inibidores , Antivirais/química , Antivirais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteína 9 Associada à CRISPR , Linhagem Celular Transformada , DNA Viral/biossíntese , DNA Viral/genética , Endonucleases/genética , Endonucleases/metabolismo , Proteína do Núcleo p24 do HIV/antagonistas & inibidores , Proteína do Núcleo p24 do HIV/biossíntese , Proteína do Núcleo p24 do HIV/genética , HIV-1/metabolismo , Humanos , Terapia de Alvo Molecular , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Linfócitos T/virologia , Replicação ViralAssuntos
Infecções por HIV , Inibidores de Integrase de HIV , HIV-1 , Farmacorresistência Viral/genética , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/farmacologia , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Mutação , Oxazinas/farmacologia , Oxazinas/uso terapêutico , Piperazinas , Piridonas/farmacologia , Piridonas/uso terapêuticoRESUMO
Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications.
Assuntos
Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , HIV-1/fisiologia , Replicação Viral , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Proteína 9 Associada à CRISPR , Linhagem Celular , Endonucleases/genética , Endonucleases/metabolismo , Marcação de Genes , Genoma Viral , Humanos , Mutação , Ligação Proteica , Provírus/genética , Edição de RNA , RNA Guia de CinetoplastídeosRESUMO
The 5' leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication.
Assuntos
Genoma Viral/genética , HIV-1/genética , Motivos de Nucleotídeos/genética , RNA Viral/genética , Replicação Viral/genética , Pareamento de Bases , Sequência de Bases , Northern Blotting , Linhagem Celular Tumoral , Dimerização , Células HEK293 , HIV-1/química , HIV-1/metabolismo , Humanos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/patologia , Linfócitos T/virologia , Transcrição GênicaRESUMO
A recent study by Fennessey et al. (Retrovirology 12:49, 2015) described the optimization of a popular SIV clone by removal of four suboptimal point mutations. One of these mutations is present in a non-coding part of the viral genome and is probed in that study in more detail because of some fascinating properties. This primer binding site (PBS) mutation reverts rapidly to the wild-type sequence, which the authors interpret as indicating that this mutation exerts a profound fitness impact. The authors proposed the involvement of a cellular DNA repair mechanism in the reversion. Furthermore, it was suggested that premature termination of reverse transcription can explain why some of the viral progeny still contained the mutant sequence. However, we argue that all these special properties are a direct consequence of the unique nature of the viral PBS motif. The PBS binds the tRNA primer for reverse transcription and the viral progeny inherits either the sequence of the cellular tRNA or the PBS sequence of the viral RNA genome. The presence of a variant tRNA species explains the rapid appearance and disappearance of a variant PBS sequence.
Assuntos
Aptidão Genética , Genoma Viral , HIV-1/genética , Mutação Puntual , Sequências Reguladoras de Ácido Ribonucleico , Vírus da Imunodeficiência Símia/genética , Sequência de Bases , Sítios de Ligação , HIV-1/fisiologia , Humanos , RNA de Transferência de Lisina/genética , Transcrição Reversa , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral/genéticaRESUMO
BACKGROUND: Intracellular defense proteins, also referred to as restriction factors, are capable of interfering with different steps of the viral life cycle. Among these, we have shown that Tripartite motif 22 (TRIM22) suppresses basal as well as phorbol ester-induced HIV-1 long terminal repeat (LTR)-mediated transcription, independently of its E3 ubiquitin ligase activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to the U3 region and Tat interaction with the TAR region of the HIV-1 LTR. As basal HIV-1 transcription is driven by the transcription factor specificity protein 1 (Sp1), we have investigated whether TRIM22 could interfere with Sp1-driven transcriptional activation of the HIV-1 LTR. FINDINGS: 293T cells, devoid of endogenous TRIM22 expression, were transfected with a TRIM22-expressing plasmid together with reporter plasmids driven by the HIV-1 LTR promoter either containing or lacking Sp1 binding sites or with reporter plasmids driven by non-viral promoter sequences either containing or lacking the three Sp1 binding sites from the HIV-1 LTR. These reporter assays showed that TRIM22 efficiently inhibited Sp1-driven transcription. Knocking down TRIM22 expression in the CD4(+) SupT1 T cell line increased the replication of Sp1-dependent HIV-1 variants. TRIM22 did not interact with Sp1, but prevented binding of Sp1 to the HIV-1 promoter, as demonstrated in protein-DNA pull down and chromatin immunoprecipitation assays. CONCLUSION: TRIM22 acts as a suppressor of basal HIV-1 LTR-driven transcription by preventing Sp1 binding to the HIV-1 promoter.
Assuntos
HIV-1/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Sítios de Ligação , Linfócitos T CD4-Positivos/virologia , Proteínas de Ligação a DNA/metabolismo , Regulação Viral da Expressão Gênica , Genes Reporter , Células HEK293 , Repetição Terminal Longa de HIV , HIV-1/fisiologia , Humanos , Antígenos de Histocompatibilidade Menor , Proteínas Repressoras/deficiência , Deleção de Sequência , Fator de Transcrição Sp1/genética , Proteínas com Motivo Tripartido , Latência Viral , Replicação Viral/genéticaRESUMO
Human immunodeficiency virus type 1 (HIV-1) splicing has to be strictly controlled to ensure the balanced production of the unspliced and all differently spliced viral RNAs. Splicing at the major 59 splice site (59ss) that is used for the synthesis of all spliced RNAs is modulated by the local RNA structure and binding of regulatory SR proteins. Here, we demonstrate that the suboptimal sequence complementarity between this 59ss and U1 small nuclear RNA (snRNA) also contributes to prevent excessive splicing. Analysis of a large set of HIV-1 sequences revealed that all three regulatory features of the 59ss region (RNA structure, SR protein binding and sequence complementarity with U1 snRNA) are highly conserved amongst virus isolates, which supports their importance. Combined mutations that destabilize the local RNA structure, remove binding sites for inhibitory SR proteins and optimize the U1 snRNA complementarity resulted in almost complete splicing and accordingly reduced virus replication.
Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/genética , Sítios de Splice de RNA , Splicing de RNA , RNA Viral/genética , Sequência de Bases , Sequência Conservada , HIV-1/química , HIV-1/fisiologia , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Viral/química , Replicação ViralRESUMO
The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing regulatory (SR) proteins, in particular SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins.
Assuntos
HIV-1/fisiologia , Conformação de Ácido Nucleico , Sítios de Splice de RNA , Splicing de RNA , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Genes Reporter , HIV-1/genética , Humanos , Luciferases/análise , Luciferases/genéticaRESUMO
The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the in vivo SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.
Assuntos
Regiões 5' não Traduzidas/genética , HIV-1/genética , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico/genética , Sequência de Bases , Análise Mutacional de DNA , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Sequências Repetidas Invertidas/genética , Dados de Sequência Molecular , Técnica de Seleção de AptâmerosRESUMO
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.
RESUMO
BACKGROUND: Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV. RESULTS: Using a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIV-rtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable. Twelve rhesus macaques were infected with a peak plasma vRNA on average two log10 lower than in 6 macaques infected with unconditionally replication-competent SIVΔnef. Consistent with the attenuated phenotype of the viruses the majority of animals displayed low or undetectable levels of viraemia by 42-84 days after infection. Next, comparison of circulating T cells before and after chronic infection with parental SIVΔnef revealed a profound global polarisation toward CD28-CCR7- T-effector memory 2 (TEM2) cells within CD95+CD4+ and CD95+CD8+ populations. Critically, a similar effect was seen in the CD95+ CD4+ population and to somewhat lesser extent in the CD95+ CD8+ population of SIV-rtTAΔnef chronically infected macaques that were maintained on doxycycline, but was not seen in animals from which doxycycline had been withdrawn. The proportions of gut-homing T-central memory (TCM) and TEM defined by the expression of α4ß7 and CD95 and differential expression of CD28 were increased in CD4 and CD8 cells under replication competent conditions and gut-homing CD4 TCM were also significantly increased under non-permissive conditions. TEM2 polarisation was seen in the small intestines of animals under replication permissive conditions but the effect was less pronounced than in the circulation. Intracellular cytokine staining of circulating SIV-specific T cells for IL-2, IFN-γ, TNF-α and IL-17 showed that the extent of polyfunctionality in CD4 and CD8 T cells was associated with replication permissivity; however, signature patterns of cytokine combinations were not distinguishable between groups of macaques. CONCLUSION: Taken together our results show that the global T memory cell compartment is profoundly skewed towards a mature effector phenotype by attenuated SIV. Results with the replication-conditional mutant suggest that maintenance of this effect, that may be important in vaccine design, might require persistence of replicating virus.
Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Linfócitos T/imunologia , Animais , Anti-Infecciosos/farmacologia , Doxiciclina/farmacologia , Memória Imunológica/fisiologia , Intestino Delgado/virologia , Macaca mulatta , Fenótipo , RNA Viral/genética , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/fisiologia , Superinfecção/prevenção & controle , Regulação para Cima , Vacinas Atenuadas/imunologia , Viremia , Replicação ViralRESUMO
The mRNAs encoding the Rev and Env proteins of simian immunodeficiency virus (SIV) are unique because upstream translation start codons are present that may modulate the expression of these viral proteins. This is true for the regular mRNAs, but we also report novel mRNA splicing variants that encode up to five upstream AUG (uAUG) codons. Their influence on Rev and Env translation was measured by mutational inactivation in reporter constructs and in the SIVmac239 strain. An intricate regulatory mechanism was disclosed that allows the virus to express a balanced amount of these two proteins. This insight also allows the design of vector constructs that efficiently express these proteins.