ABSTRACT
Human APOBEC3H (A3H) is a cytidine deaminase that inhibits HIV-1 replication. To evade this restriction, the HIV-1 Vif protein binds A3H and mediates its proteasomal degradation. To date, little information on the Vif-A3H interface has been available. To decipher how both proteins interact, we first mapped the Vif-binding site on A3H by functionally testing a large set of A3H mutants in single-cycle infectivity and replication assays. Our data show that the two A3H α-helixes α3 and α4 represent the Vif-binding site of A3H. We next used viral adaptation and a set of Vif mutants to identify novel, reciprocal Vif variants that rescued viral infectivity in the presence of two Vif-resistant A3H mutants. These A3H-Vif interaction points were used to generate the first A3H-Vif structure model, which revealed that the A3H helixes α3 and α4 interact with the Vif ß-sheet (ß2-ß5). This model is in good agreement with previously reported Vif and A3H amino acids important for interaction. Based on the predicted A3H-Vif interface, we tested additional points of contact, which validated our model. Moreover, these experiments showed that the A3H and A3G binding sites on HIV-1 Vif are largely distinct, with both host proteins interacting with Vif ß-strand 2. Taken together, this virus-host interface model explains previously reported data and will help to identify novel drug targets to combat HIV-1 infection.IMPORTANCE HIV-1 needs to overcome several intracellular restriction factors in order to replicate efficiently. The human APOBEC3 locus encodes seven proteins, of which A3D, A3F, A3G, and A3H restrict HIV-1. HIV encodes the Vif protein, which binds to the APOBEC3 proteins and leads to their proteasomal degradation. No HIV-1 Vif-APOBEC3 costructure exists to date despite extensive research. We and others previously generated HIV-1 Vif costructure models with A3G and A3F by mapping specific contact points between both proteins. Here, we applied a similar approach to HIV-1 Vif and A3H and successfully generated a Vif-A3H interaction model. Importantly, we find that the HIV-1 Vif-A3H interface is distinct from the Vif-A3G and Vif-A3F interfaces, with a small Vif region being important for recognition of both A3G and A3H. Our Vif-A3H structure model informs on how both proteins interact and could guide toward approaches to block the Vif-A3H interface to target HIV replication.
Subject(s)
Aminohydrolases/chemistry , HIV-1/physiology , vif Gene Products, Human Immunodeficiency Virus/chemistry , Base Sequence , Binding Sites , HEK293 Cells , Humans , Molecular Docking Simulation , Protein Binding , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Sequence Analysis, DNA , Virus ReplicationABSTRACT
HIV-1, the cause of AIDS, is composed of four phylogenetic lineages, groups M, N, O, and P, each of which resulted from an independent cross-species transmission event of simian immunodeficiency viruses (SIVs) infecting African apes. Although groups M and N have been traced to geographically distinct chimpanzee communities in southern Cameroon, the reservoirs of groups O and P remain unknown. Here, we screened fecal samples from western lowland (n = 2,611), eastern lowland (n = 103), and mountain (n = 218) gorillas for gorilla SIV (SIVgor) antibodies and nucleic acids. Despite testing wild troops throughout southern Cameroon (n = 14), northern Gabon (n = 16), the Democratic Republic of Congo (n = 2), and Uganda (n = 1), SIVgor was identified at only four sites in southern Cameroon, with prevalences ranging from 0.8-22%. Amplification of partial and full-length SIVgor sequences revealed extensive genetic diversity, but all SIVgor strains were derived from a single lineage within the chimpanzee SIV (SIVcpz) radiation. Two fully sequenced gorilla viruses from southwestern Cameroon were very closely related to, and likely represent the source population of, HIV-1 group P. Most of the genome of a third SIVgor strain, from central Cameroon, was very closely related to HIV-1 group O, again pointing to gorillas as the immediate source. Functional analyses identified the cytidine deaminase APOBEC3G as a barrier for chimpanzee-to-gorilla, but not gorilla-to-human, virus transmission. These data indicate that HIV-1 group O, which spreads epidemically in west central Africa and is estimated to have infected around 100,000 people, originated by cross-species transmission from western lowland gorillas.
Subject(s)
Epidemics , Gorilla gorilla/virology , HIV-1/physiology , Simian Acquired Immunodeficiency Syndrome/epidemiology , Simian Acquired Immunodeficiency Syndrome/virology , Animals , Animals, Wild/virology , Antibodies, Viral/immunology , Biological Evolution , Cameroon/epidemiology , Cytidine Deaminase/metabolism , Feces/virology , Genetic Variation , Genome/genetics , Geography , Humans , Molecular Sequence Data , Phylogeny , Proteolysis , Sequence Analysis, DNA , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunologyABSTRACT
Cellular restriction factors, which render cells intrinsically resistant to viruses, potentially impose genetic barriers to cross-species transmission and emergence of viral pathogens in nature. One such factor is APOBEC3G. To overcome APOBEC3G-mediated restriction, many lentiviruses encode Vif, a protein that targets APOBEC3G for degradation. As with many restriction factor genes, primate APOBEC3G displays strong signatures of positive selection. This is interpreted as evidence that the primate APOBEC3G locus reflects a long-term evolutionary "arms-race" between retroviruses and their primate hosts. Here, we provide direct evidence that APOBEC3G has functioned as a barrier to cross-species transmission, selecting for viral resistance during emergence of the AIDS-causing pathogen SIVmac in captive colonies of Asian macaques in the 1970s. Specifically, we found that rhesus macaques have multiple, functionally distinct APOBEC3G alleles, and that emergence of SIVmac and simian AIDS required adaptation of the virus to evade APOBEC3G-mediated restriction. Our evidence includes the first comparative analysis of APOBEC3G polymorphism and function in both a reservoir and recipient host species (sooty mangabeys and rhesus macaques, respectively), and identification of adaptations unique to Vif proteins of the SIVmac lineage that specifically antagonize rhesus APOBEC3G alleles. By demonstrating that interspecies variation in a known restriction factor selected for viral counter-adaptations in the context of a documented case of cross-species transmission, our results lend strong support to the evolutionary "arms-race" hypothesis. Importantly, our study confirms that APOBEC3G divergence can be a critical determinant of interspecies transmission and emergence of primate lentiviruses, including viruses with the potential to infect and spread in human populations.
Subject(s)
Acquired Immunodeficiency Syndrome/enzymology , Cytidine Deaminase/metabolism , Polymorphism, Genetic , Simian Acquired Immunodeficiency Syndrome/enzymology , APOBEC-3G Deaminase , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/transmission , Animals , Cercocebus atys , Chlorocebus aethiops , Cytidine Deaminase/genetics , HeLa Cells , Humans , Macaca , Simian Acquired Immunodeficiency Syndrome/genetics , Simian Acquired Immunodeficiency Syndrome/transmission , Species SpecificityABSTRACT
BACKGROUND: Molecular latency allows HIV-1 to persist in resting memory CD4+ T-cells as transcriptionally silent provirus integrated into host chromosomal DNA. Multiple transcriptional regulatory mechanisms for HIV-1 latency have been described in the context of progressive epigenetic silencing and maintenance. However, our understanding of the determinants critical for the establishment of latency in newly infected cells is limited. RESULTS: In this study, we used a recently described, doubly fluorescent HIV-1 latency model to dissect the role of proviral integration sites and cellular activation state on direct non-productive infections at the single cell level. Proviral integration site mapping of infected Jurkat T-cells revealed that productively and non-productively infected cells are indistinguishable in terms of genomic landmarks, surrounding epigenetic landscapes, and proviral orientation relative to host genes. However, direct non-productive infections were inversely correlated with both cellular activation state and NFκB activity. Furthermore, modulating NFκB with either small molecules or by conditional overexpression of NFκB subunits was sufficient to alter the propensity of HIV-1 to directly enter a non-productive latent state in newly infected cells. Importantly, this modulatory effect was limited to a short time window post-infection. CONCLUSIONS: Taken together, our data suggest that cellular activation state and NFκB activity during the time of infection, but not the site of proviral integration, are important regulators of direct HIV-1 non-productive infections.
Subject(s)
HIV-1/immunology , HIV-1/physiology , NF-kappa B/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/virology , Virus Latency , Virus Replication , Humans , Jurkat Cells , Virus IntegrationABSTRACT
HIV-1 latency poses a major barrier to viral eradication. Canonically, latency is thought to arise from progressive epigenetic silencing of active infections. However, little is known about when and how long terminal repeat (LTR)-silent infections arise since the majority of the current latency models cannot differentiate between initial (LTR-silent) and secondary (progressive silencing) latency. In this study, we constructed and characterized a novel, double-labeled HIV-1 vector (Red-Green-HIV-1 [RGH]) that allows for detection of infected cells independently of LTR activity. Infection of Jurkat T cells and other cell lines with RGH suggests that the majority of integrated proviruses were LTR-silent early postinfection. Furthermore, the LTR-silent infections were transcriptionally competent, as the proviruses could be reactivated by a variety of T cell signaling agonists. Moreover, we used the double-labeled vector system to compare LTRs from seven different subtypes with respect to LTR silencing and reactivation. These experiments indicated that subtype D and F LTRs were more sensitive to silencing, whereas the subtype AE LTR was largely insensitive. Lastly, infection of activated human primary CD4(+) T cells yielded LTR-silent as well as productive infections. Taken together, our data, generated using the newly developed RGH vector as a sensitive tool to analyze HIV-1 latency on a single-cell level, show that the majority of HIV-1 infections are latent early postinfection.
Subject(s)
HIV-1/physiology , Virus Latency , Genes, Reporter , Humans , Jurkat Cells , Staining and Labeling/methods , T-Lymphocytes/virology , Virology/methodsABSTRACT
APOBEC3G (A3G) is a cytidine deaminase that restricts human immunodeficiency virus type 1 (HIV-1) and other lentiviruses. Most of these viruses encode a Vif protein that directly binds A3G and leads to its proteasomal degradation. Both Vif proteins of HIV-1 and African green monkey simian immunodeficiency virus (SIVagm) bind residue 128 of A3G. However, this position does not control the A3G degradation by Vif variants derived from HIV-2 and SIVmac, which both originated from SIV of sooty mangabey monkeys (SIVsmm), suggesting that the A3G binding site for Vif proteins of the SIVsmm/HIV-2 lineage differs from that of HIV-1. To map the SIVsmm Vif binding site of A3G, we performed immunoprecipitations of individual A3G domains, Vif/A3G degradation assays and a detailed mutational analysis of human A3G. We show that A3G residue 129, but not the adjacent position 128, confers susceptibility to degradation by SIVsmm Vif. An artificial A3G mutant, the P129D mutant, was resistant to degradation by diverse Vifs from HIV-1, HIV-2, SIVagm, and chimpanzee SIV (SIVcpz), suggesting a conserved lentiviral Vif binding site. Gorilla A3G naturally contains a glutamine (Q) at position 129, which makes its A3G resistant to Vifs from diverse lineages. We speculate that gorilla A3G serves as a barrier against SIVcpz strains. In summary, we show that Vif proteins from distinct lineages bind to the same A3G loop, which includes positions 128 and 129. The multiple adaptations within this loop among diverse primates underscore the importance of counteracting A3G in lentiviral evolution.
Subject(s)
Cytidine Deaminase/metabolism , Gene Products, vif/metabolism , Host-Pathogen Interactions , Lentivirus/physiology , APOBEC-3G Deaminase , Animals , Binding Sites , DNA Mutational Analysis , Humans , Immunoprecipitation , Lentivirus/isolation & purification , Molecular Sequence Data , Primates , Protein Binding , Proteolysis , Sequence Analysis, DNAABSTRACT
The APOBEC3 family comprises seven cytidine deaminases (APOBEC3A [A3A] to A3H), which are expressed to various degrees in HIV-1 susceptible cells. The HIV-1 Vif protein counteracts APOBEC3 restriction by mediating its degradation by the proteasome. We hypothesized that Vif proteins from various HIV-1 subtypes differ in their abilities to counteract different APOBEC3 proteins. Seventeen Vif alleles from seven HIV-1 subtypes were tested for their abilities to degrade and counteract A3G, A3F, and A3H haplotype II (hapII). We show that most Vif alleles neutralize A3G and A3F efficiently but display differences with respect to the inhibition of A3H hapII. The majority of non-subtype B Vif alleles tested presented some activity against A3H hapII, with two subtype F Vif variants being highly effective in counteracting A3H hapII. The residues required for activity were mapped to two residues in the amino-terminal region of Vif (positions 39F and 48H). Coimmunoprecipitations showed that these two amino acids were necessary for association of Vif with A3H hapII. These findings suggest that the A3H hapII binding site in Vif is distinct from the regions important for A3G and A3F recognition and that it requires specific amino acids at positions 39 and 48. The differential Vif activity spectra, especially against A3H hapII, suggest adaptation to APOBEC3 repertoires representative of different human ancestries. Phenotypic assessment of anti-APOBEC3 activity of Vif variants against several cytidine deaminases will help reveal the requirement for successful replication in vivo and ultimately point to interventions targeting the Vif-APOBEC3 interface.
Subject(s)
Aminohydrolases/metabolism , Cytidine Deaminase/metabolism , Cytosine Deaminase/metabolism , HIV Infections/enzymology , HIV-1/classification , HIV-1/metabolism , vif Gene Products, Human Immunodeficiency Virus/metabolism , APOBEC-3G Deaminase , Amino Acid Sequence , Aminohydrolases/chemistry , Aminohydrolases/genetics , Cell Line , Cytidine Deaminase/chemistry , Cytidine Deaminase/genetics , Cytosine Deaminase/chemistry , Cytosine Deaminase/genetics , HIV Infections/genetics , HIV Infections/virology , HIV-1/chemistry , HIV-1/genetics , Humans , Molecular Sequence Data , Phylogeny , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Sequence Alignment , vif Gene Products, Human Immunodeficiency Virus/chemistry , vif Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.
Subject(s)
Aminohydrolases/immunology , Cytidine Deaminase/immunology , Human T-lymphotropic virus 1/immunology , Proteins/immunology , Aminohydrolases/genetics , Aminohydrolases/metabolism , Catalytic Domain , Cell Line , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , HIV-1/growth & development , HIV-1/immunology , Haplotypes , Human T-lymphotropic virus 1/growth & development , Humans , Minor Histocompatibility Antigens , Mutant Proteins/genetics , Mutant Proteins/immunology , Mutant Proteins/metabolism , Proteins/genetics , Proteins/metabolismABSTRACT
Several members of the human APOBEC3 family of cytidine deaminases can potently restrict retroviruses such as HIV-1. The single-domain APOBEC3H (A3H) is encoded by four haplotypes, of which only A3H haplotype II-RDD (hapII-RDD) restricts HIV-1 efficiently. The goal of this study was to elucidate the mechanisms underlying the differences in antiviral activity among A3H haplotypes. The naturally occurring A3H hapI-GKE and hapII-RDD variants differ at three amino acid positions. A panel of six site-directed mutants containing combinations of the three variable residues was used to determine A3H protein expression, requirements of A3H virion incorporation, and A3H-Gag interactions. The catalytic activity of each A3H protein was assessed directly by using an Escherichia coli mutator assay. We found that the incorporation efficiencies of A3H variants into HIV-1 virions were comparable despite major differences in cellular expression. An assessment of the enzymes' catalytic activities showed that the deaminase activity of each A3H variant correlated with protein expression, suggesting similar enzymatic efficiencies. Surprisingly, virion incorporation experiments using Gag deletion mutants demonstrated that A3H haplotypes interacted with different Gag regions. A3H hapII-RDD associated with nucleocapsid in an RNA-dependent manner, whereas A3H hapI-GKE associated with the C-terminal part of matrix and the N-terminal capsid domain. Our results show that the A3H hapII-RDD interaction with nucleocapsid is critical for its antiviral activity and that the inability of A3H hapI-GKE to interact with nucleocapsid underlies its limited antiviral potential. Thus, the antiviral activity of A3H haplotypes is determined by its incorporation into the viral core, in proximity to the reverse transcription complex.
Subject(s)
Aminohydrolases/immunology , Aminohydrolases/metabolism , HIV-1/immunology , HIV-1/physiology , Virus Assembly , gag Gene Products, Human Immunodeficiency Virus/metabolism , Aminohydrolases/genetics , Cell Line , DNA Mutational Analysis , Haplotypes , Humans , Mutagenesis, Site-Directed , Mutation, Missense , Protein BindingABSTRACT
Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.
Subject(s)
Cytosine Deaminase/immunology , HIV-1/immunology , Lymphocytes/virology , Virus Replication , Cells, Cultured , HIV-1/physiology , HumansABSTRACT
Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that potently inhibit exogenous and endogenous retroviruses. The impact of single nucleotide polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human APOBEC3H is currently unknown. In this study, we show that APOBEC3H transcripts derived from human peripheral blood mononuclear cells are polymorphic in sequence and subject to alternative splicing. We found that APOBEC3H variants encoding a SNP cluster (G105R, K121D and E178D, hapII-RDD) restricted human immunodeficiency virus type 1 (HIV-1) more efficiently than wild-type APOBEC3H (hapI-GKE). All APOBEC3H variants tested were resistant to HIV-1 Vif, the viral protein that efficiently counteracts APOBEC3G/3F activity. Alternative splicing of APOBEC3H was common and resulted in variants with distinct C-terminal regions and variable antiretroviral activities. Splice variants of hapI-GKE displayed a wide range of antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that uniformly and efficiently restricted viral infectivity (>20-fold). Site-directed mutagenesis identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading to an average additional 10-fold increase in antiviral activity. APOBEC3H variants were catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. HIV-1 mutagenesis as a mode of action for APOBEC3H is suggested by the decrease of restriction observed with a cytidine deaminase domain mutant and the inverse correlation between G-to-A mutations and infectivity. Thus, the anti-HIV activity of APOBEC3H seems to be regulated by a combination of genomic variation and alternative splicing. Since prevalence of hapII-RDD is high in populations of African descent, these findings raise the possibility that some individuals may harbor effective as well as HIV-1 Vif-resistant intracellular antiviral defense mechanisms.
Subject(s)
Alternative Splicing , Cytosine Deaminase/genetics , Cytosine Deaminase/immunology , HIV-1/immunology , Polymorphism, Single Nucleotide , Virus Replication/immunology , Aminohydrolases , Cell Line , Humans , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
The TAR hairpin of the human immunodeficiency virus type 1 (HIV-1) RNA genome is essential for virus replication. TAR forms the binding site for the transcriptional trans-activator protein Tat and multiple additional TAR functions have been proposed. We previously constructed an HIV-1 variant in which the TAR-Tat transcription control mechanism is replaced by the components of the Tet-ON regulatory system. In this context, the surprising finding was that TAR can be truncated or even deleted, but partial TAR deletions that destabilize the stem structure cause a severe replication defect. In this study, we demonstrate that the HIV-1 RNA genome requires a stable hairpin at its 5'-end because unpaired TAR sequences affect the proper folding of the untranslated leader RNA. Consequently, multiple leader-encoded functions are affected by partial TAR deletions. Upon evolution of such mutant viruses, the replication capacity was repaired through the acquisition of additional TAR mutations that restore the local RNA folding, thus preventing the detrimental effect on the leader conformation.
Subject(s)
5' Untranslated Regions/chemistry , HIV Long Terminal Repeat , HIV-1/genetics , RNA, Viral/chemistry , Base Sequence , Dimerization , HIV-1/physiology , Molecular Sequence Data , Nucleic Acid Conformation , Poly A/chemistry , Sequence Deletion , Virus ReplicationABSTRACT
AIMS AND OBJECTIVES: The aim of this study was to develop an observational scale to measure the social well-being of nursing home residents, by assessing not only the social behaviour of the resident towards others, but also the behaviour of others towards the resident. BACKGROUND: Traditionally, aspects of the social well-being of nursing home residents are assessed according to the social activities and interactions where they engage. Although these are important indicators of social well-being, other important indicators may include the positive social behaviour of others towards the resident (e.g. confirming the resident's behaviour or showing affection). DESIGN: A cross-sectional descriptive survey design. METHOD: From the perspective of human social needs, items relating to fulfillment of the needs for affection, behavioural confirmation and status were formulated and tested. This took place in three nursing homes in the Netherlands that provide somatic and psycho-geriatric care. RESULTS: The study (sample n = 306) yielded a short and reliable scale, the Social Well-being Of Nursing home residents-scale, with separate sub-scales (three items each) for fulfillment of the three social needs. CONCLUSIONS: These first results indicate that overall social well-being and its sub-dimensions can be measured with this new observational scale, although its validity needs to be confirmed. Including the social behaviour of others towards the resident may have provided a more comprehensive measure of the social well-being of nursing home residents. RELEVANCE TO CLINICAL PRACTICE: This measure may help to underscore the importance of the social behaviour of others (e.g. caregivers) for the overall social well-being of residents and with that assist care-providers in nursing homes to improve the social well-being of the residents.
Subject(s)
Inpatients/psychology , Nursing Homes , Social Behavior , Cross-Sectional Studies , Humans , Netherlands , Reproducibility of ResultsABSTRACT
Genomic RNA circularization has been proposed for several RNA viruses. In this study, we examined if the 5' and 3' ends of the 9-kb HIV-1 RNA genome can interact. In vitro assays demonstrated a specific interaction between transcripts encompassing the 5' and 3' terminal 1 kb, suggesting that the HIV-1 RNA genome can circularize. Truncation of the transcripts indicated that the 5'-3' interaction is formed by 600-700 nt in the gag open reading frame and the terminal 123 nt of the genomic RNA. Detailed RNA structure probing indicates that sequences flanking the 3' TAR hairpin interact with complementary sequences in the gag gene. Phylogenetic analysis indicates that all HIV-1 subtypes can form the 5'/3' interaction despite considerable sequence divergence, suggesting an important role of RNA circularization in the HIV-1 replication cycle.
Subject(s)
Genome, Viral , HIV-1/genetics , RNA, Viral/chemistry , RNA/chemistry , 3' Untranslated Regions/chemistry , 5' Untranslated Regions/chemistry , Base Sequence , Genes, gag , HIV-1/classification , Molecular Sequence Data , Phylogeny , RNA, Circular , TemperatureABSTRACT
Initiation of reverse transcription of a retroviral RNA genome is strictly regulated. The tRNA primer binds to the primer binding site (PBS), and subsequent priming is triggered by the primer activation signal (PAS) that also pairs with the tRNA. We observed that in vitro reverse transcription initiation of the HIV-1 leader RNA varies in efficiency among 3'-end truncated transcripts, despite the presence of both PBS and PAS motifs. As the HIV-1 leader RNA can adopt two different foldings, we investigated if the conformational state of the transcripts did influence the efficiency of reverse transcription initiation. However, mutant transcripts that exclusively fold one or the other structure were similarly active, thereby excluding the possibility of regulation of reverse transcription initiation by the structure riboswitch. We next set out to determine the availability of the PAS element. This sequence motif enhances the efficiency of reverse transcription initiation, but its activity is regulated because the PAS motif is initially base paired within the wild-type template. We measured that the initiation efficiency on different templates correlates directly with accessibility of the PAS motif. Furthermore, changes in PAS are critical to facilitate a primer-switch to a new tRNA species, demonstrating the importance of this enhancer element.
Subject(s)
HIV Reverse Transcriptase/metabolism , HIV-1/genetics , RNA, Viral/chemistry , RNA/chemistry , Regulatory Sequences, Ribonucleic Acid , Reverse Transcription , 5' Untranslated Regions/chemistry , Base Sequence , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer, Lys/chemistry , Templates, GeneticABSTRACT
BACKGROUND: Vaccination of macaques with live attenuated simian immunodeficiency virus (SIV) provides significant protection against the wild-type virus. The use of a live attenuated human immunodeficiency virus (HIV) as AIDS vaccine in humans is however considered unsafe because of the risk that the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. We earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (dox). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient dox administration. Since the effectiveness and safety of such a conditionally live virus vaccine should be tested in macaques, we constructed a similar dox-dependent SIV variant. The Tat-TAR transcription control mechanism in this virus was inactivated through mutation and functionally replaced by the dox-inducible Tet-On regulatory system. This SIV-rtTA variant replicated in a dox-dependent manner in T cell lines, but not as efficiently as the parental SIVmac239 strain. Since macaque studies will likely require an efficiently replicating variant, we set out to optimize SIV-rtTA through in vitro viral evolution. RESULTS: Upon long-term culturing of SIV-rtTA, additional nucleotide substitutions were observed in TAR that affect the structure of this RNA element but that do not restore Tat binding. We demonstrate that the bulge and loop mutations that we had introduced in the TAR element of SIV-rtTA to inactivate the Tat-TAR mechanism, shifted the equilibrium between two alternative conformations of TAR. The additional TAR mutations observed in the evolved variants partially or completely restored this equilibrium, which suggests that the balance between the two TAR conformations is important for efficient viral replication. Moreover, SIV-rtTA acquired mutations in the U3 promoter region. We demonstrate that these TAR and U3 changes improve viral replication in T-cell lines and macaque peripheral blood mononuclear cells (PBMC) but do not affect dox-control. CONCLUSION: The dox-dependent SIV-rtTA variant was optimized by viral evolution, yielding variants that can be used to test the conditionally live virus vaccine approach and as a tool in SIV biology studies and vaccine research.
Subject(s)
Doxycycline/pharmacology , Gene Expression Regulation/drug effects , Gene Expression/drug effects , Simian Immunodeficiency Virus/growth & development , Simian Immunodeficiency Virus/genetics , Virus Replication/drug effects , Animals , Base Sequence , Cell Line , Cells, Cultured , Evolution, Molecular , Genes, Viral , Humans , Leukocytes, Mononuclear/virology , Macaca , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , SAIDS Vaccines/genetics , Sequence Alignment , Serial PassageABSTRACT
The objective of this study was to improve validity and reliability estimates of the Index for Social Engagement (ISE) for long-term care. After exploring content validity and internal consistency in Dutch and Canadian data, two ISE items were dropped, and two new items were added. Reliability of this Revised ISE (RISE) was tested in 189 nursing home residents. It appeared that the RISE has enhanced reliability estimates, especially in residents with cognitive impairment. The RISE for long-term care improves the existing index by including additional dimensions of social engagement and by increasing the reliability of results for residents with cognitive impairment.
Subject(s)
Aged/psychology , Geriatric Assessment/methods , Long-Term Care/psychology , Nursing Assessment/methods , Social Behavior , Aged, 80 and over , Cognition Disorders/psychology , Female , Humans , Interpersonal Relations , Male , Netherlands , Nursing Assessment/standards , Nursing Evaluation Research , Ontario , Principal Component Analysis , Psychometrics , Sensitivity and SpecificityABSTRACT
HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.
Subject(s)
Anti-HIV Agents/pharmacology , HIV-1/genetics , RNA Interference , RNA, Small Interfering/pharmacology , RNA, Viral/chemistry , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Base Sequence , Cell Line , Gene Products, nef/antagonists & inhibitors , Gene Products, nef/genetics , Genome, Viral , Humans , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , RNA Stability , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , RNA, Viral/drug effects , RNA, Viral/genetics , nef Gene Products, Human Immunodeficiency VirusABSTRACT
BACKGROUND AND OBJECTIVE: Generalizability of clinical predictors for mortality from lower respiratory infection (LRI) in nursing home residents has not been assessed for residents with dementia. STUDY DESIGN AND SETTING: In prospective cohort studies of LRI in 61 nursing homes in the Netherlands (n = 541) and 36 nursing homes in Missouri, USA (n = 564), we examined 14-day and 1- and 3-month mortality in residents with dementia who were treated with antibiotics. RESULTS: A logistic model predicting 14-day mortality derived from Dutch data included eating dependency, elevated pulse, decreased alertness, respiratory difficulty, insufficient fluid intake, high respiratory rate, male gender, and pressure sores. After adjusting coefficients with the heuristic shrinkage factor, the 14-day model showed good discrimination and calibration in both datasets. The apparent c-statistic for the original Dutch model was 0.80 (after correction for optimism, it was 0.75); the c-statistic was 0.74 in the U.S. validation population. The models predicting 1- and 3-month mortality showed moderate performance. A scoring system for estimating 14-day mortality performed equally well as the original model. CONCLUSION: We identified a set of credible clinical predictors that are easily assessed and demonstrated validity in identifying residents at low risk of dying from LRI across different nursing home populations. This tool should inform decision-making for families and doctors.
Subject(s)
Cross Infection/mortality , Dementia/mortality , Homes for the Aged , Nursing Homes , Respiratory Tract Infections/mortality , Aged , Anti-Bacterial Agents/therapeutic use , Cross Infection/complications , Cross Infection/drug therapy , Dementia/complications , Dementia/drug therapy , Female , Health Status Indicators , Humans , Logistic Models , Male , Netherlands , Prospective Studies , Respiratory Tract Infections/complications , Respiratory Tract Infections/drug therapy , Risk Assessment/methods , United StatesABSTRACT
Studies on the prediction of survival have mainly focused on hospital and hospice patients suffering from cancer. The aim of this study was to describe the predicted vs. the actual survival in terminally ill, mainly noncancer patients in Dutch nursing homes (NHs). A prospective cohort study was conducted in 16 NHs representative for The Netherlands. A total of 515 NH patients with a maximum life expectancy of 6 weeks, as assessed by an NH physician, were included. NH physicians were accurate in more than 90% of their prognoses for terminally ill--mainly noncancer--NH patients, when death occurred within 7 days. For a longer period of time, their predictions became inaccurate. In the category of patients who were expected to die within 8-21 days, predictions were accurate in 16.0%, and in the category of patients expected to die within 22-42 days, this was 13.0%. Predictions in these categories were mainly optimistic (patient died earlier) in 68.6% and 52.2%, respectively. The findings of this study suggest that accurate prediction of survival of (mainly) noncancer patients in NHs is only possible when death is imminent and seems to be dependent on an intimate knowledge of patients. Prognostication over a longer period of time tends to be less accurate, and, therefore, continues to be a challenging task for NH physicians.