Isoform-specific RNA structure determination using Nano-DMS-MaP.

RNA structure determination is essential to understand how RNA carries out its diverse biological functions. In cells, RNA isoforms are readily expressed with partial variations within their sequences due, for example, to alternative splicing, heterogeneity in the transcription start site, RNA processing or differential termination/polyadenylation. Nanopore dimethyl sulfate mutational profiling (Nano-DMS-MaP) is a method for in situ isoform-specific RNA structure determination. Unlike similar methods that rely on short sequencing reads, Nano-DMS-MaP employs nanopore sequencing to resolve the structures of long and highly similar RNA molecules to reveal their previously hidden structural differences. This Protocol describes the development and applications of Nano-DMS-MaP and outlines the main considerations for designing and implementing a successful experiment: from bench to data analysis. In cell probing experiments can be carried out by an experienced molecular biologist in 3-4 d. Data analysis requires good knowledge of command line tools and Python scripts and requires a further 3-5 d.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

Nano-DMS-MaP allows isoform-specific RNA structure determination.

Genome-wide measurements of RNA structure can be obtained using reagents that react with unpaired bases, leading to adducts that can be identified by mutational profiling on next-generation sequencing machines. One drawback of these experiments is that short sequencing reads can rarely be mapped to specific transcript isoforms. Consequently, information is acquired as a population average in regions that are shared between transcripts, thus blurring the underlying structural landscape. Here, we present nanopore dimethylsulfate mutational profiling (Nano-DMS-MaP)-a method that exploits long-read sequencing to provide isoform-resolved structural information of highly similar RNA molecules. We demonstrate the value of Nano-DMS-MaP by resolving the complex structural landscape of human immunodeficiency virus-1 transcripts in infected cells. We show that unspliced and spliced transcripts have distinct structures at the packaging site within the common 5' untranslated region, likely explaining why spliced viral RNAs are excluded from viral particles. Thus, Nano-DMS-MaP is a straightforward method to resolve biologically important transcript-specific RNA structures that were previously hidden in short-read ensemble analyses.

A dsRNA-binding mutant reveals only a minor role of exonuclease activity in interferon antagonism by the arenavirus nucleoprotein.

The arenavirus nucleoprotein (NP) plays an important role in the virus' ability to block interferon (IFN) production, and its exonuclease function appears to contribute to this activity. However, efforts to analyze this contribution are complicated by the functional overlap between the exonuclease active site and a neighboring region involved in IKKε-binding and subsequent inhibition of IRF3 activation, which also plays an important role in IFN production. To circumvent this issue, we mutated a residue located away from the active site that is involved in binding of the dsRNA substrate being targeted for exonuclease digestion, i.e. H426A. We found that expression of Tacaribe virus (TCRV) NP containing this RNA-binding H426A mutation was still able to efficiently block IFN-ß promoter activity in response to Sendai virus infection, despite being strongly impaired in its exonuclease activity. This was in contrast to a conventional exonuclease active site mutant (E388A), which was impaired with respect to both exonuclease activity and IFN antagonism. Importantly, growth of a recombinant virus encoding the RNA-binding mutation (rTCRV-H426A) was similar to wild-type in IFN-deficient cells, unlike the active site mutant (rTCRV-E388A), which was already markedly impaired in these cells. Further, in IFN-competent cells, the TCRV-H426A RNA-binding mutant showed more robust growth and delayed IFN-ß mRNA upregulation compared to the TCRV-E388A active site mutant. Taken together, this novel mutational approach, which allows us to now dissect the different contributions of the NP exonuclease activity and IKKε-binding/IRF3 inhibition to IFN antagonism, clearly suggests that conventional exonuclease mutants targeting the active site overestimate the contribution of the exonuclease function, and that rather other IFN antagonistic functions of NP play the dominant role in IFN-antagonism.

Short- and long-range interactions in the HIV-1 5' UTR regulate genome dimerization and packaging.

RNA dimerization is the noncovalent association of two human immunodeficiency virus-1 (HIV-1) genomes. It is a conserved step in the HIV-1 life cycle and assumed to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. Here, we developed functional analysis of RNA structure-sequencing (FARS-seq) to comprehensively identify sequences and structures within the HIV-1 5' untranslated region (UTR) that regulate this critical step. Using FARS-seq, we found nucleotides important for dimerization throughout the HIV-1 5' UTR and identified distinct structural conformations in monomeric and dimeric RNA. In the dimeric RNA, key functional domains, such as stem-loop 1 (SL1), polyadenylation signal (polyA) and primer binding site (PBS), folded into independent structural motifs. In the monomeric RNA, SL1 was reconfigured into long- and short-range base pairings with polyA and PBS, respectively. We show that these interactions disrupt genome packaging, and additionally show that the PBS-SL1 interaction unexpectedly couples the PBS with dimerization and Pr55Gag binding. Altogether, our data provide insights into late stages of HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.

RNA Structures and Their Role in Selective Genome Packaging.

To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.

Acid ceramidase of macrophages traps herpes simplex virus in multivesicular bodies and protects from severe disease.

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1-/- mice results in replication of HSV-1 and Asah1-/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies.

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species.

Rapid Identification of Major Histocompatibility Complex Class I Haplotypes Using Deep Sequencing in an Endangered Old World Monkey.

Immunogenetic data from wild primate populations have been difficult to obtain, due to logistic and methodological constraints. We applied a well-characterized deep sequencing method for MHC I typing, developed for macaques, to a population of wild red colobus to assess the feasibility of identifying MHC I-A/B haplotypes. Ten individuals produced sufficient data from blood and tissue samples to assign haplotypes. Eighty-two sequences were classified as red colobus MHC I alleles distributed across six MHC I loci. Individuals averaged ~13k reads across six MHC I loci, with 83% of all alleles representing either MHC I-A or MHC I-B loci. This study not only represents an important advance in the identification and genotyping of MHC in the endangered red colobus but also shows the potential for using this approach in other endangered wild primates.

A novel SIV gag-specific CD4(+)T-cell clone suppresses SIVmac239 replication in CD4(+)T cells revealing the interplay between antiviral effector cells and their infected targets.

To study CD4(+)T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4(+)T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8(+)T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1ß, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4(+)T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4(+)effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4(+)T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4(+)T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4(+)T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.

The TB-specific CD4(+) T cell immune repertoire in both cynomolgus and rhesus macaques largely overlap with humans.

Non-human primate (NHP) models of tuberculosis (TB) immunity and pathogenesis, especially rhesus and cynomolgus macaques, are particularly attractive because of the high similarity of the human and macaque immune systems. However, little is known about the MHC class II epitopes recognized in macaques, thus hindering the establishment of immune correlates of immunopathology and protective vaccination. We characterized immune responses in rhesus macaques vaccinated against and/or infected with Mycobacterium tuberculosis (Mtb), to a panel of antigens currently in human vaccine trials. We defined 54 new immunodominant CD4(+) T cell epitopes, and noted that antigens immunodominant in humans are also immunodominant in rhesus macaques, including Rv3875 (ESAT-6) and Rv3874 (CFP10). Pedigree and inferred restriction analysis demonstrated that this phenomenon was not due to common ancestry or inbreeding, but rather presentation by common alleles, as well as, promiscuous binding. Experiments using a second cohort of rhesus macaques demonstrated that a pool of epitopes defined in the previous experiments can be used to detect T cell responses in over 75% of individual monkeys. Additionally, 100% of cynomolgus macaques, irrespective of their latent or active TB status, responded to rhesus and human defined epitope pools. Thus, these findings reveal an unexpected general repertoire overlap between MHC class II epitopes recognized in both species of macaques and in humans, showing that epitope pools defined in humans can also be used to characterize macaque responses, despite differences in species and antigen exposure. The results have general implications for the evaluation of new vaccines and diagnostics in NHPs, and immediate applicability in the setting of macaque models of TB.

Linking pig-tailed macaque major histocompatibility complex class I haplotypes and cytotoxic T lymphocyte escape mutations in simian immunodeficiency virus infection.

UNLABELLED: The influence of major histocompatibility complex class I (MHC-I) alleles on human immunodeficiency virus (HIV) diversity in humans has been well characterized at the population level. MHC-I alleles likely affect viral diversity in the simian immunodeficiency virus (SIV)-infected pig-tailed macaque (Macaca nemestrina) model, but this is poorly characterized. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIV(mac251) genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIV(mac251) at 4 to 10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms at both individual mutations and groups of mutations spanning 10-amino-acid segments were linked using in-house bioinformatics pipelines, since cytotoxic T lymphocyte (CTL) escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays, we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described to be CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and strategies for treatment of HIV infection. IMPORTANCE: Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV, and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV type 1-infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing nearly identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaques to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques, and this enables a more refined level of analysis of future vaccine design and strategies for treatment of HIV infection.

Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one.

Deep sequencing has revolutionized major histocompatibility complex (MHC) class I analysis of nonhuman primates by enabling high-throughput, economical, and comprehensive genotyping. Full-length MHC class I cDNA sequences, which are required to generate reagents such as MHC-peptide tetramers, cannot be directly obtained by short read deep sequencing. We combined data from two next-generation sequencing platforms to discover novel full-length MHC class I mRNA/cDNA transcripts in Chinese rhesus macaques. We first genotyped macaques by Roche/454 pyrosequencing using a 530-bp amplicon spanning the densely polymorphic exons 2 through 4 of the MHC class I loci that encode the peptide-binding region. We then mapped short paired-end 250 bp Illumina sequence reads spanning the full-length transcript to each 530-bp amplicon at high stringency and used paired-end information to reconstruct full-length allele sequences. We characterized 65 full-length sequences from six Chinese rhesus macaques. Overall, approximately 70 % of the alleles distinguished in these six animals contained new sequence information, including 29 novel transcripts. The flexibility of this approach should make full-length MHC class I allele genotyping accessible for any nonhuman primate population of interest. We are currently optimizing this method for full-length characterization of other highly polymorphic, duplicated loci such as the MHC class II DRB and killer immunoglobulin-like receptors. We anticipate that this method will facilitate rapid expansion and near completion of sequence libraries of polymorphic loci, such as MHC class I, within a few years.

Haplessly hoping: macaque major histocompatibility complex made easy.

Major histocompatibility complex (MHC) gene products control the repertoire of T cell responses that an individual may create against pathogens and foreign tissues. This text will review the current understanding of MHC genetics in nonhuman primates, with a focus on Mauritian-origin cynomolgus macaques (Macaca fascicularis) and Indian-origin rhesus macaques (Macaca mulatta). These closely related macaque species provide important experimental models for studies of infectious disease pathogenesis, vaccine development, and transplantation research. Recent advances resulting from the application of several cost effective, high-throughput approaches, with deep sequencing technologies have revolutionized our ability to perform MHC genotyping of large macaque cohorts. Pyrosequencing of cDNA amplicons with a Roche/454 GS Junior instrument, provides excellent resolution of MHC class I allelic variants with semi-quantitative estimates of relative levels of transcript abundance. Introduction of the Illumina MiSeq platform significantly increased the sample throughput, since the sample loading workflow is considerably less labor intensive, and each instrument run yields approximately 100-fold more sequence data. Extension of these sequencing methods from cDNA to genomic DNA amplicons further streamlines the experimental workflow and opened opportunities for retrospective MHC genotyping of banked DNA samples. To facilitate the reporting of MHC genotypes, and comparisons between groups of macaques, this text also introduces an intuitive series of abbreviated rhesus MHC haplotype designations based on a major Mamu-A or Mamu-B transcript characteristic for ancestral allele combinations. The authors believe that the use of MHC-defined macaques promises to improve the reproducibility, and predictability of results from pre-clinical studies for translation to humans.

Major histocompatibility complex class I haplotype diversity in Chinese rhesus macaques.

The use of Chinese-origin rhesus macaques (Macaca mulatta) for infectious disease immunity research is increasing despite the relative lack of major histocompatibility complex (MHC) class I immunogenetics information available for this population. We determined transcript-based MHC class I haplotypes for 385 Chinese rhesus macaques from five different experimental cohorts, providing a concise representation of the full complement of MHC class I major alleles expressed by each animal. In total, 123 Mamu-A and Mamu-B haplotypes were defined in the full Chinese rhesus macaque cohort. We then performed an analysis of haplotype frequencies across the experimental cohorts of Chinese rhesus macaques, as well as a comparison against a group of 96 Indian rhesus macaques. Notably, 35 of the 51 Mamu-A and Mamu-B haplotypes observed in Indian rhesus macaques were also detected in the Chinese population, with 85% of the 385 Chinese-origin rhesus macaques expressing at least one of these class I haplotypes. This unexpected conservation of Indian rhesus macaque MHC class I haplotypes in the Chinese rhesus macaque population suggests that immunologic insights originally gleaned from studies using Indian rhesus macaques may be more applicable to Chinese rhesus macaques than previously appreciated and may provide an opportunity for studies of CD8(+) T-cell responses between populations. It may also be possible to extend these studies across multiple species of macaques, as we found evidence of shared ancestral haplotypes between Chinese rhesus and Mauritian cynomolgus macaques.

Organic extracts of urban aerosol (< or =PM2.5) enhance rBet v 1-induced upregulation of CD63 in basophils from birch pollen-allergic individuals.

Epidemiological studies have linked the high prevalence rates of IgE-mediated allergic diseases to an increase in exposure to traffic-related air pollutants such as diesel exhaust particles (DEPs). There is growing experimental evidence that organic compounds of DEPs, predominantly polycyclic aromatic hydrocarbons (PAHs), participate in the development and maintenance of allergic airway diseases. In this study we investigated the impact of organic extracts of urban aerosol (AERex) containing various PAH concentrations on the activation of human basophils. Whole blood samples from six birch pollen-allergic and five control subjects were repeatedly incubated in the presence of AERex with or without recombinant Bet v 1 (rBet v 1). Basophils were analyzed for CD63 expression as a measure of basophil activation by using multiparameter flow cytometry. Basophils, when exposed in vitro to AERex and rBet v 1, expressed CD63 significantly more than with antigen activation alone. AERex synergized with rBet v 1 in a dose-dependent manner, but did not activate basophils from nonallergic donors. AERex effect on CD63 upregulation was found in blood samples of all patients and did not occur in the absence of rBet v 1. Strongest basophil activation was monitored upon stimulation with AERex comprising the highest PAH content. The capability of AERex to increase activation of basophils from birch pollen-allergic subjects at ambient concentrations suggests an important role of organic compounds of airborne particles in the aggravation of IgE-mediated allergic diseases. This could be a new aspect of regulation of unspecific promoting stimuli in clinical manifestation of allergic inflammation.