Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1_NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.     

Induction of AIDS in Rhesus Monkeys by a Recombinant Simian Immunodeficiency Virus Expressing nef of Human Immunodeficiency Virus Type 1

A nef gene is present in all primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus of macaque monkeys (SIVmac). However, the nef genes of HIV-1 and SIVmac exhibit minimal sequence identity, and not all properties are shared by the two. Nef sequences of SIVmac239 were replaced by four independent nef alleles of HIV-1 in a context that was optimal for expression. The sources of the HIV-1 nef sequences included NL 4-3, a variant NL 4-3 gene derived from a recombinant-infected rhesus monkey, a patient nef allele, and a nef consensus sequence. Of 16 rhesus monkeys infected with these SHIVnef chimeras, 9 maintained high viral loads for prolonged periods, as observed with the parental SIVmac239, and 6 have died with AIDS 52 to 110 weeks postinfection. Persistent high loads were observed at similar frequencies with the four different SIV recombinants that expressed these independent HIV-1 nef alleles. Infection with other recombinant SHIVnef constructions resulted in sequence changes in infected monkeys that either created an open nef reading frame or optimized the HIV-1 nef translational context. The HIV-1 nef gene was uniformly retained in all SHIVnef-infected monkeys. These results demonstrate that HIV-1 nef can substitute for SIVmac nef in vivo to produce a pathogenic infection. However, the model suffers from an inability to consistently obtain persisting high viral loads in 100% of the infected animals, as is observed with the parental SIVmac239.     

Simian immunodeficiency virus from the sooty mangabey and rhesus macaque is modified with O-linked carbohydrate

Although stretches of serine and threonine are sometimes sites for O-linked carbohydrate attachment, specific sequence and structural determinants for O-linked attachment remain ill defined. The gp120 envelope protein of SIVmac239 contains a serine-threonine-rich stretch of amino acids at positions 128 to 139. Here we show that lectin protein from jackfruit seed (jacalin), which binds to non- and monosialylated core 1 O-linked carbohydrate, potently inhibited the replication of SIVmac239. Selection of a jacalin-resistant SIVmac239 variant population resulted in virus with specific substitutions within amino acids 128 to 139. Cloned simian immunodeficiency virus (SIV) variants with substitutions in the 128-to-139 region had infectivities equivalent to, or within 1 log unit of, that of SIVmac239 and were resistant to the inhibitory effects of jacalin. Characterization of the SIVmac239 gp120 O-linked glycome showed the presence of core 1 and core 2 O-linked carbohydrate; a 128-to-139-substituted variant gp120 from jacalin-resistant SIV lacked O-linked carbohydrate. Unlike that of SIVmac239, the replication of HIV-1 strain NL4-3 was resistant to inhibition by jacalin. Purified gp120s from four SIVmac and SIVsm strains bound jacalin strongly in an enzyme-linked immunosorbent assay, while nine different HIV-1 gp120s, two SIVcpz gp120s, and 128-to-139-substituted SIVmac239 gp120 did not bind jacalin. The ability or inability to bind jacalin thus correlated with the presence of the serine-threonine-rich stretch in the SIVmac and SIVsm gp120s and the absence of such stretches in the SIVcpz and HIV-1 gp120s. Consistent with sequence predictions, two HIV-2 gp120s bound jacalin, while one did not. These data demonstrate the presence of non- and monosialylated core 1 O-linked carbohydrate on the gp120s of SIVmac and SIVsm and the lack of these modifications on HIV-1 and SIVcpz gp120s.     

RNA structures facilitate recombination-mediated gene swapping in HIV-1

Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.     

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.     

A highly conserved RNA folding region coincident with the Rev response element of primate immunodeficiency viruses.

A series of unusual folding regions (UFR) immediately 3' to the cleavage site of the outer membrane protein (OMP) and transmembrane protein (TMP) were detected in the envelope gene RNA of the human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV) by an extensive Monte Carlo simulation. These RNA secondary structures were predicted to be both highly stable and statistically significant. In the calculation, twenty-five different sequence isolates of HIV-1, three isolates of HIV-2 and eight sequences of SIV were included. Although significant sequence divergence occurs in the env coding regions of these viruses, a distinct UFR of 234-nt is consistently located ten nucleotides 3' to the cleavage site of the OMP/TMP in HIV-1, and a 216-nt UFR occurs forty-six and forty-nine nucleotides downstream from the OMP/TMP cleavage site of HIV-2 and SIV, respectively. Compensatory base changes in the helical stem regions of these conserved RNA secondary structures are identified. These results support the hypothesis that these special RNA folding regions are functionally important and suggest that the role of this sequence as the Rev response element (RRE) is mediated by secondary structure as well as primary RNA sequence.     

Automatic detection of conserved RNA structure elements in complete RNA virus genomes.

We propose a new method for detecting conserved RNA secondary structures in a family of related RNA sequences. Our method is based on a combination of thermodynamic structure prediction and phylogenetic comparison. In contrast to purely phylogenetic methods, our algorithm can be used for small data sets of approximately 10 sequences, efficiently exploiting the information contained in the sequence variability. The procedure constructs a prediction only for those parts of sequences that are consistent with a single conserved structure. Our implementation produces reasonable consensus structures without user interference. As an example we have analysed the complete HIV-1 and hepatitis C virus (HCV) genomes as well as the small segment of hantavirus. Our method confirms the known structures in HIV-1 and predicts previously unknown conserved RNA secondary structures in HCV.     

The association of Nef with a cellular serine/threonine kinase and its enhancement of infectivity are viral isolate dependent.

The nef genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) encode a 27- to 34-kDa myristoylated protein which induces downregulation of CD4 surface levels and enhances virus infectivity. In adult macaques, Nef has been implicated in pathogenesis and disease progression. Both HIV-1 SF2 Nef and SIVmac239 Nef have been shown to associate with a cellular serine/threonine kinase. We tested five functional Nef isolates to examine whether this kinase association is a property conserved among different isolates. HIV-1 SF2 and 248 and SIVmac239 Nef proteins were found associated with the kinase. HIV-1 NL4-3 and 233 Nef proteins were found weakly associated or not associated with the kinase. All five Nef isolates efficiently downregulated CD4 cell surface expression, suggesting that the association with this cellular kinase is not required for Nef to downregulate CD4. Comparison of the SF2 and NL4-3 isolates shows a differential ability of Nef to enhance infectivity that suggests a possible correlation between kinase association and enhancement of infectivity.     

HIV-1 but not SIVmac239 induces higher interferon-α antiviral state in chronic infected northern pig-tailed macaques (Macaca leonina)

Studies have shown that interferon (IFN)-α has an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in the acute infection stage, but its role in chronic infection is still unclear. We previously established a nonpathogenic HIV-1 and pathogenic simian immunodeficiency virus (SIV) model in northern pig-tailed macaques (NPMs, Macaca leonina). In the current study, we detected viral RNA and DNA in various tissues (axillary lymph nodes (LNs), inguinal LNs, and spleen) in HIV-1_NL4-3- and SIV_mac239-infected NPM during the chronic stage of infection. Results indicated that the levels of viral DNA and RNA were higher in the tested tissues (LNs and spleen) of the SIV_mac239-infected NPMs than in the HIV-1_NL4-3 infected NPMs. Furthermore, IFN-α expression was higher in the HIV-infected tissues than in the SIV-infected controls. The HIV restriction factors induced by IFN-α (i.e., tetherin and MX2), as well as inflammatory factors IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), were analyzed using real-time polymerase chain reaction (PCR) and immunofluorescence staining assays. Results showed that their expression levels were much higher in the HIV-infected tissues than in the SIV-infected controls. These findings were confirmed by in vitro experiments on healthy NPM peripheral blood mononuclear cells infected with HIV-1_NL4-3, which showed lower viral replication, higher IFN-α expression, and an antiviral status. This study demonstrated that HIV-1 infection, but not SIV_mac239 infection, in NPMs caused higher expression of IFN-α and induced a higher antiviral status. This may be one of the reasons why HIV-1 cannot replicate at a high level or develop into AIDS in NPMs.     

Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities.

The prediction of the complete matrix of base pairing probabilities was applied to the 3'' noncoding region (NCR) of flavivirus genomes. This approach identifies not only well-defined secondary structure elements, but also regions of high structural flexibility. Flaviviruses, many of which are important human pathogens, have a common genomic organization, but exhibit a significant degree of RNA sequence diversity in the functionally important 3''-NCR. We demonstrate the presence of secondary structures shared by all flaviviruses, as well as structural features that are characteristic for groups of viruses within the genus reflecting the established classification scheme. The significance of most of the predicted structures is corroborated by compensatory mutations. The availability of infectious clones for several flaviviruses will allow the assessment of these structural elements in processes of the viral life cycle, such as replication and assembly.     

Empirical comparison of cross-platform normalization methods for gene expression data

Background

The prediction of secondary structure, i.e. the set of canonical base pairs between nucleotides, is a first step in developing an understanding of the function of an RNA sequence. The most accurate computational methods predict conserved structures for a set of homologous RNA sequences. These methods usually suffer from high computational complexity. In this paper, TurboFold, a novel and efficient method for secondary structure prediction for multiple RNA sequences, is presented.

Results

TurboFold takes, as input, a set of homologous RNA sequences and outputs estimates of the base pairing probabilities for each sequence. The base pairing probabilities for a sequence are estimated by combining intrinsic information, derived from the sequence itself via the nearest neighbor thermodynamic model, with extrinsic information, derived from the other sequences in the input set. For a given sequence, the extrinsic information is computed by using pairwise-sequence-alignment-based probabilities for co-incidence with each of the other sequences, along with estimated base pairing probabilities, from the previous iteration, for the other sequences. The extrinsic information is introduced as free energy modifications for base pairing in a partition function computation based on the nearest neighbor thermodynamic model. This process yields updated estimates of base pairing probability. The updated base pairing probabilities in turn are used to recompute extrinsic information, resulting in the overall iterative estimation procedure that defines TurboFold. TurboFold is benchmarked on a number of ncRNA datasets and compared against alternative secondary structure prediction methods. The iterative procedure in TurboFold is shown to improve estimates of base pairing probability with each iteration, though only small gains are obtained beyond three iterations. Secondary structures composed of base pairs with estimated probabilities higher than a significance threshold are shown to be more accurate for TurboFold than for alternative methods that estimate base pairing probabilities. TurboFold-MEA, which uses base pairing probabilities from TurboFold in a maximum expected accuracy algorithm for secondary structure prediction, has accuracy comparable to the best performing secondary structure prediction methods. The computational and memory requirements for TurboFold are modest and, in terms of sequence length and number of sequences, scale much more favorably than joint alignment and folding algorithms.

Conclusions

TurboFold is an iterative probabilistic method for predicting secondary structures for multiple RNA sequences that efficiently and accurately combines the information from the comparative analysis between sequences with the thermodynamic folding model. Unlike most other multi-sequence structure prediction methods, TurboFold does not enforce strict commonality of structures and is therefore useful for predicting structures for homologous sequences that have diverged significantly. TurboFold can be downloaded as part of the RNAstructure package at http://rna.urmc.rochester.edu.     

Simian immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV-1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive plasma

To date, only a small number of anti-human immunodeficiency virus type 1 (HIV-1) monoclonal antibodies (MAbs) with relatively broad neutralizing activity have been isolated from infected individuals. Adequate techniques for defining how frequently antibodies of these specificities arise in HIV-infected people have been lacking, although it is generally assumed that such antibodies are rare. In order to create an epitope-specific neutralization assay, we introduced well-characterized HIV-1 epitopes into the heterologous context of simian immunodeficiency virus (SIV). Specifically, epitope recognition sequences for the 2F5, 4E10, and 447-52D anti-HIV-1 neutralizing monoclonal antibodies were introduced into the corresponding regions of SIVmac239 by site-directed mutagenesis. Variants with 2F5 or 4E10 recognition sequences in gp41 retained replication competence and were used for neutralization assays. The parental SIVmac239 and the neutralization-sensitive SIVmac316 were not neutralized by the 2F5 and 4E10 MAbs, nor were they neutralized significantly by any of the 96 HIV-1-positive human plasma samples that were tested. The SIV239-2F5 and SIV239-4E10 variants were specifically neutralized by the 2F5 and 4E10 MAbs, respectively, at concentrations within the range of what has been reported previously for HIV-1 primary isolates (J. M. Binley et al., J. Virol. 78:13232-13252, 2004). The SIV239-2F5 and SIV239-4E10 epitope-engrafted variants were used as biological screens for the presence of neutralizing activity of these specificities. None of the 92 HIV-1-positive human plasma samples that were tested exhibited significant neutralization of SIV239-2F5. One plasma sample exhibited >90% neutralization of SIV239-4E10, but this activity was not competed by a 4E10 target peptide and was not present in concentrated immunoglobulin G (IgG) or IgA fractions. We thus confirm by direct analysis that neutralizing activities of the 2F5 and 4E10 specificities are either rare among HIV-1-positive individuals or, if present, represent only a very small fraction of the total neutralizing activity in any given plasma sample. We further conclude that the structures of gp41 from SIVmac239 and HIV-1 are sufficiently similar such that epitopes engrafted into SIVmac239 can be readily recognized by the cognate anti-HIV-1 monoclonal antibodies.     

Neutralization sensitivity of cell culture-passaged simian immunodeficiency virus.

CEMx174- and C8166-45-based cell lines which contain a secreted alkaline phosphatase (SEAP) reporter gene under the control of a tat-responsive promoter derived from either SIVmac239 or HIV-1(NL4-3) were constructed. Basal levels of SEAP activity from these cell lines were low but were greatly stimulated upon transfection of tat expression plasmids. Infection of these cell lines with simian immunodeficiency virus (SIV) or human immunodeficiency virus type 1 (HIV-1) resulted in a dramatic increase in SEAP production within 48 to 72 h that directly correlated with the amount of infecting virus. When combined with chemiluminescent measurement of SEAP activity in the cell-free supernatant, these cells formed the basis of a rapid, sensitive, and quantitative assay for SIV and HIV infectivity and neutralization. Eight of eight primary isolates of HIV-1 that were tested induced readily measurable SEAP activity in this system. While serum neutralization of cloned SIVmac239 was difficult to detect with other assays, neutralization of SIVmac239 was readily detected at low titers with this new assay system. The neutralization sensitivities of two stocks of SIVmac251 with different cell culture passage histories were tested by using sera from SIV-infected monkeys. The primary stock of SIVmac251 had been passaged only twice through primary cultures of rhesus monkey peripheral blood mononuclear cells, while the laboratory-adapted stock had been extensively passaged through the MT4 immortalized T-cell line. The primary stock of SIVmac251 was much more resistant to neutralization by a battery of polyclonal sera from SIV-infected monkeys than was the laboratory-adapted virus. Thus, SIVmac appears to be similar to HIV-1 in that extensive laboratory passage through T-cell lines resulted in a virus that is much more sensitive to serum neutralization.     

Simian immunodeficiency virus mutants resistant to serum neutralization arise during persistent infection of rhesus monkeys.

We previously described the pattern of sequence variation in gp120 following persistent infection of rhesus monkeys with the pathogenic simian immunodeficiency virus SIVmac239 molecular clone (D.P.W. Burns and R.C. Desrosiers, J. Virol. 65:1843, 1991). Sequence changes were confined largely to five variable regions (V1 to V5), four of which correspond to human immunodeficiency virus type 1 (HIV-1) gp120 variable regions. Remarkably, 182 of 186 nucleotide substitutions that were documented in these variable regions resulted in amino acid changes. This is an extremely nonrandom pattern, which suggests selective pressure driving amino acid changes in discrete variable domains. In the present study, we investigated whether neutralizing-antibody responses are one selective force responsible at least in part for the observed pattern of sequence variation. Variant env sequences called 1-12 and 8-22 obtained 69 and 93 weeks after infection of a rhesus monkey with cloned SIVmac239 were recombined into the parental SIVmac239 genome, and variant viruses were generated by transfection of cultured cells with cloned DNA. The 1-12 and 8-22 recombinants differ from the parental SIVmac239 at 18 amino acid positions in gp120 and at 5 and 10 amino acid positions, respectively, in gp41. Sequential sera from the monkey infected with cloned SIVmac239 from which the 1-12 and 8-22 variants were isolated showed much higher neutralizing antibody titers to cloned SIVmac239 than to the cloned 1-12 and 8-22 variants. For example, at 55 weeks postinfection the neutralizing antibody titer against SIVmac239 was 640 while those to the variant viruses were 40 and less than 20. Two other rhesus monkeys infected with cloned SIVmac239 showed a similar pattern. Rhesus monkeys were also experimentally infected with the cloned variants so that the type-specific nature of the neutralizing antibody responses could be verified. Indeed, each of these monkeys showed neutralizing-antibody responses of much higher titer to the homologous variant used for infection. These experiments unambiguously demonstrate that SIV mutants resistant to serum neutralization arise during the course of persistent infection of rhesus monkeys.     

Secondary structure model for mouse beta Maj globin mRNA derived from enzymatic digestion data, comparative sequence and computer analysis.

A model for the secondary structure of mouse beta Maj globin messenger RNA is presented based on enzymatic digestion data, comparative sequence and computer analysis. Using 5'-32P-end-labeled beta globin mRNA as a substrate, single-stranded regions were determined with S1 and T1 nucleases and double-stranded regions with V1 ribonuclease from cobra venom. The structure data obtained for ca. 75% of the molecule was introduced into a computer algorithm which predicts secondary structures of minimum free energy consistent with the enzymatic data. Two prominent base paired regions independently derived by phylogenetic analysis were also present in the computer generated structure lending support for the model. An interesting feature of the model is the presence of long-range base pairing interactions which permit the beta globin mRNA to fold back on itself, thereby bringing the 5'- and 3'-noncoding regions within close proximity. This feature is consistent with data from other laboratories suggesting an interaction of the 5'- and 3'-domains in the mammalian globin mRNAs.     

The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures.

We analyzed the leader region of human immunodeficiency virus type 1 (HIV-1) RNA to decipher the nature of the cis-acting E/psi element required for encapsidation of viral RNA into virus particles. Our data indicate that, for RNA encapsidation, there are at least two functional subregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is located between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognition signals for encapsidation. To determine whether specific putative RNA secondary structures serve as the signal(s) for encapsidation, we constructed primary base substitution mutations that would be expected to destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mutants allowed the identification of two discrete hairpins that facilitate RNA encapsidation in vivo. Thus, the HIV-1 E/psi region is a multipartite element composed of specific and functional RNA secondary structures. Compensation of the primary mutations by the second-site mutations could not be attained in trans. This indicates that interstrand base pairing between these two stem regions within the hairpins does not appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competent HIV-1 strains suggests that a subset of the hydrogen-bonded base pairs within the stems of the hairpins is likely to be required for function in cis.