A premature termination codon mutation at the C terminus of foamy virus Gag downregulates the levels of spliced pol mRNA

Foamy viruses (FV) comprise a subfamily of retroviruses. Orthoretroviruses, such as human immunodeficiency virus type 1, synthesize Gag and Pol from unspliced genomic RNA. However, FV Pol is expressed from a spliced mRNA independently of Gag. FV pol splicing uses a 3′ splice site located at the 3′ end of gag, resulting in a shared exon between gag and pol. Previously, our laboratory showed that C-terminal Gag premature termination codon (PTC) mutations in the 3′ shared exon led to greatly decreased levels of Pol protein (C. R. Stenbak and M. L. Linial, J. Virol. 78:9423-9430, 2004). To further characterize these mutants, we quantitated the levels of unspliced gag and spliced pol mRNAs using a real-time PCR assay. In some of the PTC mutants, the levels of spliced pol mRNA were reduced as much as 30-fold, whereas levels of unspliced gag RNA were not affected. Substitutions of a missense codon in place of a PTC restored normal levels of spliced pol mRNA. Disrupting Upf proteins involved in nonsense-mediated mRNA decay (NMD) did not affect Pol protein expression. Introduction of an exonic splicing enhancer downstream of the PTC mutation restored pol splicing to the wild-type level. Taken together, our results show that the PTC mutation itself is responsible for decreased levels of pol mRNA but that mechanisms other than NMD might be involved in downregulating Pol expression. The results also suggest that normal pol splicing utilizes a suboptimal splice site seen for other spliced mRNAs in most retroviruses, in that introduced exonic enhancer elements can increase splicing efficiency.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.     

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

Mobile LTR-retroelements comprising retroviruses and LTR-retrotransposons form a large part of eukaryotic genomes. Their mode of replication and abundance favour the notion that they are major actors in eukaryote evolution. The Gypsy retroelement can spread in the germ line of the fruit fly Drosophila melanogaster via both env-independent and env-dependent processes. Thus, Gypsy is both an active retrotransposon and an infectious retrovirus resembling the gammaretrovirus MuLV. However, unlike gammaretroviruses, the Gypsy Gag structural precursor is not processed into Matrix, Capsid and Nucleocapsid (NC) proteins. In contrast, it has features in common with Gag of the ancient yeast TY1 retroelement. These characteristics of Gypsy make it a very interesting model to study replication of a retroelement at the frontier between ancient retrotransposons and retroviruses. We investigated Gypsy replication using an in vitro model system and transfection of insect cells. Results show that an unstructured domain of Gypsy Gag has all the properties of a retroviral NC. This NC-like peptide forms ribonucleoparticle-like complexes upon binding Gypsy RNA and directs the annealing of primer tRNA^Lys,2 to two distinct primer binding sites (PBS) at the genome 5′ and 3′ ends. Only the 5′ PBS is indispensable for cDNA synthesis in vitro and in Drosophila cells.     

A novel role for gag as a cis-acting element regulating RNA structure,dimerization and packaging in HIV-1 lentiviral vectors

Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5′ leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5–gag interaction and a more stable structure with a U5–AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5′ region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.     

HIV-1 predisposed to acquiring resistance to maraviroc (MVC) and other CCR5 antagonists in vitro has an inherent, low-level ability to utilize MVC-bound CCR5 for entry

In 2009, a newly discovered human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), was reported by Lombardi et al. in 67% of persons from the US with chronic fatigue syndrome (CFS) by PCR detection of gag sequences. Although six subsequent studies have been negative for XMRV, CFS was defined more broadly using only the CDC or Oxford criteria and samples from the US were limited in geographic diversity, both potentially reducing the chances of identifying XMRV positive CFS cases. A seventh study recently found polytropic MuLV sequences, but not XMRV, in a high proportion of persons with CFS. Here we tested blood specimens from 45 CFS cases and 42 persons without CFS from over 20 states in the United States for both XMRV and MuLV. The CFS patients all had a minimum of 6 months of post-exertional malaise and a high degree of disability, the same key symptoms described in the Lombardi et al. study. Using highly sensitive and generic DNA and RNA PCR tests, and a new Western blot assay employing purified whole XMRV as antigen, we found no evidence of XMRV or MuLV in all 45 CFS cases and in the 42 persons without CFS. Our findings, together with previous negative reports, do not suggest an association of XMRV or MuLV in the majority of CFS cases.     

Characterization of a cis-Acting Sequence in the pol Region Required To Transfer Human Foamy Virus Vectors

To identify cis-acting elements in the foamy virus (FV) RNA pregenome, we developed a transient-vector-production system based on cotransfection of indicator gene-bearing vector and gag-pol and env expression plasmids. Two elements which were critical for vector transfer were found and mapped approximately. The first element was located in the RU5 leader and the 5′ gag region (approximately up to position 650 of the viral RNA). The second element was located in an approximately 2-kb sequence in the 3′ pol region. Although small 5′ and 3′ deletions, as well as internal deletions of the latter element, were tolerated, both elements were found to be absolutely required for vector transfer. The functional characterization of the pol region-located cis-acting element revealed that it is essential for efficient incorporation or the stability of particle-associated virion RNA. Furthermore, virions derived from a vector lacking this sequence were found to be deficient in the cleavage of the Gag protein by the Pol precursor protease. Our results suggest that during the formation of infectious virions, complex interactions between FV Gag and Pol and the viral RNA take place.     

Evolution of the HIV-1 nef gene in HLA-B*57 Positive Elite Suppressors

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.     

A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5′ untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIV_Mac coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role.     

A 5′UTR-Spliced mRNA Isoform Is Specialized for Enhanced HIV-2 gag Translation

Full-length unspliced genomic RNA plays critical roles in HIV replication, serving both as mRNA for the synthesis of the key viral polyproteins Gag and Gag-Pol and as genomic RNA for encapsidation into assembling viral particles. We show that a second gag mRNA species that differs from the genomic RNA molecule by the absence of an intron in the 5′ untranslated region (5′UTR) is produced during HIV-2 replication in cell culture and in infected patients. We developed a cotransfection system in which epitopically tagged Gag proteins can be traced back to their mRNA origins in the translation pool. We show that a disproportionate amount of Gag is translated from 5′UTR intron-spliced mRNAs, demonstrating a role for the 5′UTR intron in the regulation of gag translation. To further characterize the effects of the HIV-2 5′UTR on translation, we fused wild-type, spliced, or mutant leader RNA constructs to a luciferase reporter gene and assayed their translation in reticulocyte lysates. These assays confirmed that leaders lacking the 5′UTR intron increased translational efficiency compared to that of the unspliced leader. In addition, we found that removal or mutagenesis of the C-box, a pyrimidine-rich sequence located in the 5′UTR intron and previously shown to affect RNA dimerization, also strongly influenced translational efficiency. These results suggest that the splicing of both the 5′UTR intron and the C-box element have key roles in regulation of HIV-2 gag translation in vitro and in vivo.     

Grossly Defective nef Gene Sequences in a Human Immunodeficiency Virus Type 1-Seropositive Long-Term Nonprogressor

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4⁺ T-cell counts (>1,000 CD4 cells/μl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5′ long terminal repeat (5′LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5′LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5′LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.     

Identification and Mutational Analysis of a Rej Response Element in Jaagsiekte Sheep Retrovirus RNA

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5′ end of env, and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3′ end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus that causes ovine pulmonary adenocarcinoma, an infectious lung tumor of sheep (10, 29). Ovine pulmonary adenocarcinoma has morphological resemblance to a human lung cancer, bronchioloalveolar carcinoma, which is only weakly associated with cigarette smoking. In recent years, complete infectious and oncogenic molecular clones of JSRV have been isolated (30). We and others found that the JSRV envelope (Env) protein also functions as an oncogene in that it can induce morphological transformation of fibroblast and epithelial cell lines in culture and tumors in animals (1, 24, 34). Further studies have demonstrated that amino acids in the cytoplasmic tail of the Env transmembrane (TM) protein are important for transformation, as are multiple domains in the surface (SU) protein (17, 18).The nuclear export of mRNA is a critical step in gene expression. All retroviruses employ unspliced genome-length RNA as mRNA for synthesis of Gag and Pol proteins, while splicing yields mRNA(s) for Env (and other) proteins (15). Thus, genome-length mRNA for Gag and Pol is equivalent to an unspliced precursor for Env mRNA. A key issue for retroviruses is how they transport unspliced genome-length RNA to the cytoplasm. This is accomplished by two general mechanisms. The human immunodeficiency virus type 1 (HIV-1) Rev protein (encoded by a doubly spliced mRNA) specifically binds to a Rev-responsive element (RRE), located in RNA of the env gene. The Rev/RRE complex recruits the cellular CRM1/Xpo1 protein (as well as other cellular proteins), which results in transport of this RNA-protein complex to the cytoplasm (7). Similarly, human T-cell leukemia virus type 1 (HTLV-1) Rex protein binds a Rex-responsive element on viral RNA, resulting in export via the CRM1 pathway (21). The betaretroviruses mouse mammary tumor virus (MMTV) and human endogenous retrovirus K (HERV-K) also encode analogous regulatory proteins (Rem and Rec, respectively) (19, 22, 27).In contrast, the betaretroviruses Mason-Pfizer monkey virus (MPMV) and simian retrovirus (SRV) contain constitutive RNA export elements (constitutive transport elements [CTEs]) that facilitate nuclear export of unspliced RNA (4, 41). The MPMV CTE is located between env and the 3′ long terminal repeat (LTR); it binds to the cellular trans-acting factor NXF1/Tap, which directs nuclear export of the RNA-protein complex to the cytoplasm (14). Rous sarcoma virus and the related avian leukosis viruses contain direct repeat sequences flanking the src gene or in the 3′ untranslated region of their RNA (28). Structure-function analyses of these RNA-exporting elements revealed specific stem-loop structures that are important for activity and for binding of the host cell factors (3).Like other betaretroviruses, JSRV contains the standard genes gag, pro, pol, and env. In addition we recently found that JSRV also encodes a regulatory factor, Rej (17a). Rej is reminiscent of MMTV Rem and HERV-K Rec in that it is encoded in the 5′ end of env and it is required for efficient synthesis of Gag protein. We found that Rej is required for translation of unspliced viral RNA, and in 293T cells it also enhances accumulation of cytoplasmic unspliced viral RNA in the cytoplasm. In the results presented here, we show that JSRV RNA also contains a Rej-responsive element (RejRE) in the 3′ end of env that is required for translation of Gag protein and efficient export or accumulation of unspliced viral RNA in the cytoplasm in 293T cells. Mutational analyses of RejRE based on M-fold suggest that both primary sequences and secondary structures in this region play important roles in nuclear export or accumulation of unspliced viral RNA in the cytoplasm and Gag synthesis. This accumulation is independent of Tap but dependent on CRM1. Moreover, Rej protein was exported from the nucleus to the cytoplasm in cells expressing wild-type JSRV RNA but not RejRE mutants, suggesting binding of Rej protein to the RejRE.     

Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection

Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-β-cyclodextrin (2OHpβCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpβCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.     

The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop

Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem–loops and a conserved long-range interaction between heptanucleotide sequences 5′-CCCUGUC-3′ in R/U5 and 5′-GACAGGG-3′ in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5′ UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.     

cis-Acting Sequences Required for Simian Foamy Virus Type 1 Vectors

We have constructed a series of vectors based on simian foamy virus type 1 (SFV-1) to define the minimum cis-acting elements required for gene transfer. To characterize these vectors, we inserted the coding sequence of the bacterial lacZ gene linked to the cytomegalovirus immediate-early gene promoter. Introduction of a deletion mutation in the leader region between the 5′ long terminal repeat and the start of the gag gene at position 1659 to 1694 completely abrogated gene transfer by the SFV-1 vector. Deletion of 39 nucleotides from position 1692 to 1731 in the leader region resulted in a significant reduction in the transducing-particle titer. Furthermore, we have identified a second cis-acting element located at the 3′ end of the pol gene between position 6486 and 6975 to be critical for SFV-1 vector transduction. These results identify the two important cis-acting elements required for SFV-1 vector construction, and the finding of a cis-acting element in the pol gene is unique among retroviruses.     

The Bovine Leukemia Virus Encapsidation Signal Is Composed of RNA Secondary Structures

The encapsidation signal of bovine leukemia virus (BLV) was previously shown by deletion analysis to be discontinuous and to extend into the 5′ end of the gag gene (L. Mansky et al., J. Virol. 69:3282–3289, 1995). The global minimum-energy optimal folding for the entire BLV RNA, including the previously mapped primary and secondary encapsidation signal regions, was analyzed. Two stable stem-loop structures (located just downstream of the gag start codon) were predicted within the primary signal region, and one stable stem-loop structure (in the gag gene) was predicted in the secondary signal region. Based on these predicted structures, we introduced a series of mutations into the primary and secondary encapsidation signals in order to explore the sequence and structural information contained within these regions. The replication efficiency and levels of cytoplasmic and virion RNA were analyzed for these mutants. Mutations that disrupted either or both of the predicted stem-loop structures of the primary signal reduced the replication efficiency by factors of 7 and 40, respectively; similar reductions in RNA encapsidation efficiency were observed. The mutant with both stem-loop structures disrupted had a phenotype similar to that of a mutant containing a deletion of the entire primary signal region. Mutations that disrupted the predicted stem-loop structure of the secondary signal led to similar reductions (factors of 4 to 6) in both the replication and RNA encapsidation efficiencies. The introduction of compensatory mutations into mutants from both the primary and secondary signal regions, which restored the predicted stem-loop structures, led to levels of replication and RNA encapsidation comparable to those of virus containing the wild-type encapsidation signal. Replacement of the BLV RNA region containing the primary and secondary encapsidation signals with a similar region from human T-cell leukemia virus (HTLV) type 1 or type 2 led to virus replication at three-quarters or one-fifth of the level of the parental virus, respectively. The results from both the compensatory mutants and BLV-HTLV chimeras indicate that the encapsidation sequences are recognized largely by their secondary or tertiary structures.