A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.

Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.

The structure of FIV reverse transcriptase and its implications for non-nucleoside inhibitor resistance.

Reverse transcriptase (RT) is the target for the majority of anti-HIV-1 drugs. As with all anti-AIDS treatments, continued success of RT inhibitors is persistently disrupted by the occurrence of resistance mutations. To explore latent resistance mechanisms potentially accessible to therapeutically challenged HIV-1 viruses, we examined RT from the related feline immunodeficiency virus (FIV). FIV closely parallels HIV-1 in its replication and pathogenicity, however, is resistant to all non-nucleoside inhibitors (NNRTI). The intrinsic resistance of FIV RT is particularly interesting since FIV harbors the Y181 and Y188 sensitivity residues absent in both HIV-2 and SIV. Unlike RT from HIV-2 or SIV, previous efforts have failed to make FIV RT susceptible to NNRTIs concluding that the structure or flexibility of the feline enzyme must be profoundly different. We report the first crystal structure of FIV RT and, being the first structure of an RT from a non-primate lentivirus, enrich the structural and species repertoires available for RT. The structure demonstrates that while the NNRTI binding pocket is conserved, minor subtleties at the entryway can render the FIV RT pocket more restricted and unfavorable for effective NNRTI binding. Measuring NNRTI binding affinity to FIV RT shows that the "closed" pocket configuration inhibits NNRTI binding. Mutating the loop residues rimming the entryway of FIV RT pocket allows for NNRTI binding, however, it does not confer sensitivity to these inhibitors. This reveals a further layer of resistance caused by inherent FIV RT variances that could have enhanced the dissociation of bound inhibitors, or, perhaps, modulated protein plasticity to overcome inhibitory effects of bound NNRTIs. The more "closed" conformation of FIV RT pocket can provide a template for the development of innovative drugs that could unlock the constrained pocket, and the resilient mutant version of the enzyme can offer a fresh model for the study of NNRTI-resistance mechanisms overlooked in HIV-1.

Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.

The Role of Reverse Transcriptase in the Origin of Life.

It has been suggested that RNA polymerase ribozyme displaying reverse transcriptase and integrase activities has played a vital role in the origin of life on Earth. Here, we present a hypothesis that formation of universal ancestral units of all living organisms - retroelements - in the evolution was mediated by reverse transcriptase. The propensity of retroelements to mutations and their insertion capacity have formed a basis for the origin of complex DNA structures - primary genomes - that have given rise to archaea, eukaryotes, bacteria, and viruses. Conserved properties of retroelements have been preserved throughout the evolution; their modifications have facilitated the emergence of mechanisms for the interactions between proteins and nucleic acids. Life has evolved due to insertional mutagenesis and competition of autonomously replicating polynucleotides that allowed to preserve structures with adaptive properties. We hypothesize that natural selection of mechanisms for the defense against insertions based on the ribonuclease activity of reverse transcriptase ribozyme has led to the emergence of all universal enzymatic systems for the processing of RNA molecules. These systems have been and still remain the key sources of structural and functional transformations of genomes in the course of evolution. The data presented in this review suggest that the process of translation, which unifies the nucleic acid and protein worlds, has developed as a modification of the defense mechanisms against insertions. Polypeptides formed by this defense system have potentiated the activity of ribozymes in the composition of ribonucleoproteins (RNPs) and even functionally replaced them as more efficient catalysts of biological reactions. Here, we analyze the mechanisms of retroelement involvement in the structural and regulatory transformations of eukaryotic genomes supposedly reflecting the adaptive principles that had originated during the beginning of life on Earth. Simultaneously with the evolution of existing proteins, retroelements have served as sources of new ribozymes, such as long non-coding RNAs. These ribozymes can function in complexes with proteins in the composition of RNPs, as well as display independent catalytic and translational activities; their genes have a potential for the transformation into protein-coding genes. Hence, the conserved principles of RNA, DNA, and proteins interregulation formed at the time of life origin on Earth have been used throughout the evolution.

L1 Retrotransposons Are Transcriptionally Active in Hippocampus of Rat Brain.

LINE1 (L1) is an autonomous, non-LTR retrotransposon and the L1 family of retrotransposons constitute around 17%, 20% and 23% in the human, mouse and rat genomes respectively. Under normal physiological conditions, the retroelements remain by and large transcriptionally silent but are activated in response to biotic and abiotic stress conditions and during perturbation in cellular metabolism. They have also been shown to be transiently activated under certain developmental programs. Using RT-PCR, we show that the L1 elements are transcriptionally active in the hippocampus region of the brain of four-month-old rat under normal conditions without any apparent stress. Twenty non-redundant LINE1-specific reverse transcriptase (RTase) sequences form ORF2 region were isolated, cloned and sequenced. Full length L1 element sequences complementary to the isolated sequences were retrieved from the L1 database. In silico analysis was used to determine the presence of these retroelements proximal (up to 10 kb) to the genes transcriptionally active in the hippocampus. Many important genes were found to be in close proximity of the transcriptionally active L1 elements. Transcriptional activation of the elements possibly affects the expression of the neighbouring genes.

[Positive and negative regulation of transcription from HIV provirus].

The RNA genome of retroviruses including human immunodeficiency virus type 1 (HIV-1) will be converted into DNA, called "propvirus". This proviral DNA will be integrated into host cell genome and behave like host genes. Since the step at which the viral RNA genome is converted into DNA will not allow any increase of viral genetic information because of the presence of RNaseH activity inherent to the reverse transcriptase and is responsible for the degradation of viral RNA in forming the DNA:RNA hybrid as the intermediate molecule for this conversion. However, during transcription from proviral DNA into viral RNA, hundreds and even thousands of mRNA encoding viral information will be synthesized by the action of host cellular RNA polymerase II, thus producing a large amount of progeny viral particles after translation and assembly. HIV is unique in that it contains virus-specific transcriptional activator called Tat.

Self-priming of reverse transcriptase impairs strand-specific detection of dengue virus RNA.

Dengue virus infection is the most frequent arthropod-borne infection affecting humans in the world. Our understanding of the pathophysiological events leading to mild or severe outcomes of the disease remains limited by the fact that viral target cells in the human body are poorly characterized. One of the most sensitive strategies for detecting cells supporting active replication of this positive-strand RNA virus is the search for the replicative intermediate, an antigenome of negative polarity, by RT-PCR. However, a phenomenon described as 'false priming' of the reverse transcriptase (RT) prevents strand-specific detection. The results of the current study showed that this event corresponds to cDNA synthesis that is independent of any primer addition. This property was general to all RNAs tested and was not associated with small free nucleic acids, such as tRNAs and microRNAs. Rather, it corresponded to initiation of cDNA synthesis from the 3' end of the RNA template, and a model is proposed in which the template RNA snaps back upon itself and creates a transient RNA primer suitable for the RT. Such a property would explain why many assays proposed for detection of a replicative intermediate are not specific, and may help in the development of a molecular biology protocol that could allow replication studies of RNA viruses of human interest, such as dengue virus, hepatitis C virus and enteroviruses.

Ty3 nuclear entry is initiated by viruslike particle docking on GLFG nucleoporins.

Yeast retrotransposons form intracellular particles within which replication occurs. Because fungal nuclear membranes do not break down during mitosis, similar to retroviruses infecting nondividing cells, the cDNA produced must be translocated through nuclear pore complexes. The Saccharomyces cerevisiae long terminal repeat retrotransposon Ty3 assembles its Gag3 and Gag3-Pol3 precursor polyproteins into viruslike particles in association with perinuclear P-body foci. These perinuclear clusters of Ty3 viruslike particles localized to sites of clustered nuclear pore complexes (NPCs) in a nup120Delta mutant, indicating that Ty3 particles and NPCs interact physically. The NPC channels are lined with nucleoporins (Nups) with extended FG (Phe-Gly) motif repeat domains, further classified as FG, FxFG, or GLFG repeat types. These domains mediate partitioning of proteins between the cytoplasm and the nucleus. Here we have systematically examined the requirements for FG repeat domains in Ty3 nuclear transport. The GLFG domains interacted in vitro with virus-like particle Gag3, and this interaction was disrupted by mutations in the amino-terminal domain of Gag3, which is predicted to lie on the external surface of the particles. Accordingly, Ty3 transposition was decreased in strains with the GLFG repeats deleted. The spacer-nucleocapsid domain of Gag3, which is predicted to be internal to the particle, interacted with GLFG repeats and nucleocapsid localized to the nucleus. We conclude that Ty3 particle docking on nuclear pores is facilitated by interactions between Gag3 and GLFG Nups and that nuclear entry of the preintegration complex is further promoted by nuclear localization signals within the nucleocapsid and integrase.

Ty3 nucleocapsid controls localization of particle assembly.

Expression of the budding yeast retrotransposon Ty3 results in production of viruslike particles (VLPs) and retrotransposition. The Ty3 major structural protein, Gag3, similar to retrovirus Gag, is processed into capsid, spacer, and nucleocapsid (NC) during VLP maturation. The 57-amino-acid Ty3 NC protein has 17 basic amino acids and contains one copy of the CX(2)CX(4)HX(4)C zinc-binding motif found in retrovirus NC proteins. Ty3 RNA, protein, and VLPs accumulate in clusters associated with RNA processing bodies (P bodies). This study investigated the role of the NC domain in Ty3-P body clustering and VLP assembly. Fifteen Ty3 NC Ala substitution and deletion mutants were examined using transposition, immunoblot, RNA protection, cDNA synthesis, and multimerization assays. Localization of Ty3 proteins and VLPs was characterized microscopically. Substitutions of each of the conserved residues of the zinc-binding motif resulted in the loss of Ty3 RNA packaging. Substitution of the first two of four conserved residues in this motif caused the loss of Ty3 RNA and protein clustering with P bodies and disrupted particle formation. NC was shown to be a mediator of formation of Ty3 RNA foci and association of Ty3 RNA and protein with P bodies. Mutations that disrupted these NC functions resulted in various degrees of Gag3 nuclear localization and a spectrum of different particle states. Our findings are consistent with the model that Ty3 assembly is associated with P-body components. We hypothesize that the NC domain acts as a molecular switch to control Gag3 conformational states that affect both assembly and localization.

Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis.

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (10(7)-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells.

Progress in understanding the biology of the human mutagen LINE-1.

Long interspersed nucleotide element (LINE)-1 retrotransposon (L1) has emerged as the largest contributor to mammalian genome mass, responsible for over 35% of the human genome. Differences in the number and activity levels of L1s contribute to interindividual variation in humans, both by affecting an individual's likelihood of acquiring new L1-mediated mutations, as well as by differentially modifying gene expression. Here, we report on recent progress in understanding L1 biology, with a focus on mechanisms of L1-mediated disease. We discuss known details of L1 life cycle, including L1 structure, transcriptional regulation, and the mechanisms of translation and retrotransposition. Current views on cell type specificity, timing, and control of retrotransposition are put forth. Finally, we discuss the role of L1 as a mutagen, using the latest findings in L1 biology to illuminate molecular mechanisms of L1-mediated gene disruption.

Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.

R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3' untranslated region of the R2 element. If this RNA template ends at the precise 3' end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3' junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3' junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3' end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3' end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.

Hepatitis B virus replication.

Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, epsilon, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the epsilon RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

In vivo RNA localization of I factor, a non-LTR retrotransposon, requires a cis-acting signal in ORF2 and ORF1 protein.

According to the current model of non-LTR retrotransposon (NLR) mobilization, co-expression of the RNA transposition intermediate, and the proteins it encodes (ORF1p and ORF2p), is a requisite for the formation of cytoplasmic ribonucleoprotein complexes which contain necessary elements to complete a retrotransposition cycle later in the nucleus. To understand these early processes of NLR mobilization, here we analyzed in vivo the protein and RNA expression patterns of the I factor, a model NLR in Drosophila. We show that ORF1p and I factor RNA, specifically produced during transposition, are co-expressed and tightly co-localize with a specific pattern (Loc+) exclusively in the cytoplasm of germ cells permissive for retrotransposition. Using an ORF2 mutated I factor, we show that ORF2p plays no role in the Loc+ patterning. With deletion derivatives of an I factor we define an RNA localization signal required to display the Loc+ pattern. Finally, by complementation experiments we show that ORF1p is necessary for the efficient localization of I factor RNA. Our data suggest that ORF1p is involved in proper folding and stabilization of I factor RNA for efficient targeting, through Loc+ patterning, to the nuclear neighborhood where downstream steps of the retrotransposition process occur.

Systemic antiviral silencing in plants.

RNA silencing controls numerous developmental processes in eukaryotic organisms from fungi, plants, to animals. In plants as well as in animals, this system of RNA regulation functions as part of an immune response against invading viruses. From transitive RNA silencing to virus-induced gene silencing (VIGS), the systemic effects are proven to be the core of RNA silencing. This article reviews the latest advances in view of the effect of cellular RDR6, an RNA-dependent RNA polymerase (RdRp), on systemic RNA silencing, systemic virus silencing, and discusses the abilities of viral suppressors in modulating RNA silencing efficiency to establish effective infection.

A single amino acid residue of RNA-dependent RNA polymerase in the Potato virus X genome determines the symptoms in Nicotiana plants.

A Potato virus X (PVX) strain, PVX-OS, causes a necrotic mosaic in Nicotiana benthamiana and ring spot mosaic in N. tabacum cv. SamsunNN. By contrast, strain PVX-BS causes a mild mosaic in N. benthamiana and systemic asymptomatic infection in N. tabacum cv. SamsunNN. To investigate the viral determinant of this difference, we produced various infectious cDNA clones chimeric between these PVX genomes and clones with point mutations introduced by site-directed mutagenesis. Inoculation tests with these clones mapped the symptom determinant in Nicotiana plants to the 1422 amino acid residue in the region of the C-terminus of RNA-dependent RNA polymerase (RdRp). Western blot analysis and local lesion assay indicated that virus accumulation in the infected leaves was similar for these PVX strains, suggesting that the symptom difference was not due to virus accumulation.

Mature reverse transcriptase (p66/p51) is responsible for low levels of viral DNA found in human immunodeficiency virus type 1 (HIV-1).

Reverse transcription of the HIV RNA genome is thought to occur in the host cell cytoplasm after viral adsorption. However, viral DNA has been isolated in cell-free virus particles. We have quantitated by polymerase chain reaction (PCR) amplification the amount of viral DNA in virions as compared to RNA. Virus produced by proviral DNA transfections of cos-7 cells or by chronically-infected H9 cells; neither of which express the cell surface CD4 receptor, contained at least 1000 times more viral RNA than DNA. In contrast, only 60 times more RNA than DNA was present in virus particles produced by transfection of Jurkat cells, which were CD4-positive and thus potentially susceptible to superinfection. Protease-defective virus, carrying only the precursor of reverse transcriptase (RT) p160gag-pol, contained virtually no detectable DNA. These results indicate that only mature RT (p66/p51) and not its precursor (p160gag-pol) is responsible for the presence of viral DNA in HIV.

Testing the reverse transcriptase model of somatic mutation.

Somatic hypermutation of the variable (V) regions of rearranged immunoglobulin genes leads to antibody affinity maturation. Although this process has been extensively studied, the mechanisms responsible for these multiple point mutations are still elusive. One mechanism that was proposed over 10 years ago by Steele and Pollard was that an intrinsic reverse transcriptase (RT) copies the nascent mRNA creating the large number of observed point mutations due to its high error rate. A cDNA copy of the mutated V region would then replace the endogenous DNA through a gene conversion-like event, thus integrating these point mutations into the genome. This model of hypermutation would account for the very high mutation rate, the presence of hotspots, strand bias, the requirement for transcription and localization of mutation within the immunoglobulin V region. Using AZT and ddC to inhibit endogenous RTs, we have assayed for somatic mutation using a murine in vivo model. Somatic mutation occurred at similar frequencies and with the same characteristics with or without treatment of RT inhibitors, suggesting that standard reverse transcription is not required for antibody V region hypermutation in the mouse.