Initiation Of Minus-strand Synthesis Research Articles

Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA–RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (−) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (−) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (−) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates.

A Novel Interaction of tRNALys,3 with the Feline Immunodeficiency Virus RNA Genome Governs Initiation of Minus Strand DNA Synthesis

Complementarity between nucleotides at the 5' terminus of tRNA(Lys,3) and the U5-IR loop of the feline immunodeficiency virus RNA genome suggests a novel intermolecular interaction controls initiation of minus strand synthesis in a manner analogous to other retroviral systems. Base pairing of this tRNA-viral RNA duplex was confirmed by nuclease mapping of the RNA genome containing full-length or 5'-deleted variants of tRNA(Lys,3) hybridized to the primer-binding site. A major pause in RNA-dependent DNA synthesis occurred 14 nucleotides ahead of the primer-binding site with natural and synthetic tRNA(Lys,3) primers, indicating it was not a consequence of tRNA base modifications. The majority of the paused complexes resulted in dissociation of the reverse transcriptase from the template/primer, as demonstrated by an assay limited to a single binding event. Hybridization of a tRNA mutant whose 5' nucleotides are deleted relieved pausing at this position and subsequently allowed high level DNA synthesis. Additional experiments with tRNA-DNA chimeric primers were used to localize the stage of minus strand synthesis at which the tRNA-viral RNA interaction was disrupted. Finally, replacing nucleotides of the feline immunodeficiency virus U5-IR loop with the (A)(4) sequence of its human immunodeficiency virus (HIV)-1 counterpart also relieved pausing, but did not induce pausing immediately downstream of the primer-binding site previously noted during initiation of HIV-1 DNA synthesis. These combined observations provide further evidence of cis-acting sequences immediately adjacent to the primer-binding site controlling initiation of minus strand DNA synthesis in retroviruses and retrotransposons.

Structural Studies of Model RNA Helices with Relevance to Aminoacyl-tRNA Synthetase Specificity and HIV Reverse Transcription

We describe high-resolution crystal structures of synthetic nucleic-acid fragments determined as part of an effort to understand determinants of sequence-specific protein binding on the level of double-helix structure. In a first set of experiments, 7-base-pair RNA duplexes representing the acceptor-stem helix of Escherichia coli tRNAAla and variants thereof were characterized at atomic resolution. The structures revealed a standard A-form double helix locally perturbed by a G·U wobble base pair at sequence position 3/70 of the tRNA. The G·U pair shows a characteristic hydration pattern which must be considered an integral part of the double-helix structure. It does not seem to exert a global effect on the duplex structure. A second experiment concerned the chimeric DNA-RNA hybrid structure formed transiently during initiation of minus-strand synthesis by the reverse transcriptase of HIV-1. The crystal structure of an 8-base-pair duplex with an RNA template strand derived from HIV-1 and a complementary strand representing the junction between the tRNALys,3 RNA primer and the newly synthesized DNA strand was solved at a resolution of 1.9 Å. As before, the double helix was found to adopt standard A-type conformation with only local variations of backbone conformation. Based on the global helix structure as present in the crystal, it remains difficult to explain the preference of the reverse-transcriptase-associated RNAse H activity for certain sites of the template strand. Structural plasticity near the main cleavage site in suggested to govern cutting preferences. In both systems investigated, structural studies by NMR spectroscopy were carried out by others in parallel. In both cases, the solution structures are in partial disagreement with the crystallographic results by describing a significantly higher level of deviation from the canonical A-conformation.

A branched stem-loop structure in the M-site of bacteriophage Qβ RNA is important for template recognition by Qβ replicase holoenzyme

An internal site on bacteriophage Qβ RNA, the M-site (map position 2545 to 2867), was recently shown by us to be required for the efficient initiation of minus strand synthesis by Qβ replicase. In a more detailed mutational analysis, we show here that the essential elements within the M-site consist of two successive stem-loop structures followed by a bulge loop of unpaired purines, located at nucleotides 2696 to 2754 on the tip of a long, imperfectly base-paired stalk. Mutational changes affecting the sequences of paired or unpaired nucleotides in this segment reduced the template efficiency only mildly. The only severe effects were observed when one of the helical stems or the unpaired bulge was completely deleted or substantially shortened. We conclude that the three-dimensional backbone arrangement of these three elements constitutes the feature recognized by replicase. The role of the long stalk remains undetermined, because mutations that either stabilized or disrupted its base-pairing barely affected template activity, and even deletion of a major portion of one of its strands did not cause complete inactivation. Earlier evidence had implicated protein S1 (the α subunit of replicase) as the mediator of the M-site interaction. The lack of an active M-site on the Qβ RNA template has the same quantitative and qualitative effects on template recognition as the absence of the S1 protein from replicase in the presence of wild-type RNA. We therefore believe that the M-site interaction explains most of the role of S1 protein in the replication of Qβ RNA by replicase.

Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase.

A turnip yellow mosaic virus RNA-dependent RNA polymerase activity was used to study the template requirements for in vitro minus strand synthesis, which is initiated specifically opposite the 3'-CCA that terminates the 3'-tRNA-like structure. A deletion survey confirmed earlier results suggesting the absence of minus strand promoter elements upstream of the pseudoknotted acceptor stem and 3'-terminus. Reiteration of this 27-nt domain provided two competing initiation sites. By varying the added downstream element, it was shown that the pseudoknotted domain could be functionally replaced by various simple stem/loops, although with some decrease in activity. The addition of varying numbers of consecutive -CCA- triplets to the 3' end of the tRNA-like structure resulted in accurate initiation from each added triplet. A similar spectrum of initiations occurred with an unstructured RNA consisting of 12 consecutive -CCA- triplets and no additional viral sequence. Substitution mutations revealed no influence on minus strand synthesis of the identity of the nucleotide immediately upstream of a -CC- initiation site, but a preference for a purine immediately downstream. The introduction of secondary structure into the linear template showed that the usage of potential -CCR- initiation sites is influenced by nonspecific secondary structure. We conclude that specificity arises from the requirement that a -CCR- sequence be sterically accessible. This mechanism is only applicable to interactions that do not involve RNA unwinding during site selection, but may be used commonly in positive strand RNA virus replication and be applicable to other RNA-protein interactions.

The role of the pseudoknot at the 3' end of turnip yellow mosaic virus RNA in minus-strand synthesis by the viral RNA-dependent RNA polymerase

The tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro. Deletions in the 5'-to-3' direction up to the pseudoknot structure did not result in a decrease of transcription efficiency. However, transcription efficiency was reduced twofold when a fragment of 21 nucleotides, comprising the 3'-terminal hairpin, was used as a template. tRNA(Phe) from yeast, Escherichia coli 5S rRNA, and the 3'-terminal 208 nucleotides of alfalfa mosaic virus RNA 3 could not be transcribed by the RNA-dependent RNA polymerase (RdRp) of TYMV. Various mutations in the sequences of loop regions L1 and L2 or of stem region S1 of the pseudoknot were tested to further investigate the importance of the pseudoknot structure. The results were compared with those obtained in an earlier study on aminoacylation with the same mutants (R. M. W. Mans, M. H. van Steeg, P. W. G. Verlaan, C. W. A. Pleij, and L. Bosch, J. Mol. Biol. 223:221-232; 1992). Mutants which still harbor a stable pseudoknot, as proven by probing its structure, have a transcription efficiency very close to that of the wild-type virus. Disruption of the pseudoknot structure, however, gives rise to a drop in transcription efficiency to about 50%. No indications of base-specific interactions between L1, L2, or S1 of the pseudoknot and the RdRp were found.

Interaction of Retroviral Reverse Transcriptase with Template–Primer Duplexes during Replication

Conversion of the single-stranded RNA of an invading retrovirus into double-stranded proviral DNA is catalyzed in a multi-step process by a single virus-coded enzyme, reverse transcriptase (RT). Achieving this requires a combination of DNA polymerase abd ribonuclease H (RNase H) activities, which are located at the amino and carboxy terminus of the enzyme, respectively. Moreover, proviral DNA synthesis requires that three structurally-distinct nucleic acid duplexes are accommodated by this enzyme, namely (a) A-form RNA (initiation of minus strand synthesis), non-A, non-B RNA/DNA hybrid (minus strand synthesis and initiation of plus strand synthesis) and B-form duplex DNA (plus strand synthesis). This review summarizes our current understanding of the manner in which retroviral RT interacts with this diverse array of nucleic acid duplexes, exploiting in many cases mutants unable to catalyze a specific event. These studies illustrate that seemingly 'simple' events such as tRNA-primed initiation of minus strand synthesis are considerably more complex, involving intermolecular tRNA-viral RNA interactions outside the primer binding site. Moreover, RNase H activity, generally thought to catalyze non-specific degradation of the RNA-DNA replicative intermediate, is required for highly specialized events including DNA strand transfer and polypurine selection. Finally, a unique structure near the center of HIV proviral DNA, the central termination sequence, serves to halt the replication machinery in a manner analogous to termination of transcription. As these highly specialized events are better understood at the molecular level, they may open new avenues of therapeutic intervention in the continuing effort to stem the progression of HIV infection and AIDS.