Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z eff ) and nonelectrostatic (i.e., specific) component of binding, K d(1M) . Gag binds to Psi RNA with a dramatically reduced K d(1M) and lower Z eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z eff . These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

The innate immune response plays a key role as the primary host defense against invading pathogens including viruses. We have previously shown that treatment of human monocyte-derived macrophages with EBV-encoded dUTPase induces the expression of proinflammatory cytokines through the activation of NF-κB. However, the receptor responsible for EBV-encoded dUTPase-mediated biological effects is not known. In this study, we demonstrate that the purified EBV-encoded dUTPase activates NF-κB in a dose-dependent manner through TLR2 and requires the recruitment of the adaptor molecule MyD88 but not CD14. Furthermore, activation of NF-κB was abrogated by anti-TLR2, anti-EBV-encoded dUTPase blocking Abs and the overexpression of a dominant negative construct of MyD88 in human embryonic kidney 293 cells expressing TLR2. In addition, treatment of human monocyte-derived macrophages with the anti-EBV-encoded dUTPase Ab 7D6 or the anti-TLR2 Ab blocked the production of IL-6 by the EBV-encoded dUTPase. To our knowledge, this is the first report demonstrating that a nonstructural protein encoded by EBV is a pathogen-associated molecular pattern and that it has immunomodulatory functions. Although additional studies are necessary to define the signaling pathways activated by the EBV-encoded dUTPase and to determine its role in modulating immune responses to EBV infection, our results suggest that the dUTPase could be a potential target for the development of novel therapeutic agents against infections caused by EBV.

Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA 3 Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA 3 Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA 3 Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.

Plants synthesize a diversity of tannin structures but little is known about whether these different types have different oxidative activities in herbivores. Oxidative activities of hydrolyzable and condensed tannins were compared at pH 10 with two methods: EPR spectrometry was used to quantify semiquinone radicals in anoxic conditions and a spectrophotometric assay was used to measure the rate of browning of phenolics oxidized in ambient oxygen conditions. A little-studied group of hydrolyzable tannins (ellagitannins) contained the most active tannins examined, forming high concentrations of semiquinone radicals and browning at the highest rates. On average, galloyl glucoses and high-molecular-weight gallotannins had intermediate to low oxidative activities. Condensed tannins generally formed low levels of semiquinone radicals and browned most slowly. The results suggest that ellagitannin-rich plants have active oxidative defenses against herbivores, such as caterpillars, whereas the opposite may hold true for plants that contain predominantly condensed tannins or high-molecular-weight gallotannins.

Significance A highly conserved region of the HIV-1 RNA genome is responsible for regulating numerous steps of the retroviral life cycle, including initiation of reverse transcription. A complete understanding of the mechanisms controlling HIV-1 replication requires structural characterization of this RNA; unfortunately, however, its large size and conformational flexibility makes common methods of solving structures, such as X-ray crystallography and NMR, exceedingly difficult. The present study uses a solution technique, small-angle X-ray scattering coupled with computational molecular modeling, to characterize three ∼100-nucleotide RNAs that play central roles in HIV-1 replication. One of these domains mimics the L-shaped fold of tRNA, providing a structural basis for understanding how this genomic RNA coordinates interactions with a tRNA-binding host factor to facilitate initiation of reverse transcription.

Retrovirus particles are constructed from a single virus-encoded protein, termed Gag. Given that assembly is an essential step in the viral replication cycle, it is a potential target for antiviral therapy. However, such an approach has not yet been exploited because of the lack of fundamental knowledge concerning the structures and interactions responsible for assembly. Assembling an infectious particle entails a remarkably diverse array of interactions, both specific and nonspecific, between Gag proteins and RNAs.These interactions are essential for the construction of the particle, for packaging the viral RNA into the particle, and for placement of the primer for viral DNA synthesis. Recent results have provided some new insights into each of these interactions. In the case of HIV-1 Gag, it is clear that more than one domain of the protein contributes to Gag-RNA interaction.

Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasiequivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression.

The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA Lys3. Host cell tRNA Lys is selectively packaged into HIV-1 through a specific interaction between the major tRNA Lys -binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primerbinding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA Lys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA Lys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA Lys3 in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA Lys to increase the efficiency of tRNA Lys3 annealing to viral RNA.