Siddhartha A.K. Datta

Nucleic acid binding and chaperone properties of HIV-1 Gag and nucleocapsid proteins

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ∼10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7s capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gags stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

In vitro characterization of the interaction between HIV-1 gag and human lysyl-tRNA synthetase

Human immunodeficiency virus type 1 (HIV-1) viral assembly is mediated by multiple protein-protein and protein-nucleic acid interactions. Human tRNALys3 is used as the primer for HIV reverse transcription, and HIV Gag and GagPol are required for packaging of the tRNA into virions. Human lysyl-tRNA synthetase (LysRS) is also specifically packaged into HIV, suggesting a role for LysRS in tRNA packaging. Gag alone is sufficient for packaging of LysRS, and these two proteins have been shown to interact in vitro using glutathione S-transferase pull-down assays. In vitro pull-down assays using truncated constructs have also revealed that residues important for homodimerization of Gag and LysRS are critical for the Gag/LysRS interaction. In this work, we report further in vitro characterization of the interaction between HIV Gag and human LysRS using affinity pull-down assays, fluorescence anisotropy measurements and gel chromatography. An equilibrium binding constant of 310 ± 80 nm was measured for the Gag/LysRS interaction. We also show that capsid alone binds to LysRS with a similar affinity as full-length Gag. Point mutations that disrupt the homodimerization of LysRS and Gag in vitro do not affect their interaction. These results suggest that dimerization of each protein per se is not required for the interaction but that residues involved in forming the homodimer interfaces contribute to heterodimer formation. Gel chromatography studies further support the formation of a Gag/LysRS heterodimer.

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid.

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.

Assembly Properties of Human Immunodeficiency Virus Type 1 Gag-Leucine Zipper Chimeras: Implications for Retrovirus Assembly

ABSTRACT Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant “Gag-Zipper” proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the “dimerizing” Gag-Zipper protein misassembles into very small particles, while the “trimerizing” protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the “dimerizing” Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.

Diverse interactions of retroviral Gag proteins with RNAs

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 of 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.

Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding

ABSTRACT 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 tRNA3 Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3 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 tRNA3 Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gags 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 Gags binding mode. We propose that MAs interactions with the PM trigger the switch between these two binding modes and stimulate Gags chaperone function, which may be important for the regulation of events such as tRNA primer annealing.

RNA Aptamers Directed to Human Immunodeficiency Virus Type 1 Gag Polyprotein Bind to the Matrix and Nucleocapsid Domains and Inhibit Virus Production

ABSTRACT Gag orchestrates the assembly and release of human immunodeficiency virus type 1 (HIV-1) particles. We explored here the potential of anti-Gag RNA aptamers to inhibit HIV-1 replication. In vitro, RNA aptamers raised against an HIV-1 Gag protein, lacking the N-terminal myristate and the C-terminal p6 (DP6-Gag), could bind to matrix protein (MA), nucleocapsid protein (NC), or entire DP6-Gag protein. Upon cotransfection with pNL4-3.Luc molecular clone into 293T cells, six of the aptamers caused mild inhibition (2- to 3-fold) in the extracellular capsid levels, and one aptamer displayed 20-fold inhibition. The reduction was not due to a release defect but reflected Gag mRNA levels. We hypothesized that the aptamers influence genomic RNA levels via perturbation of specific Gag-genomic RNA interactions. Binding studies revealed that the “NC-binders” specifically compete with the packaging signal (ψ) of HIV-1 for binding to DP6-Gag. Therefore, we tested the ability of two NC-binders to inhibit viruses containing ψ-region deletions (ΔSL1 or ΔSL3) and found that the NC-binders were no longer able to inhibit Gag synthesis. The inability of these aptamers to inhibit ψ-deleted viruses correlated with the absence of competition with the corresponding ψ transcripts lacking SL1 or SL3 for binding DP6-Gag in vitro. These results indicate that the NC-binding aptamers disrupt Gag-genomic RNA interaction and negatively affect genomic RNA transcription, processing, or stability. Our results reveal an essential interaction between HIV-1 Gag and the ψ-region that may be distinct from that which occurs during the encapsidation of genomic RNA. Thus, anti-Gag aptamers can be an effective tool to perturb Gag-genomic RNA interactions.

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs—only four nucleotides per genomic RNA—reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions.

Preparation of recombinant HIV-1 gag protein and assembly of virus-like particles in vitro.

The mechanism of assembly of retroviruses is not fully understood. Purification of retroviral Gag protein and studying its solution state and assembly properties might provide insights into retroviral assembly mechanisms. Here we describe a rapid method for the purification of Gag and its subsequent assembly into virus-like particles in a defined system in vitro. The purification scheme does not use affinity tags, but purifies the native protein by virtue of its high affinity for phosphocellulose, a property presumably related to the affinity of Gag proteins for nucleic acids.