An unusual topological structure of the HIV-1 Rev response element.

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.

HIV-1 Gag protein with or without p6 specifically dimerizes on the viral RNA packaging signal.

The HIV-1 Gag protein is responsible for genomic RNA (gRNA) packaging and immature viral particle assembly. Although the presence of gRNA in virions is required for viral infectivity, in its absence, Gag can assemble around cellular RNAs and form particles resembling gRNA-containing particles. When gRNA is expressed, it is selectively packaged despite the presence of excess host RNA, but how it is selectively packaged is not understood. Specific recognition of a gRNA packaging signal (Psi) has been proposed to stimulate the efficient nucleation of viral assembly. However, the heterogeneity of Gag-RNA interactions renders capturing this transient nucleation complex using traditional structural biology approaches challenging. Here, we used native MS to investigate RNA binding of wild-type (WT) Gag and Gag lacking the p6 domain (GagΔp6). Both proteins bind to Psi RNA primarily as dimers, but to a control RNA primarily as monomers. The dimeric complexes on Psi RNA require an intact dimer interface within Gag. GagΔp6 binds to Psi RNA with high specificity in vitro and also selectively packages gRNA in particles produced in mammalian cells. These studies provide direct support for the idea that Gag binding to Psi specifically promotes nucleation of Gag-Gag interactions at the early stages of immature viral particle assembly in a p6-independent manner.

Structure and architecture of immature and mature murine leukemia virus capsids.

Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein-protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.

Nucleic acid-induced dimerization of HIV-1 Gag protein.

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. In vitro, it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element.

The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other.IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A" shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly.IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication.

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation.

UNLABELLED: By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals--electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE: Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

UNLABELLED: HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE: Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus.

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein.

UNLABELLED: Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCE: Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex.

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

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.

Biochemical and biological studies of mouse APOBEC3.

UNLABELLED: Many murine leukemia viruses (MLVs) are partially resistant to restriction by mouse APOBEC3 (mA3) and essentially fully resistant to induction of G-to-A mutations by mA3. In contrast, Vif-deficient HIV-1 (ΔVif HIV-1) is profoundly restricted by mA3, and the restriction includes high levels of G-to-A mutation. Human APOBEC3G (hA3G), unlike mA3, is fully active against MLVs. We produced a glutathione S-transferase-mA3 fusion protein in insect cells and demonstrated that it possesses cytidine deaminase activity, as expected. This activity is localized within the N-terminal domain of this 2-domain protein; the C-terminal domain is enzymatically inactive but required for mA3 encapsidation into retrovirus particles. We found that a specific arginine residue and several aromatic residues, as well as the zinc-coordinating cysteines in the C-terminal domain, are necessary for mA3 packaging; a structural model of this domain suggests that these residues line a potential nucleic acid-binding interface. Mutation of a few potential phosphorylation sites in mA3 drastically reduces its antiviral activity by impairing either deaminase activity or its encapsidation. mA3 deaminates short single-stranded DNA oligonucleotides preferentially toward their 3' ends, whereas hA3G exhibits the opposite polarity. However, when packaged into infectious ΔVif HIV-1 virions, both mA3 and hA3G preferentially induce deaminations toward the 5' end of minus-strand viral DNA, presumably because of the sequence of events during reverse transcription in vivo. Despite the fact that mA3 in MLV particles does not induce detectable deaminations upon infection, its deaminase activity is easily detected in virus lysates. We still do not understand how MLV resists mA3-induced G-to-A mutation. IMPORTANCE: One way that mammalian cells defend themselves against infection by retroviruses is with APOBEC3 proteins. These proteins convert cytidine bases to uridine bases in retroviral DNA. However, mouse APOBEC3 protein blocks infection by murine leukemia viruses without catalyzing this base change, and the mechanism of inhibition is not understood in this case. We have produced recombinant mouse APOBEC3 protein for the first time and characterized it here in a number of ways. Our mutational studies shed light on the mechanism by which mouse APOBEC3 protein is incorporated into retrovirus particles. While mouse APOBEC3 does not catalyze base changes in murine leukemia virus DNA, it can be recovered from these virus particles in enzymatically active form; it is still not clear why it fails to induce base changes when these viruses infect new cells.

An immature retroviral RNA genome resembles a kinetically trapped intermediate state.

UNLABELLED: Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. IMPORTANCE: The structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.

A conformational transition observed in single HIV-1 Gag molecules during in vitro assembly of virus-like particles.

UNLABELLED: The conformational changes within single HIV-1 Gag molecules that occur during assembly into immature viruses are poorly understood. Using an in vitro assembly assay, it has been proposed that HIV-1 Gag undergoes a conformational transition from a compact conformation in solution to an extended rod-like conformation in virus-like particles (VLPs). Here we used single-molecule Förster resonance energy transfer (smFRET) to test this model by directly probing the conformation of single HIV-1 Gag molecules. We demonstrate that monomeric HIV-1 Gag lacking the p6 domain and the N-terminal myristoyl moiety is found in solution predominantly in a compact conformation. Gag in this conformation, and in the presence of nucleic acid, assembles into 30-nm-diameter particles. However, with the addition of inositol hexakisphosphate, Gag adopts a linear conformation and assembles into full-sized ∼100-to-150-nm-diameter VLPs. Parallel fluorescence correlation spectroscopy measurements show that this conformational transition occurs early in the assembly process when Gag oligomers are small, perhaps as early as upon dimerization. Thus, smFRET measurements confirm that HIV-1 Gag transitions from a compact to a linear conformation during the formation of VLPs. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid. IMPORTANCE: The establishment of single-molecule fluorescence techniques reveals the conformational state of individual HIV-1 Gag molecules prior to and during in vitro assembly into virus-like particles. The data demonstrate that Gag in distinct conformations forms particles with different morphologies. In the compact conformation, in the presence of nucleic acid, Gag forms spherical particles of a diameter of approximately 30 nm. In the extended conformation, Gag forms spherical virus-like particles of approximately 100-nm diameter. The adoption of the extended conformation required the presence of inositol hexakisphosphate in addition to nucleic acid. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid.

Differential inhibitory effects of cyanovirin-N, griffithsin, and scytovirin on entry mediated by envelopes of gammaretroviruses and deltaretroviruses.

The antiviral lectins griffithsin (GRFT), cyanovirin-N (CV-N), and scytovirin (SVN), which inhibit several enveloped viruses, including lentiviruses, were examined for their ability to inhibit entry mediated by Env proteins of delta- and gammaretroviruses. The glycoproteins from human T-cell leukemia virus type 1 (HTLV-1) were resistant to the antiviral effects of all three lectins. For gammaretroviruses, CV-N inhibited entry mediated by some but not all of the envelopes examined, whereas GRFT and SVN displayed only little or no effect.

Tribute to Dr. Judith G. Levin (1934-2023).

Dr. Judith G. Levin passed away in Teaneck, NJ, USA, on 8 December 2023 [...].

Essential functions of inositol hexakisphosphate (IP6) in murine leukemia virus replication.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.IMPORTANCEInositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.