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.

Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell.

Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of ∼20Å, native MLV Env presents as a hollow trimer (height ∼85Å, diameter ∼120Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by ∼35Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition.

Dynamics and restriction of murine leukemia virus cores in mitotic and interphase cells.

BACKGROUND: Murine leukemia viruses (MLVs) naturally infect unsynchronized T and B lymphocytes, thus, the incoming virus encounters both interphase and mitotic cells. While it is well accepted that MLV requires cell division to complete its replication cycle, it is not known if ab initio infection of mitotic cells can result in productive infection. This question is highly relevant since the milieu of mitotic cells is markedly different from this of interphase cells; e.g. lacking radial microtubule network and intact nuclear envelope. To follow MLV infection in mitotic and interphase cells in real-time, we employed our recently developed infectious MLV particles with labeled cores, cellular models expressing fluorescence markers of different intracellular compartments and protocols for reversible mitotic arrest of MLV-susceptible cells. RESULTS: Multi-wavelength live cell imaging was employed to simultaneously visualize GFP-labeled MLV cores, DiD-labeled viral or cellular membranes, and fluorescently-labeled microtubules or chromosomes. Cells were imaged either at interphase or upon mitotic arrest with microtubule poisons. Analysis of virus localization and trajectories revealed entry by endocytosis at interphase and mitosis, and correlation between viral mobility parameters and presence or absence of polymerized interphase microtubules. The success of infection of viruses that entered cells in mitosis was evidenced by their ability to reverse transcribe, their targeting to condensed chromosomes in the absence of radial microtubule network, and gene expression upon exit from mitosis. Comparison of infection by N, B or NB -tropic viruses in interphase and mitotic human cells revealed reduced restriction of the N-tropic virus, for infection initiated in mitosis. CONCLUSIONS: The milieu of the mitotic cells supports all necessary requirements for early stages of MLV infection. Such milieu is suboptimal for restriction of N-tropic viruses, most likely by TRIM5α.

Removal of xenotropic murine leukemia virus by nanocellulose based filter paper.

The removal of xenotrpic murine leukemia virus (xMuLV) by size-exclusion filter paper composed of 100% naturally derived cellulose was validated. The filter paper was produced using cellulose nanofibers derived from Cladophora sp. algae. The filter paper was characterized using atomic force microscopy, scanning electron microscopy, helium pycnometry, and model tracer (100 nm latex beads and 50 nm gold nanoparticles) retention tests. Following the filtration of xMuLV spiked solutions, LRV ≥5.25 log10 TCID50 was observed, as limited by the virus titre in the feed solution and sensitivity of the tissue infectivity test. The results of the validation study suggest that the nanocellulose filter paper is useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins.

The N-terminus of murine leukaemia virus p12 protein is required for mature core stability.

The murine leukaemia virus (MLV) gag gene encodes a small protein called p12 that is essential for the early steps of viral replication. The N- and C-terminal regions of p12 are sequentially acting domains, both required for p12 function. Defects in the C-terminal domain can be overcome by introducing a chromatin binding motif into the protein. However, the function of the N-terminal domain remains unknown. Here, we undertook a detailed analysis of the effects of p12 mutation on incoming viral cores. We found that both reverse transcription complexes and isolated mature cores from N-terminal p12 mutants have altered capsid complexes compared to wild type virions. Electron microscopy revealed that mature N-terminal p12 mutant cores have different morphologies, although immature cores appear normal. Moreover, in immunofluorescent studies, both p12 and capsid proteins were lost rapidly from N-terminal p12 mutant viral cores after entry into target cells. Importantly, we determined that p12 binds directly to the MLV capsid lattice. However, we could not detect binding of an N-terminally altered p12 to capsid. Altogether, our data imply that p12 stabilises the mature MLV core, preventing premature loss of capsid, and that this is mediated by direct binding of p12 to the capsid shell. In this manner, p12 is also retained in the pre-integration complex where it facilitates tethering to mitotic chromosomes. These data also explain our previous observations that modifications to the N-terminus of p12 alter the ability of particles to abrogate restriction by TRIM5alpha and Fv1, factors that recognise viral capsid lattices.

Design of hybrid lipid/retroviral-like particle gene delivery vectors.

Recombinant retroviruses provide highly efficient gene delivery and the potential for stable gene expression. The retroviral envelope protein, however, is the source of significant disadvantages such as immunogenicity, poor stability (half-life of transduction activity of 5-7 h at 37 °C for amphotropic murine leukemia virus), and difficult production and purification. To address these problems, we report the construction of efficient hybrid vectors through the association of murine leukemia virus (MLV)-like particles (M-VLP) with synthetic liposomes comprising DOTAP, DOPE, and cholesterol (φ/M-VLP). We conclude that the lipid composition is a significant determinant of the transfection efficiency and uptake of φ/M-VLP in HEK293 cells with favorable compositions for transfections being those with low DOTAP, low DOPE, and high cholesterol content. Cellular uptake, however, was dependent on DOTAP content alone. By extrusion of liposomes prior to vector assembly, the size of these hybrid vectors could also be decreased to ≈300 nm, as confirmed via DLS and TEM. φ/M-VLP were also robust on storage in terms of vector size and transfection efficiency and provided stable transgene expression over a period of three weeks. We conclude that the noncovalent combination of biocompatible synthetic lipids with inactive retroviral particles to form a highly efficient hybrid vector is a significant extension to the development of novel gene delivery platforms.

Consideration of the three-dimensional structure of core shells (capsids) in spherical retroviruses.

The problem of three-dimensional organization of retroviral cores has been a matter of interest for the past 30 years. The general opinion in favor of icosahedral symmetry based on electron microscopy observations was questioned when cryo-electron microscopy failed to provide convincing evidence in its favor. More recent studies by cryo-electron microscopy, X-ray crystallography and in vitro assembly of the CA domain of Human immuno deficiency virus (HIV), Murine leukemia virus (MuLV) and Rous sarcoma virus (RSV) threw new light on the organization of retroviral cores. In this communication we report how we produced a three-dimensional (3D) model of MuLV core using data from CA assembly on a lipid film [Ganser, B.K., Cheng, A., Sundquist, W.I., Yeager, M., 2003. Three-dimensional structure of the M-MuLV CA protein on a lipid monolayer: a general model for retroviral capsid assembly. EMBO J. 22, 2886-2892]. The resulting structure revealed that the molecular organization of the core shell is specific and the presence of a 5,3,2 rotational symmetry of the 3D model provides support for icosahedral shape of MuLV cores. The model made it possible to determine the diameter of the cores and calculate the number of CA copies as well as the molecular mass of a core of specific diameter. Thus MuLV cores 68 (or 81.6) nm in diameter consist of 1500 (or 2160) copies of CA. About 12% of molecules from fullerene-like Gag shells versus 71% of molecules of closely packed (core-like). Gag shells were not incorporated into the core shells (capsids). Our 3D models received support from X-ray data of MuLV CA NTD domain published by Mortuza et al. [Mortuza, G., Haire, L.F., Stevens, A., Smerdon, S.J., Stoye, J.P., Taylor, I.A., 2004. High resolution structure of a retroviral capsid hexameric amino-terminal domain. Nature 431, 481-485].

Mechanical properties of murine leukemia virus particles: effect of maturation.

After budding from the host cell, retroviruses undergo a process of internal reorganization called maturation, which is prerequisite to infectivity. Viral maturation is accompanied by dramatic morphological changes, which are poorly understood in physical/mechanistic terms. Here, we study the mechanical properties of live mature and immature murine leukemia virus particles by indentation-type experiments conducted with an atomic force microscope tip. We find that both mature and immature particles have an elastic shell. Strikingly, the virus shell is twofold stiffer in the immature (0.68 N/m) than the mature (0.31 N/m) form. However, finite-element simulation shows that the average Young's modulus of the immature form is more than fourfold lower than that of the mature form. This finding suggests that per length unit, the protein-protein interactions in the mature shell are stronger than those in the immature shell. We also show that the mature virus shell is brittle, since it can be broken by application of large loading forces, by firm attachment to a substrate, or by repeated application of force. Our results are the first analysis of the mechanical properties of an animal virus, and demonstrate a linkage between virus morphology and mechanical properties.

Intracellular assembly and budding of the Murine Leukemia Virus in infected cells.

BACKGROUND: Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle assembly describe the recruitment of the host vacuolar protein sorting machinery to the cell surface to induce the budding of new particles. Previous fluorescence microscopy study reported the vesicular traffic of the MLV components (Gag, Env and RNA). Here, electron microscopy (EM) associated with immunolabeling approaches were used to go deeply into the assembly of the "prototypic" MLV in chronically infected NIH3T3 cells. RESULTS: Beside the virus budding events seen at the cell surface of infected cells, we observed that intracellular budding events could also occur inside the intracellular vacuoles in which many VLPs accumulated. EM in situ hybridization and immunolabeling analyses confirmed that these latter were MLV particles. Similar intracellular particles were detected in cells expressing MLV Gag alone. Compartments containing the MLV particles were identified as late endosomes using Lamp1 endosomal/lysosomal marker and BSA-gold pulse-chase experiments. In addition, infectious activity was detected in lysates of infected cells. CONCLUSION: Altogether, our results showed that assembly of MLV could occur in part in intracellular compartments of infected murine cells and participate in the production of infectious viruses. These observations suggested that MLV budding could present similarities with the particular intracellular budding of HIV in infected macrophages.

Mutant murine leukemia virus Gag proteins lacking proline at the N-terminus of the capsid domain block infectivity in virions containing wild-type Gag.

We have investigated the properties of murine leukemia virus Gag mutants in which the p12-CA cleavage site is altered. In one mutant, the cleavage is blocked; in the other, the conserved proline at the N-terminus of CA has been replaced with glycine. No infectivity was detected in either mutant. Mutant particles cannot synthesize full-length DNA upon infecting permissive cells. Particles composed of a mixture of wild-type and mutant proteins have severely impaired infectivity. These mixed particles are defective in their ability to synthesize DNA upon infection, but this defect is less severe than the loss of infectivity. Thus, proteins lacking the correct N-terminus of CA inhibit DNA synthesis and also interfere with formation or integration of a full-length, normal provirus. The results imply that CA proteins function as part of a large, highly organized structure in reverse transcription and apparently at a later step as well.

Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells.

Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.

Cooperative effect of gag proteins p12 and capsid during early events of murine leukemia virus replication.

The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

Atomic force microscopy investigation of isolated virions of murine leukemia virus.

Virions of mouse leukemia virus spread on glass substrates were visualized by atomic force microscopy. The size distribution mode was 145 nm, significantly larger than that for human immunodeficiency virus particles. The distribution of particle sizes is broad, indicating that no two particles are likely identical in content or surface features. Virions possess knoblike protrusions, which may represent vestiges of budding from cell membranes. Particles which split open allowed imaging of intact cores with diameters of 65 nm. They also permitted estimation of viral shell thickness (35 to 40 nm) and showed the presence of a distinct trough between the shell and the core surface.

Atomic force microscopy imaging of retroviruses: human immunodeficiency virus and murine leukemia virus.

Retroviruses are membrane-enveloped, RNA-containing viruses that produce a wide range of threatening diseases in higher animals. Among these are human immunodeficiency virus (HIV), which produces acquired immune deficiency syndrome (AIDS) in humans, and murine leukemia virus (MuLV), which produces leukemias in rodents. We have obtained the first atomic force microscopy (AFM) images of these two retroviruses, both isolated from culture media and emerging from infected cell surfaces. The HIV virions are 127 nm diameter on average, and those of MuLV are 145 nm, although there are wide distributions about the means. The AFM images show the arrangement of the envelope protein, responsible for host cell entry, on the surfaces of both virions. Disruption of the viruses using detergents or physical means allowed us to visualize interior structures, including the outer shells of both MuLV and HIV, the cores of MuLV, and the nucleic acid of HIV complexed with core proteins. Using immunolabeling techniques borrowed from electron microscopy, we were able to demonstrate the binding of gold-labeled antibodies directed against the envelope protein of MuLV. The AFM images are revealing, not only in terms of surface topology, but in terms of interior features as well, and they reveal the eccentricities and uniqueness of individual virus particles rather than yielding the average member of the population. Further application of AFM to viruses associated with other pathologies may ultimately have a significant impact on the diagnosis and treatment of virus-promoted diseases.

Replication-competent hybrids between murine leukemia virus and foamy virus.

Replication-competent chimeric retroviruses constructed of members of the two subfamilies of Retroviridae, orthoretroviruses and spumaretroviruses, specifically murine leukemia viruses (MuLV) bearing hybrid MuLV-foamy virus (FV) envelope (env) genes, were characterized. All viruses had the cytoplasmic tail of the MuLV transmembrane protein. In ESL-1, a truncated MuLV leader peptide (LP) was fused to the complete extracellular portion of FV Env, and ESL-2 to -4 contained the complete MuLV-LP followed by N-terminally truncated FV Env decreasing in size. ESL-1 to -4 had an extended host cell range compared to MuLV, induced a cytopathology reminiscent of FVs, and exhibited an ultrastructure that combined the features of the condensed core of MuLV with the prominent surface knobs of FVs. Replication of ESL-2 to -4 resulted in the acquisition of a stop codon at the N terminus of the chimeric Env proteins. This mutation rendered the MuLV-LP nonfunctional and indicated that the truncated FV-LP was sufficient to direct Env synthesis into the secretory pathway. Compared to the parental viruses, the chimeras replicated with only moderate cell-free titers.

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV).

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.

Abundant defective viral particles budding from microglia in the course of retroviral spongiform encephalopathy.

A pathogenetic hallmark of retroviral neurodegeneration is the affinity of neurovirulent retroviruses for microglia cells, while degenerating neurons are excluded from retroviral infections. Microglia isolated ex vivo from rats peripherally infected with a neurovirulent retrovirus released abundant mature type C virions; however, infectivity associated with microglia was very low. In microglia, viral transcription was unaffected but envelope proteins were insufficiently cleaved into mature viral proteins and were not detected on the microglia cell surface. These microglia-specific defects in envelope protein translocation and processing not only may have prevented formation of infectious virus particles but also may have caused further cellular defects in microglia with the consequence of indirect neuronal damage. It is conceivable that similar events play a role in neuro-AIDS.

Ruthenium red preserves glycoprotein peplomers of C-type retroviruses for transmission electron microscopy.

Peplomers, the glycoprotein projections of the outer viral envelope, are distinctive for many viruses. Peplomers of retroviral C-type particles are fragile and are not preserved in standard preparations for transmission electron microscopy of thin sections, whereas the peplomers of B- and D- type retroviruses are usually preserved. Ruthenium red, extensively used in transmission electron microscopy to enhance the preservation of glycosylated proteins, was used in the preparation of three retrovirus-producing lymphoblastoid cell lines: murine SC-1 cells producing the C-type murine leukemia retrovirus LP-BM5 that causes immunodeficiency, human DG-75 cells producing a murine leukemia retrovirus, and human C5/MJ cells producing human T-cell lymphotropic virus type I (HTLV-I). Fixation of cells was carried out with ruthenium red present in the glutaraldehyde, osmium tetroxide, and the ethanol dehydration through the 70% ethanol step. The detailed structure of peplomers of these three different viruses was well preserved.

Incorporation of Pr160(gag-pol) into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag-Pol polyprotein.

The determinants critical for the incorporation of Pr160(gag-pol) into human immunodeficiency virus type 1 (HIV-1) particles were examined by cotransfecting cells with (i) a plasmid expressing wild-type Gag protein and (ii) a series of chimeric Gag-Pol expression plasmids in which individual murine leukemia virus (MLV) Gag regions and subdomains precisely replaced their HIV-1 counterparts. The presence of the MLV MA and NC Gag regions in the chimeric Gag-Pol precursor had no detectable effect on the incorporation of Gag-Pol into progeny virions. In contrast, the entire HIV-1 CA region was required to achieve wild-type levels of Gag-Pol assembly into particles; both the CA major homology region and the adjacent C-terminal CA sequences play dominant roles in this process yet, when assayed in the context of a chimeric Gag-Pol polyprotein, restored the defect affecting Gag-Pol incorporation to approximately half of the wild-type level.

Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix.

Incorporation of envelope glycoproteins into a budding retrovirus is an essential step in the formation of an infectious virus particle. By using site-directed mutagenesis, we identified specific amino acid residues in the matrix domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein that are critical to the incorporation of HIV-1 envelope glycoproteins into virus particles. Pseudotyping analyses were used to demonstrate that two heterologous envelope glycoproteins with short cytoplasmic tails (the envelope of the amphotropic murine leukemia virus and a naturally truncated HIV-2 envelope) are efficiently incorporated into HIV-1 particles bearing the matrix mutations. Furthermore, deletion of the cytoplasmic tail of HIV-1 transmembrane envelope glycoprotein gp41 from 150 to 7 or 47 residues reversed the incorporation block imposed by the matrix mutations. These results suggest the existence of a specific functional interaction between the HIV-1 matrix and the gp41 cytoplasmic tail.