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Previous Article | Next Article 
Journal of Virology, November 2003, p. 11896-11909, Vol. 77, No. 22
0022-538X/03/$08.00+0 DOI: 10.1128/JVI.77.22.11896-11909.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Molecular Biology and Biochemistry,1 Department of Microbiology and Molecular Genetics,2 Department of Pathology, University of California—Irvine, Irvine, California 92697-39003
Received 15 May 2003/ Accepted 18 August 2003
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ABSTRACT |
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INTRODUCTION |
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The mature virion contains a cone-shaped protein capsid that is probably not based on strict icosahedral symmetry (50) but on a derivative pattern using a conical hexagonal lattice (18). The conical capsid encapsidates two strands of genomic RNA, each of about 9 kb. Also enclosed in the capsid are molecules of replication enzymes, tRNAs (30, 31), and other less well-defined cellular proteins, such as cyclophilin A (52) and Tsg101 (12). The capsid, or core, of the particle is enclosed within a membrane which is studded with envelope glycoprotein composed of gp120 heads which are noncovalently linked to embedded gp41 transmembrane stalks. Between the core and the lipid membrane is a layer of matrix protein and some accessory proteins as well. The matrix protein is attached to the inside of the membrane through embedded myristyl groups (26, 58) and by electrostatic interactions with lipid head groups (11) which may target virion assembly. Indeed, over 90% of myristoylated Gag is found within lipid rafts (51, 63). Thus, a contiguous assembly of envelope protein-lipid membrane-matrix protein forms a dense shroud about the core. The model for the mature virion is still speculative, as pointed out by Coffin et al. (10), though its features have been increasingly refined by more recent analyses (6, 18, 73). The X-ray diffraction structures of gp120, gp41, and both the matrix and the capsid proteins have been determined, but in all cases, the structures are for only truncated or severely altered forms of the molecules (8, 27, 38, 45). The entire collection of currently known protein structures from retroviruses has been reviewed by Turner and Summers (64) and Frankel and Young (16). These structures, while of substantial value in their own right, have contributed only sparingly to further delineation of the overall architecture of the mature virion.
The model for the immature virion (10, 17, 70, 73) also remains speculative and is probably a compendium of structural states. Prior to cleavage of the Gag protein by HIV protease, the immature virus is composed of radially oriented copies of the Gag polyprotein that are aligned in sectors to produce local order and paracrystalline arrays (17, 70, 73). These polyproteins are anchored to the lipid membrane at the exterior through the matrix protein domain and at the center of the quasi-micelle by protein-RNA interactions mediated by the nucleocapsid domain (6, 73). The aligned copies of the Gag protein, of which there are estimated to be about 2,100 (6, 53), are subsequently cleaved to produce the layered arrangement of the structural proteins, including the conical core (18). The physical process by which this occurs in the interior of the maturing virion is largely unknown.
To this point, transmission electron microscopy (TEM) has been virtually the only source of images of HIV virions and HIV-infected cells, though scanning electron microscopy images have also been recorded at relatively low resolutions (28, 54). Cross sections of cells which have been embedded, stained, and visualized with TEM show the presence of virions near and at cell surfaces in the processes of maturation and budding (10, 21, 23, 68). Those images are revealing and quite remarkable, but they do have certain limitations. First, the cells have been embedded in plastic and later exposed to a heavy metal stain, either or both of which might introduce distortions into the cell and virion structures. In addition, one in fact sees only the pattern of stain from which structure must be inferred. The cells are usually compacted within a centrifuge pellet so that many cells are sectioned at oblique and arbitrary angles. It is virtually impossible to reconstruct and visualize an individual cell in its entirety. While lateral resolution with TEM is potentially high, determining the height, or third dimension, for nonsymmetric objects is problematic. Finally, TEM yields a projection image in which the entire stain distribution throughout and around the specimen is projected onto a single plane. Hence, superposition of components complicates and hinders interpretation of structure.
More recently, cryo-electron microscopy has been used to visualize both immature and mature retroviruses isolated from culture medium (6, 17, 70, 73) as well as isolated HIV capsids (6, 18, 33). These studies, though technically more demanding, are free of many of the artifacts introduced by staining and dehydration and have been quite revealing. Cryo-electron microscopy does, however, still suffer from problems in interpretation due to superposition of features.
Atomic force microscopy (AFM) has both virtues and limitations as well, but they tend to be complementary to those of TEM and cryo-electron microscopy. With AFM, samples may be imaged in air or in fluids, including culture medium or buffer, in situ, or after processing according to established histological procedures (7, 34, 48). AFM is nondestructive and nonperturbing, and it can be applied to soft biological samples, particularly when tapping mode scanning is employed (24, 62). For AFM investigation, in principle, a single cell or virion is all that is required, though several hundred of either may be present on the substrate. An isolated cell or virion may be imaged in its entirety, or many individuals may be examined. While lateral resolution, limited by the finite size of the cantilever tip, is only 5 to 10 nm, the height resolution is very good, better than half a nanometer. Finally, AFM produces three-dimensional, topological images that accurately depict the surface features of the object under study. In most ways, these images resemble common light photographic images.
We have shown in previous investigations of viruses by AFM that the technique is sufficiently incisive and reproducible that even individual capsomeres can be visualized on the surfaces of both plant and animal viruses. Furthermore, it has been shown that the structures of viruses observed by AFM are entirely consistent with models derived by X-ray crystallography and cryo-electron microscopy (1, 35, 37, 42, 43, 55). In a recent paper, we showed that AFM was a valuable approach for visualizing a similar retrovirus, murine leukemia virus (MuLV). For that investigation, both isolated virions free on glass substrates and virions emerging from infected cell surfaces were imaged by AFM, as were mutant viruses which formed aberrant virions (34). Here, we extend our AFM studies to HIV and HIV-infected lymphocytes.
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MATERIALS AND METHODS |
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Preparation of virus and cells for AFM imaging. Cells were cultured for 48 h at 37°C, and supernatant fluids were clarified of cells by low-speed centrifugation followed by filtration through 0.45-µm-pore-size cellulose acetate filters. Ten milliliters of filtered supernatant was centrifuged at 25°C for 4 h at 30,000 x g. Pelleted virus was resuspended in 40 µl of RPMI 1640 and allowed to adhere to poly-L-lysine (50 µg/ml)-coated glass coverslips (Sigma) for 18 h at 37°C. Coverslips were rinsed with 5 ml of phosphate-buffered saline (PBS) and then incubated with 0.1% glutaraldehyde for 30 min followed by 1.0% osmium tetroxide incubation for 30 min. Fixed HIV was dehydrated and inactivated by washing with 30, 50, and 70% solutions of ethanol for 7 min each. For imaging of uninfected and HIV-infected cells, approximately 10,000 nonadherent HIV-infected or uninfected H9 cells were layered onto poly-L-lysine-coated coverslips at a concentration of 100,000 cells/ml and allowed to adhere at 37°C in RPMI containing 11.5% fetal bovine serum for 18 h. Adherent cells were fixed and dehydrated essentially as described for HIV virions.
Virion lysis by nonionic and ionic detergents and drug treatment. For some experiments, HIV was disrupted by use of nonionic detergents prior to AFM imaging. Before glutaraldehyde fixation, HIV which had adhered to coverslips was exposed to various concentrations of detergents (0.10 and 0.5% Tween 20, 0.5 and 2% CHAPS, or 0.1 to 2% sodium dodecyl sulfate [SDS]) for 5 min and then washed twice with PBS. For other experiments, to determine whether virion morphology was altered in immature versus mature HIV, HIVLAI was produced in the presence of nelfinavir, an HIV protease inhibitor. HIVLAI-infected H9 cells were cultured in the presence of 4 µM nelfinavir, and supernatants were cleared of mature virions after 5 h by low-speed centrifugation of cells, which were then washed with PBS. Cells were resuspended in medium containing 4 µM nelfinavir and incubated for 48 h at 37°C. HIV was pelleted and processed for AFM as described above.
AFM. AFM instruments and procedures have been previously described for application to both cells (37) and viruses (36). Before imaging, fixed cells were dehydrated by washing the fluid cell with 30, 50, and 70% solutions of ethanol for 7 min each. Treatment with ethanol removes the lipids from the membranes of both cells and virions but leaves behind skeletal proteins of the membranes (2, 25) when fixation is done with glutaraldehyde alone. The use of osmium tetroxide postfixation, however, cross-links membrane lipids as well as proteins, and the lipids are therefore not removed by alcohol exposure. This additionally preserves many, if not all, membrane-associated proteins as well. After fixation, coverslips were washed with H2O and attached to metallic packs with double-sided tape. Coverslips were mounted on a J-piezoscanner of an atomic force microscope equipped with a fluid cell (Nanoscope III; Digital Instruments, Santa Barbara, Calif.).
Cantilevers with oxide-sharpened silicon nitride tips were 100 µm long. Images were collected in tapping height mode at frequencies of about 9.2 kHz, with a scanning frequency of 1 Hz. Because of the finite tip dimensions, isolated objects protruding above the background in AFM images appear broader than their true dimensions. For this reason, the dimensions of virions and their components were quantitated according to their heights above the background surface (36). Estimates of particle size given here are based on images obtained by height measurement of 100 to 200 particles on either glass substrates or on cell surfaces.
There is no evidence that the mild fixation with glutaraldehyde used here appreciably disturbs the structures of the cell and virus surfaces, at least to the resolution of these AFM studies. For example, we and others have fixed numerous protein and virus crystals with the same low concentrations of glutaraldehyde, and these crystals show no loss of resolution, increased mosaicism, or change in X-ray diffraction intensity distribution. We also, in previous studies (36), examined plant virus crystals under ethanol (even without glutaraldehyde fixation) and saw no obvious alteration in their surface structures compared to X-ray crystallography-determined structures, again to the resolution of the AFM images.
Direct evaluation of shrinkage of HIV due to fixation and/or dehydration was not possible for safety reasons, but extensive experiments using MuLV as a standard particle showed that fixation under our conditions produced no effect on dimensions. The same experiments in which particles were imaged in both water and ethanol, however, showed that dehydration of fixed virions, while not altering topological features, reproducibly resulted in a reduction in dimensions of 15% ± 2%. The particle sizes reported here have been corrected for shrinkage.
In the images presented here, the features are assigned a color indicative of their height above the substrate; hence, dark colors show very short (close to the substrate plane) features and light colors represent tall features, that is, features protruding above the substrate plane. White areas or points are features that are very high above the substrate plane in comparison to all other features on the object surface. It is important to bear in mind that AFM does not yield two-dimensional projections of objects onto a plane, as does TEM or light microscopy. Because height information is recorded as a function of a coordinate, a three-dimensional, topologically precise image is obtained of objects.
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RESULTS |
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Among the particles displayed on the glass substrate were some that were much smaller, on the order of 80 to 100 nm in diameter, and some that were much larger, on the order of 160 to 240 nm in diameter. These particles were not included in the averaging and were taken to be aberrant particles, the former likely lacking cores and the latter perhaps having multiple cores. Indeed, virions containing multiple cores have been reported by Gelderblom (20) and estimated by Briggs et al. (6) to comprise as much as 30% of the entire virus population based on cryo-electron microscopy. In addition, Briggs et al. (6) report that as many as 7% of HIV virions contain not conical but tubular cores, and these might also contribute to the variation in shape and size that we observe for virions by AFM. Qualitatively at least, the distribution of particle sizes that we observe is in good agreement with that reported by Briggs et al. (6).
The particles recovered from the medium and displayed on the glass substrate appear almost identical to those visualized on the surfaces of HIV-infected cells and are virtually indistinguishable from virions of MuLV imaged in an earlier study (34). It is also relevant to experiments described below that the background of cell and virus debris on the glass substrates, particularly protein, was remarkably low. Preparations of virus particles and the AFM images obtained, though somewhat dependent on the properties of individual AFM tips, were otherwise reproducible.
AFM of uninfected and infected cultured lymphocytes. H9 cells, both uninfected and infected with HIV, were similarly applied to poly-L-lysine-coated glass coverslips and visualized by AFM. The uninfected H9 cells were negative for HIV antigens (Fig. 2A), while essentially 100% of the HIV-infected cells were positive for HIV antigens (Fig. 2B) by immunofluorescence assay. AFM images of uninfected human lymphocytes taken from culture are shown in Fig. 3. In contrast to most other cells we have examined, such as fibroblasts and osteoclasts (37), the surfaces of both patient-isolated peripheral blood lymphocytes (data not shown) and lymphocyte cell lines tend to be far more complex. They are heavily decorated with protrusions of all sizes, microvilli, and membranous structures having a variety of forms. This is consistent with previous investigations on antigen-activated NK cells which show "numerous ruffled cell membrane projections" (5) and others describing complex microvilli formation and severe morphological changes of isolated lymphocytes (14). No two cells closely resemble one another, and there is a broad distribution of cell sizes, ranging from about 5 to 15 µm. It is consistent, as well, with results from scanning electron microscopy, yielding sizes of 8 to 10 µm (60), or with scanning AFM, yielding a size of 15 µm (49), along with varying cell shapes and surface features. This may reflect different stages of the cell cycle or different physiological states of individual cells. No particles resembling the virions seen in Fig. 1 were observed on the surfaces of uninfected cells.
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Some microvilli on infected cells, like those in Fig. 4d, are segmented, and the segments have dimensions corresponding to HIV particle sizes. This suggests that some microvilli may contain virus, and they may provide a venue for virus maturation. Viral particles budding through microvilli of infected primary monocytes have previously been shown by scanning electron microscopy and TEM (54). They are not essential, however, as most virions are observed by AFM to be budding directly from the plasma membrane.
Figure 5 is a gallery of HIV virions imaged by AFM as they emerge from, or are attached to, the surfaces of infected cells. Presumably, they represent a mixture of immature and mature particles and possibly virions in intermediate states. The particles have an average diameter of 127 nm, with a variation of 30 nm, based on measurement of 200 particles. Again, the distribution is broad, not because of measurement error, but because of the actual size diversity of the virions. A similar range of size diversity of particle diameters about the mean based on cryo-electron microscopy has been observed by Briggs et al. (6) as well. As with the free particles on a glass substrate, we again observed some particles that were considerably smaller than 127 nm which had the gross appearance of virions. These, we believe, may be particles lacking cores. Also frequently present were some anomalously large particles that otherwise had the appearance of virions. These had diameters in the range of 160 to 240 nm and probably represent virions that contained multiple cores. The external appearances of the virions having diameters of about 127 nm, the vast majority, are similar to one another and are the same as those of the free virions. The larger and smaller particles are usually more irregular in structure.
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Figure 6 shows a series of images typical of the many disruption experiments that we carried out. Recognizable HIV virions can be found, but two other products are now also present. These are damaged, split, and broken virus particles in the process of coming apart and a dense background of viral components, presumably the proteins and macromolecular components from more thoroughly degraded virions and virion cores.
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Examination of the partially disrupted particles is instructive in terms of understanding the organization of the HIV virion. First, particles are often encountered which exhibit a sector of missing protein on their surfaces, and this appears as a deep pit or cavity that penetrates into the interior of the virion. An example is shown in Fig. 6a. A possible explanation for these particles is that they have lost their nucleic acid-containing core through the opening and are now empty virions. Indeed, because of the finite size of the AFM tip, these cavities undoubtedly appear of less severity than is actually the case. Thus, although they may appear rather narrow in the micrographs, they are probably on the order of 40 to 50 nm in width and possibly large enough for a somewhat fluid capsid to pass. Judging from electron micrographs of isolated cores (33), their broad size and shape distributions suggest that the cores may indeed be quite malleable, even fluid, and could potentially escape through openings of the sizes that we observe.
Particles with missing sectors of protein also occur, but with less frequency, in populations of virus that are not treated with detergents and even in some virions seen on the surfaces of HIV-infected cells. Such particles were also observed in our studies of MuLV, and we in fact drew attention to them in our earlier paper (see Fig. 4 of reference 34). There is, we believe, an equally plausible explanation for the particles of HIV and MuLV that exhibit deep pits in their surfaces, like those seen in Fig. 1f and 6a. The envelope is formed by budding from the plasma membrane of the host cell and targeted to lipid rafts (40), which are estimated to be about 50 nm in diameter (57, 61, 65). The current model of viral budding involves binding of the 4-amino-acid Gag p6 PTAP motif to cellular Tsg101 protein and subsequent assortment via the cellular vacuolar protein-sorting pathway (13, 19, 44, 56). Pinching off the viral membrane from the host cell membrane is an ATP-dependent process which may generate a concentrated ring of both cellular and viral proteins. This pinch point, or budding scar, may repair seamlessly and components may redistribute so that no structural vestige remains on the envelope, i.e., the virion surface becomes spherically homogeneous. On the other hand, budding may result in an asymmetric distribution of proteins on the virion surface, making the point of budding more susceptible to disruption by detergents. This model is additionally consistent with particles exhibiting missing sectors, even in the absence of detergent, if the sealing process is at least occasionally imperfect.
Images of other virions were recorded that were at later stages of disruption and that had lost their interior cores completely. Examples are shown in Fig. 6b to d. Note that the virus particles do not appear to fall apart one subunit or sector at a time or bit by bit (as observed in a study of herpesvirus [55]), but they split or break like a fruit or nut, leaving behind thick "husks" or "peels" and an empty central cavity.
The remnant husks permit measurement of the thickness of the envelope of the virion surrounding the core. From the AFM images, these husks are about 35 nm thick. The cavity is about 40 to 50 nm in diameter, consistent with the reported size of the core (6, 18, 29). The thickness of the envelope is explicable only if we assume that it consists of a contiguous assembly of envelope protein, lipid membrane, and matrix protein. Indeed, this is reasonable since the gp41 envelope glycoprotein is known to be firmly embedded in the membrane on the outside, and the matrix protein is affixed to the same membrane on the inside through its myristyl modifications. There is even evidence, in fact, that the transmembrane portion of the envelope protein, gp41, may directly associate with the matrix protein through its cytoplasmic tail (72), thus creating a contiguous polypeptide linkage. Thus, the entire assembly forms a single thick-walled protective envelope for the apparently fragile, mobile, and perhaps even fluid core. Such a structure is consistent with the observation of Wilk et al. (70) that the matrix protein appears by cryo-electron microscopy to be a thin layer tightly associated with the inner face of the viral membrane.
Disruption of virions with ionic detergents. Use of high concentrations of the neutral detergent Tween 20, in the range of 2% or greater, produced complete destruction of the virions and left large amounts of macromolecular components and their aggregates on the substrate. We could, however, find no recognizable traces of the nucleic acid. Concluding that the neutral detergent was simply inadequate to completely dissociate protein-RNA complexes and reveal the nucleic acid, we explored the exposure of virions to SDS, a strong ionic detergent.
At concentrations of SDS of >0.1%, virions were disrupted completely and none were present on the substrate, although large aggregates of viral components were again observed. At high concentrations (>0.5%) of SDS, however, the aggregates were largely dissociated, and threads, often heavily decorated with residual proteins, became visible by AFM. Some examples are shown in Fig. 7. These are consistent in appearance with previous AFM observations of extended nucleic acid-protein complexes from other viruses, such as tobacco mosaic virus and cauliflower mosaic virus (36), herpes simplex virus (55), and vaccinia virus (43). These chains of protein molecules, linked together by the nucleic acid, are characteristic and readily identified. It is significant that simple repetitive nucleoprotein chains were not seen, but chains having large proteins or protein complexes of a variety of sizes bound to them, possibly representing not only nucleocapsid proteins but also larger replicative enzymes and enzyme complexes. The resistance of the protein-nucleic acid complexes to strong detergents suggests that the components of the complexes are indeed tightly bound to one another.
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DISCUSSION |
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The surface of HIV is comprised of the receptor binding protein gp120, which is, in turn, noncovalently connected to the lipid membrane by the protein gp41. The structure of a truncated form of the transmembrane gp41 is known from X-ray crystallography (8, 67), and in the crystal is a threefold symmetrical coiled-coil trimer of six long 
-helices. The structure of a core portion of gp120, lacking several external polypeptide loops and 90% deglycosylated, has also been solved by crystallography (38), as has the homologous protein from MuLV (15). gp120 is commonly assumed to be a symmetrical trimer, principally due to its association with the trimeric gp41, but that has, in fact, never been demonstrated. On the other hand, the envelope protein of human foamy virus (HFV) has convincingly been demonstrated to be a threefold symmetrical trimer by cryo-electron microscopy, both in its free form and when it is a part of the virion (70). By homology, therefore, one might reasonably expect the corresponding proteins of other retroviruses to have the same oligomeric structure.
The prominent tufts forming the surfaces of HIV particles in the AFM images must, according to their sizes, be aggregates of gp120. We have examined many hundreds of these tufts on many hundreds of particles and have found no evidence of even approximate threefold symmetry. The variation in size and shape of the tufts suggests that in the native HIV particle, gp120 forms surface clusters; however, they do not exist as closely associated symmetrical groups but in more arbitrary and varied arrangements.
The gp120 clusters are also inconsistent in terms of average size with symmetrical trimers. Their average diameter of about 200 ± 30 Å is significantly greater than that which might be expected for a trimer of 360 kDa. The surface area subtended by a cluster is more than twice as great. In addition, the tufts are not of consistent diameters but vary among themselves by about 30 Å or more. While the heavy glycosylation may contribute to both of these features, it is unlikely to entirely explain either. It seems probable that the individual gp120 monomers at the ends of gp41 stems, like heads of flowers in a bouquet, are otherwise separated from one another and make no systematic protein-protein contacts. Associations are arbitrary with neighbors, and the groupings therefore exhibit no consistent symmetry or shape.
A disordered arrangement of unliganded gp120 monomers is consistent with their unusually flexible and mobile character, their existence in multiple conformation states (38, 71), their conformational changes on binding ligands, such as CD4 (38, 46, 71), and their heavy coatings of oligosaccharides, which would tend to impede direct contact between polypeptide chains. It is supported, as well, by the observation that recombinant gp120, when expressed in insect cells, remains monomeric and does not crystallize as trimers (38), and neither does the homologous SU envelope protein from MuLV (15).
Aside from an anticipated structural homology with the envelope protein of HFV, the primary evidence suggesting that gp120 monomers form symmetric trimers on the surfaces of HIV stems from the threefold symmetrical arrangement of the truncated elements of gp41 in their crystals (8, 67). Even this, however, may be viewed with some doubt. Frankel and Young (16) pointed out that the crystallographic structure may represent the fusion-active form of the gp41 oligomer that occurs upon binding of gp120 to the CD4 receptor. They make a persuasive argument, based on four lines of evidence, that the symmetric arrangement observed by X-ray crystallography may not exist in the free viral particle.
While it might be argued that AFM is simply not perceptive enough to resolve the symmetry of such molecular arrangements, in other studies (35-37, 41, 42, 55) AFM has been used to directly visualize symmetrical groupings of proteins on the surfaces of numerous virus particles, many of which are smaller than HIV.
The clusters of gp120 do not form spikes on the surface of HIV as is commonly described in the literature (3, 10). The clusters are hardly protrusions at all. We suggest that the spikes observed by negative-staining electron microscopy may be an artifact of the penetration of heavy metal stain between envelope proteins. Indeed, the term "spike" appears to have assumed a rather imprecise, possibly misleading definition, and might best be used with caution. In the work of Briggs et al. (6), the spikes on the surfaces of HIV virions protruded about 7.5 nm, which is not inconsistent with the height above the virion surface that we observe by AFM for the tufts of protein. On the other hand, spikes of envelope protein are described on the surfaces of HFV that extend 13.8 nm above the surface (69), nearly twice the length of those on HIV. The gp41-gp 120 combination is probably better described as mushroom-shaped, with large, exposed exterior surfaces. The number of these gp120 clusters on individual virions, taken as the average from many particle images and assuming that one-third to one-half of the virus was visible, is close to 100 ± 20. This number is similar to that obtained by TEM (20) and the 70 to 140 trimers per virion estimated for simian immunodeficiency virus (9), but it is significantly greater than the 7 to 14 trimers per virion estimated for HIV by Chertova et al. (9). There is one complication, however, that could make our estimate somewhat problematic, and that is the quantity of host cell membrane proteins that may be incorporated into viral envelopes upon budding. If the amount is comparable to that of gp120, then substantially less of the gp120 could be present. That is, some of the protein tufts we observed might represent cellular proteins. It is not known whether virus envelope protein promotes exclusion of cellular proteins from the viral membrane, nor are there reliable estimates of the amount of cellular proteins generally incorporated. Examination of cell membranes from uninfected host cells by AFM, however, reveals a distribution of protein shapes and sizes that are far more diverse than we see on the more or less uniform surfaces of viruses. In particular, there is a much higher proportion of small proteins of <10 nm on normal cell surfaces. The appearance of the virus surfaces is not, in most respects, similar to host cell surfaces, and in particular, the many small proteins are lacking. Thus, we believe that the envelopes of the virions visualized by AFM are composed predominantly of gp120, with perhaps only occasional insertion of host cell proteins.
Our observation of anomalously small particles, both on glass substrates and on cell surfaces, suggests that some viruses may lack nucleic acid, or cores, and be otherwise empty. The very large virions of 160 to 240 nm in diameter are consistent with the findings of Gelderblom (20) and Briggs et al. (6) showing that as many as 30% of all virions may contain two or even three conical cores and that the diameters of these multicapsid virions are proportionately greater. The anomalously small and large particles, as well as the large variation of wild-type particle size about the mode of the distribution, suggest that virion size is a flexible property and is largely determined by the virion contents or the process of viral budding. Indeed, no two viral particles are likely to be exactly the same in either composition or structure. This is also the conclusion of Briggs et al. (6) based on entirely independent data.
Some AFM images suggest that the core of the virion can be expelled through a channel in the envelope surrounding it, as evidenced by the observed deep pit in some virions, though the explanation that these pits in fact represent a budding scar is also plausible. Indeed, such pits are observed by TEM after depletion of cholesterol from the virion by use of ß-cyclodextrin (22).
Alternatively, the core may be lost by the splitting open of the virion, which only then falls apart in a piecemeal manner. From the thickness of the shells, or husks, which remain (about 35 nm), we conclude that they must include not only the lipid membrane and external envelope protein but the matrix protein on the interior of the membrane as well. This implies that the matrix protein is indeed firmly joined to the membrane and is physically contiguous with the envelope protein. This structure appears to be stable, as depletion of cholesterol from the membrane of HIV leads to disrupted virions from which the mature Gag proteins are lost but the envelope glycoproteins remain associated with a virion-like particle (22). Interestingly, matrix protein was lost with ß-cyclodextrin treatment, further supporting the interpretation that myristoylated matrix protein remains associated with cholesterol found in both lipid rafts and the viral membrane.
Although adding little to the existing model of the virion, the appearance of extended protein-RNA complexes does confirm a few points. The nucleic acid is not naked but is tightly complexed with proteins, as evidenced by its obscurity in the presence of mild detergents and its appearance only when treated with SDS. The proteins are probably chiefly the nucleocapsid protein, as expected, but other, larger proteins or protein complexes are also bound to the nucleic acid. These could include the enzymes that are essential for replication.
The tight association that we observe between proteins and nucleic acid in the presence of detergents is consistent with the very positively charged nucleocapsid protein binding strongly to RNA. It would be difficult to argue that such interactions, with their condensing and organizing functions, do not also take place in the central lumens of immature particles and thereby facilitate alignment of uncleaved Gag polyproteins. This supports the hypothesis of both Briggs et al. (6) and Yeager et al. (73) that assembly of virus is likely to be a template-directed process driven by protein-nucleic acid interactions at the center of the particles.
From conventional negative-staining electron microscopy and, even more, from cryo-electron microscopy, it has become increasingly evident that maturation of retroviral particles is accompanied by a dramatic redistribution of components and vast internal structural rearrangement (6, 17, 70, 73). This is brought about by HIV protease cleavage of the Gag proteins. There is no indication from previous analyses, however, that this produces any change in the size or the external appearance of the virions. Our experiments with virus infection in the presence of protease inhibitors are entirely consistent with those investigations. We also find no significant change in virion diameter when mature and immature particles are compared, nor do we find any perceptible change in their external appearance.
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ACKNOWLEDGMENTS |
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This research was supported by a contract from NASA and a grant from the NIH and in part by a grant from the California University-Wide Biotechnology Training program (J.G.V.). W.E.R. is a Burroughs-Wellcome Fund Clinical Scientist in Translational Research.
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