Atomic Force Microscopy Investigation of Human Immunodeficiency Virus (HIV) and HIV-Infected Lymphocytes

Abstract

Isolated human immunodeficiency virus (HIV) and HIV-infected human lymphocytes in culture have been imaged for the first time by atomic force microscopy (AFM). Purified virus particles spread on glass substrates are roughly spherical, reasonably uniform, though pleomorphic in appearance, and have diameters of about 120 nm. Similar particles are also seen on infected cell surfaces, but morphologies and sizes are considerably more varied, possibly a reflection of the budding process. The surfaces of HIV particles exhibit "tufts" of protein, presumably gp120, which do not physically resemble spikes. The protein tufts, which number about 100 per particle, have average diameters of about 200 Å, but with a large variance. They likely consist of arbitrary associations of small numbers of gp120 monomers on the surface. In examining several hundred virus particles, we found no evidence that the gp120 monomers form threefold symmetric trimers. Although >95% of HIV-infected H9 lymphocytic cells were producing HIV antigens by immunofluorescent assay, most lymphocytes displayed few or no virus on their surfaces, while others were almost covered by a hundred or more viruses, suggesting a dependence on cell cycle or physiology. HIV-infected cells treated with a viral protease inhibitor and their progeny viruses were also imaged by AFM and were indistinguishable from untreated virions. Isolated HIV virions were disrupted by exposure to mild neutral detergents (Tween 20 and CHAPS) at concentrations from 0.25 to 2.0%. Among the products observed were intact virions, the remnants of completely degraded virions, and partially disrupted particles that lacked sectors of surface proteins as well as virions that were split or broken open to reveal their empty interiors. Capsids containing nucleic acid were not seen, suggesting that the capsids were even more fragile than the envelope and were totally degraded and lost. From these images, a good estimate of the thickness of the envelope protein-membrane-matrix protein outer shell of the virion was obtained. Treatment with even low concentrations (<0.1%) of sodium dodecyl sulfate completely destroyed all virions but produced many interesting products, including aggregates of viral proteins with strands of nucleic acid.

Atomic Force Microscopy Analysis of Icosahedral Virus RNA

“…The images were collected in tapping mode 51,52 at frequencies of about 300 kHz. The forces were similar to those employed in earlier studies on viruses 46,53,54 and biological crystals. [55][56][57] There is considerable previous AFM work concerning double-stranded DNA 49,58,59 but, with some exceptions, 58,[60][61][62] rather little on single-stranded RNA.…”

Atomic Force Microscopy Investigation of Viruses

“…Larger icosahedral plant viruses in crystalline form were studied subsequently (15)(16)(17)(18)(19). The first AFM studies of noncrystalline viruses were retroviruses on cell surfaces (20)(21)(22), again, principally because they were immobilized by their association with cell surfaces. Single particles of larger viruses, and helical viruses, were eventually visualized by AFM, and these included tobacco mosaic virus, cauliflower mosaic virus, Tipula iridescent virus (15,23), herpes simplex virus (24), vaccinia virus (25,26), and mimivirus (9).…”

An atomic force microscopy investigation of cyanophage structure

“…The substrates were then either dried and imaged, or washed two to three times with water before analysis. Fixation with glutaraldehyde has been shown in previous studies not, to the resolution of the AFM technique, to perturb the surface structure of viral particles, and poly-l-lysine insures adherence of cells, virus particles, and their components (Kuznetsov et al, 2001(Kuznetsov et al, , 2002(Kuznetsov et al, , 2003(Kuznetsov et al, , 2004(Kuznetsov et al, , 2005a.…”

“…AFM analysis was carried out using a Nanoscope III multimode instrument (Veeco Instruments, Santa Barbara, CA). Imaging procedures were fundamentally the same as described for previous investigations of viruses (Kuznetsov et al, 2001(Kuznetsov et al, , 2002(Kuznetsov et al, , 2003; and RNA (Kuznetsov et al, 2005b;Kuznetsov and McPherson, 2006). For scanning in air, silicon tips were employed.…”