Atomic Force Microscopy Analysis of Icosahedral Virus RNA

Abstract

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 8C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.

Atomic Force Microscopy Investigation of Viruses

“…Because they were immobilized on the surfaces of crystals, conditions were suitable for direct imaging of even the small 17-30-nm diameter virions. 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.…”

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.…”

Identification of DNA and RNA from retroviruses using ribonuclease A

“…The methods employed for AFM visualization of viruses and nucleic acids have been described elsewhere (Kuznetsov Y. G. Kuznetsov and A. McPherson: AFM of retroviral nucleic acid 279 et al 2000;2001;, 2005bMalkin et al 2003;Plomp et al 2002) including a previous paper in this journal (Kuznetsov et al 2004). In the studies described here, nucleic acid was spread on freshly cleaved mica and hydrated with 0.1 M HEPES buffer, or with that buffer containing about 0.1 mg/ml RNase A at pH 7 and 25°C.…”