Size, Composition, and Structure of the Deoxyribonucleic Acid of Herpes Simplex Virus Subtypes 1 and 2

Abstract

Studies of the size, composition, and structure of the deoxyribonucleic acid (DNA) of the F and G prototypes of herpes simplex virus (HSV) subtypes 1 and 2 (HSV-1 and HSV-2) showed the following. (i) As previously reported by Goodheart et al. HSV-1 and HSV-2 DNA have a buoyant density of 1.726 and 1.728 g/cm3, corresponding to 67 and 69 guanine i cytosine moles per cent, respectively. The difference in guanine plus cytosine content of the DNA species was confirmed by the finding of a 1 C difference in Tm. (ii) The DNA from purified virus on cocentrifugation with T4 DNA in neutral sucrose density gradients sedimented at 55S, corresponding to 99 ± 5 million daltons in molecular weight. HSV-1 and HSV-2 DNA could not be differentiated with respect to size. (iii) Cosedimentation of alkali-denatured DNA from purified virus with T4 DNA on alkaline sucrose density gradients consistently yielded several bands of single-stranded HSV DNA ranging from fragments 7 x 106 daltons to intact strands 48 X 106 daltons in molecular weight. Naturally occurring herpes simplex viruses (HSV) differ in a number of biological, physical, and immunological properties (5, 7, 8, 10, 17-19, 20, 27). As determined in neutralization tests, most [but not all (31)] fresh isolates fall into two subtype groups designated as 1 (HSV-1) and 2 (HSV-2). This report concerns the properties of HSV deoxyribonucleic acid (DNA). Previous reports have estimated the molecular weight of HSV-1 DNA to be 68 x 106 (24) to 100 x 106 daltons (2) and to contain 65 to 68 per cent guanine plus cytosine (10, 11, 20,22,23). HSV-2 DNA was reported (10) to contain 70.4 moles per cent guanine plus cytosine, and nothing was known of its size or relatedness to HSV-1 DNA. In the experiments described in this paper, we have directly compared the size, composition, and structure of HSV-1 and HSV-2 DNA. MATERIALS AND METHODS Solutions. Virus buffer consisted of 0.15 M NaCl and 0.02 M tris(hydroxymethyl)aminomethane (Tris)hydrochloride, pH 7.5. Neutral DNA buffer consisted of 1 M NaCl, 0.001 M ethylenediaminetetraacetic acid (EDTA), and 0.05 M Tris-hydrochloride, pH 7.5. Alkaline DNA buffer consisted of 0.8 M NaCl, 0.3 M NaOH, and 0.001 M EDTA. Reticulocyte standard buffer (RSB) consisted of 0.01 M sodium chloride, 0.001 M magnesium chloride, 0.01 M Tris, pH 7.5. Standard saline citrate (1 X SSC) consisted of 0.15 M sodium chloride, 0.015 M sodium citrate. SDS buffer consisted of 0.1 M NaCl, 0.001 M EDTA, 0.01 M Tris,

Transformation of mouse cells after infection with ultraviolet irradiation‐inactivated herpes simplex virus type 2

“…To determine whether transformed cells harbored intracellular complete virus, transformed cell monolayers were grown in roller bottles in 3H-thymidine labelled medium and the supernatant and cellular preparations were harvested separately and used for virus purification (Kieff et al, 1971). The detection of complete HSV particles or HSV DNA was monitored by beta scintillation spectrometry and electron microscopy.…”

“…Purification of HSV-2 was carried out using previously described methods (Kieff et a/., 1971). HSV DNA was prepared as described previously (Kieff et al, 1971).…”

“…Purification of HSV-2 was carried out using previously described methods (Kieff et a/., 1971). HSV DNA was prepared as described previously (Kieff et al, 1971). Briefly, HSV-2 was grown in roller bottles of HEp2 cells infected with a MOI of 1 PFU/cell, adsorbed 2 h at 37°C and washed with serum-free medium.…”

Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA

“…Two features of HSV DNA are relevant to this study. First, HSV-1 DNA is a linear double-stranded molecule of 97 x 106 molecular weight (1,19,33). Sheldrick and Berthelot (30) reported that terminal sequences are duplicated and inverted internally, dividing the HSV-1 DNA into two components designated as L and S. Subsequent studies in this laboratory (33) showed that the L (82% of total DNA) and S (18% of total DNA) components consist of defined regions designated as a, b, c, ul, and u, and arranged in the order ab-ul-b'a' and a'c'u.-ca, respectively.…”

Development of oncolytic virotherapy for the treatment of oral cancer – At the waiting stage for clinical use

“…The virion is a spherical particle with an average diameter of 186 nm that extends to 225 nm with spikes included. The bulk of packed DNA is linear, and the genome was previously estimated to be approximately 150 kb [25]. The entry of HSV into cells involves the interaction of at least five virion surface glycoproteins with several molecules (receptors) on the cell surface such as nectins, herpesvirus entry mediator (HVEM), and heparin sulfate, and is a consequence of the fusion of the envelope with the cellular membrane [26].…”