Compartmentalization of Spermine and Spermidine in the Herpes Simplex Virion

Abstract

Enveloped particles of herpes simplex virus produced in human cells in culture contained spermidine and spermine, in a molar ratio of 1.6 4-0.2. The spermine present within the nucleocapsid is sufficient to neutralize at least 40% of the viral DNA. Disruption of the envelope with nonionic detergent and urea resulted in the selective loss of spermidine. Exogenous ornithine can function as a precursor to host and viral polyamines only before infection.Polyamines are known to bind to nucleic acids and phospholipids (1), and may be functional (2) as well as structural (3) components of eukaryotic cell organelles. Polyamines have previously been shown to occur in appreciable amounts in the DNA-containing bacteriophages T2 and T4 (4, 5). They have not been found in poliovirus, tomato bushy stunt, cucumber, and tobacco mosaic virus, all of which contain RNA and replicate in eukaryotic cells. Interestingly, another unenveloped RNA virus, turnip yellow mosaic virus, does contain polyamines, almost exclusively in the form of spermidine (6).We report here the presence of spermine and spermidine in herpes simplex virus. The virion of herpes simplex consists of a core that contains DNA (108 daltons) made in the nucleus, a multilayered capsid assembled in the nucleus from proteins made in the cytoplasm, and an envelope derived from the nuclear membrane modified by virusspecific glycoproteins (7). Of the two polyamines in the virion, spermine appears restricted to the nucleocapsid and spermidine to the viral envelope. This result is in contrast to the finding that putrescine and spermidine are present together in the nucleocapsids of T2 and T4 phages. Purification of Virus. Purified enveloped virus was prepared by a modification of a procedure developed by Spear and Roizman (manuscript in preparation). Briefly, the infected cells were swollen in hypotonic buffer and disrupted by four strokes of a Dounce homogenizer with a tight pestle. The cytoplasm was then separated from the nuclei and large debris by centrifugation, and layered onto a 3-30% (w/w) Dextran 10 (Pharmacia Fine Chemicals, Piscataway, N.J.) gradient prepared in phosphate buffer (0.01 M, pH 7.1) in place of the Tris buffer recommended in the original procedure. The gradients were then centrifuged at 20,000 rpm for 50 min at 50C in a Beckman SW25.3 rotor. A single band present at about the midpoint of the tube contained almost exclusively enveloped nucleocapsids free of host DNA (9) with relatively little contamination by membrane fragments.

Replication of Epstein-Barr Viral DNA

“…The products of lytic DNA replication are long concatemers (Bloss and Sugden 1994). They become the substrate for further processing eventually yielding cleaved, packaged, linear genome units bound in their preformed capsids with polyamines (Gibson and Roizman 1971). These "naked" viral DNAs are unmethylated, circularize in the recipient cell following infection, and eventually reside in the nucleus in which they are organized into chromatin and become methylated at cytosines in CpG dinucleotides (Shaw et al 1979;Fernandez et al 2009;Kalla et al 2010).…”

Role of ICP0 in the Strategy of Conquest of the Host Cell by Herpes Simplex Virus 1

“…In virions, the DNA is known to be bound by polyamines (56,57), leading to the question of whether viral DNA remains unencumbered from cellular proteins until it and its progeny are repackaged in virions. One scenario that incorporates a role for ICP0 is that viral DNA becomes loosely bound to proteins and that, in the absence of transcriptional activity, the rate of conversion into closed nucleosomal structures is cell type dependent.…”

pH Reduction as a Trigger for Dissociation of Herpes Simplex Virus Type 1 Scaffolds

“…DNA is an acidic molecule with a substantial negative charge, which, within cells, is normally neutralized by being bound to basic proteins or to polyamines. Although spermine is found within the capsid in HSV-1 virions and is thought to be important for close packing of the genome, it is not known when it enters the capsid, and the amounts detected are sufficient to neutralize only about half the charges on the DNA molecule (15,17). During packaging the viral DNA enters into a confined space by passing through a narrow channel, which may shield it from the cellular environment as has been suggested for the double-stranded RNA virus bluetongue virus (18).…”