| Abstract: | We recently identified 21 structural proteins in the virion of Spodoptera frugiperda ascovirus 1a (SfAV1a), a virus with a large, double-stranded DNA genome of 157 kbp, which attacks species of the lepidopteran family Noctuidae. The two most abundant virion proteins were the major capsid protein and a novel protein (P64) of 64 kDa that contained two distinct domains not known previously to occur together. The amino-terminal half of P64 (residues 1 to 263) contained four repeats (a recently recognized motif with an unknown function) of a virus-specific two-cysteine adaptor. Adjoined to this, the carboxy-terminal half of P64 (residues 279 to 455) contained 14 copies of a highly basic, tandemly repeated motif rich in arginine and serine, having an 11- to 13-amino-acid consensus sequence, SPSQRRSTS(V/K)(A/S)RR, yielding a predicted isoelectric point of 12.2 for this protein. In the present study, we demonstrate by Southwestern analysis that SfAV1a P64 was the only virion structural protein that bound DNA. Additional electrophoretic mobility shift assays showed that P64 bound SfAV1a as well as non-SfAV1a DNA. Furthermore, we show through immunogold labeling of ultrathin sections that P64 is a component of virogenic stroma and appears to be progressively incorporated into the SfAV1a DNA core during virion assembly. As no other virion structural protein bound DNA and no basic DNA-binding proteins of lower mass are encoded by the SfAV1a genome or were identified by proteomic analysis, our results suggest that P64''s function is to condense the large genome of this virus and assist in packaging this genome into its virion.Following nucleic acid synthesis, viral genomes are condensed and packaged into virions by a variety of mechanisms (17, 23, 24, 30). Essential to this process is neutralization of the negative electrostatic charge on phosphate groups of the nucleotide backbone by positively charged ions that would otherwise resist condensation of viral genomes during encapsidation. Viruses typically accomplish this by sequestering positive charges in the form of divalent cations, such as Mg2+, or through polyvalent polyamine cations, such as spermine and spermidine or other small, basic cationic proteins that bind and condense nucleic acids with high affinity (7, 14, 19, 22). An example of the latter group is histones, which are abundant in the nucleus and high in arginine and lysine content, their primary role being to condense host chromosomal DNA and package it into nucleosomes (3). During virion assembly, histones are involved in condensation of viral genomes by several DNA viruses, including polyomaviruses, papovaviruses (8, 9, 20, 27), and herpes viruses (25), and by RNA retroviruses (31).Despite their ubiquity in the nucleus, histones do not appear to be involved in genome condensation and packaging of most viruses. For example, viruses as diverse as hepatitis B virus (HBV), baculoviruses, and other DNA viruses that replicate in the nucleus encode cationic proteins rich in arginine and lysine, a characteristic they share with histones and protamines. The latter are small, arginine-rich proteins that replace histones on chromosomal DNA during the late stages of spermatogenesis (1). Similar proteins have been found in viruses. The carboxy terminus of the HBV core antigen (16), for example, and the small P6.9 (6.9-kDa) proteins of baculoviruses (38, 39) are protaminelike virion components known to bind and condense viral DNA for encapsidation.Viruses of the family Ascoviridae are highly pathogenic to larvae of the lepidopteran family Noctuidae. The virions typically are large (130 to 150 nm by 200 to 400 nm) and have complex symmetry and organization consisting of an inner particle containing a protein/DNA core that after assembly is enveloped to form the virion (11, 12). Although the ultrastructure of ascoviruses is markedly different from those of baculoviruses, iridoviruses, and entomopoxviruses (33), all of these viruses have large, double-stranded DNA (dsDNA) genomes, typically >100 kbp, that must be condensed for packaging. However, whereas the protaminelike P6.9 protein of baculoviruses is known to be a key protein involved in condensing genomic DNA and packaging it into the virion, the proteins responsible for this function in the other DNA viruses that attack insects remain unknown. Sequence analysis of the Spodoptera frugiperda ascovirus 1a (SfAV1a) genome (4), the type species, revealed no genes coding for small, protaminelike peptides that could facilitate condensation and packaging of its large genome (157 kbp). Moreover, of 21 proteins revealed by our recent proteomic analysis of the SfAV1a virion (35a), no small, cellular histones or histonelike protamines were detected. The two most abundant proteins in the SfAV1a virion were the major capsid protein (MCP) and a protein with a mass of 64 kDa (ORF048 or P64) (4, 35a). As MCPs and core DNA-condensing and -packaging proteins generally occur in amounts proportionally greater than those of other structural proteins, the relative abundance of P64, and its high pI of 12.2, suggested that it might be a key protein involved in condensing and packaging the SfAV1a genome. In the present study, we characterized P64 and show that it is a novel protein containing two distinct domains with two distinct motifs. The amino-terminal portion contains four repeats of a well-conserved virus-specific two-cysteine adaptor motif (pfam08793.1) (18), whereas the carboxy-terminal portion contains 14 copies of a previously uncharacterized motif rich in arginine and serine residues (SPSQRRSTS[V/K][A/S]RR) and, to a lesser extent, several lysine residues. Through a combination of DNA-binding and gel shift assays, along with immunogold labeling electron microscopy, we demonstrate that P64 is a major virion protein in the SfAV1a DNA/protein core and may be involved in packaging this virus'' genome into the virion. |
