Biological and Structural Studies with an Adenovirus Type 2 Temperature-Sensitive Mutant Defective for Uncoating

Abstract

We compared some of the biological and structural features of an adenovirus type 2 temperature-sensitive mutant (tsl) defective for maturation cleavages and uncoating with wild-type (WT) virus. The cleavage defect caused tsl to produce virions at 39° that contained five precursor proteins (pTP, UK, PVI, PVΠ, PVIII). Coinfection of cells with such tsl virions and a variety of mutants or WT virus not only failed to complement tsl but actually depressed the infection by the second virus. The uncoating defect could only be overcome by multiplicity-dependent leakiness. The structure of the tsl virion was compared with that of WT virus by iodination with chloramine-T, chloroglycoluril and lactoperoxidase, by cross-linking, and by digestion with proteases. Aside from the presence of precursor proteins and the greater stability of tsl virions, no other differences were found that could account for the uncoating defect. Therefore, we postulate that this defect was caused by the greater stability imparted to the virion by precursor proteins PVI, PVΠ and PVIII.

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating

“…Elegant biophysics, cellular and molecular biology studies have given insight into the maturation process on the one hand (22,26,50) and the sequential uncoating on the other hand (8,9,12,13). The entry defect of ts1 had previously been related with increased stability (5,8,51). However, the structural and physical determinants conferring extra stability to the immature virion had not been characterized in detail nor had the structural changes undergone during uncoating been observed at single virion levels in threedimensional maps.…”

Different modes of inhibition of human adenovirus proteinase, probably a cysteine proteinase, by bovine pancreatic trypsin inhibitor

“…Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; Cbz, benzyloxycarbonyl; dTdP, 2,2'-dithiodipyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; DNS-GGACK, dansyl-L-glutarnylglycyl-Larginyl chloromethyl ketone; DTT, dithiothreitol; E-64, L-transepoxysuccinylleucylamido(4-guanidino)butane; HOAc, acetic acid; IAA, iodoacetic acid; pVIc, ll-amino-acid cofactor from the Cterminus of virion precursor protein pVI; PAGE, polyacrylamide gel electrophoresis; PCMB, p-chloromercuribenzoate; PMSF, phenylmethanesulfonyl fluoride; <Glu, pyroglutamic acid; rEP, recombinant endoproteinase; SBTI, soybean trypsin inhibitor; SDS, sodium dodecyl sulfate; TLCK, L-l-chloro-3-(4-tosylamido)-7-amino-2-heptanone HC1; TPCK, L-l-chloro-3-(4-tosylamido)°4-phenyl-2-butanone; ts-1, temperature-sensitive mutant H2ts-1 to cells but fail to initiate a productive infection [3,4]. The mutation in ts-1 was identified as a single base-pair change in a 204 codon open reading frame (L3 23-kDa) at the 3' end of the L3 family of late messages [5].…”

The active adenovirus protease is the intact L3 23K protein

“…The expression of this protein using Escherichia coli and baculovirus systems has lent support to this proposal (Anderson, 1990;Webster et al, 1993). The precursor proteins in adenovirus are required for virus assembly, indicating that the protease must be subject to some form of control in order to prevent premature proteolysis interfering with construction of the virus capsid (Hannan et al, 1983), and a number of theories have been put forward in this regard. Chatterjee & Flint (1987) presented evidence to suggest that the 23K protein is both phosphorylated and proteolytically activated whilst Houde & Weber (1990) went further and proposed that the adenovirus protease is autocatalytically cleaved at Ala(46)-Gly(47).…”