Late in the infectious cycle, structural proteins are preferentially translated and rapidly transported to the nucleus, with a lag period of 3 to 6 minutes after release from the polyribosomes (D’Halluin, 1995). The uptake of proteins by the nucleus is extremely selective, often requiring nuclear localisation signals (reviewed by Garcia-Bustos etal.,
1991). Several adenovirus proteins, namely, fibre (Shing Hong and Engler, 1991), ElA13s (Lyons et al., 1987), DBP (Morin et al., 1989), pTP (Zhao and Padmanabhan, 1988) and V (Russell and Kemp, 1995), contain arginine and lysine rich sequences that constitute these signals. The nuclear transport of viral proteins lacking NLS sequences may be facilitated by an association with proteins that do, as is the case with Ad-Pol which is chaperoned with pTP.
The first step in adenovirus assembly is the formation of hexon capsomers, which requires the L3 encoded protein II (hexon) and the L4 lOOkd protein. The L4 lOOkd protein binds to hexon in stoichiometric amounts while the latter is still attached to polyribosomes. The L4 lOOkd protein facilitates trimerisation of hexon monomers shortly after translation, then dissociates from the complex. Although the L4 lOOkd protein may be involved in transporting the hexon to the nucleus (Cepko and Sharp, 1983), there is evidence that the L3 encoded protein, pVI, is required for this function (Morin and Boulanger, 1984). On reaching the nucleus, hexon capsomers appear to be able to self assemble to form groups of nine (GON) hexon molecules which assemble to form the 20 sides of the virion molecule, while penton capsomers will assemble to form the 12 vertices. There is evidence that hexon capsomeres can assemble to form virus-like particles, and that formation of penton base may be a rate limiting step in the assembly process (Boudin et al., 1979). In contrast to hexon, the L2 encoded penton, and L5 encoded fibre proteins oligomerise in the absence of other viral proteins (Karayan etal., 1994).
After the construction of hexon shells, the assembly of virus particles proceeds through an ordered series of events (figure 1.7) which have been studied thoroughly using viral temperature sensitive mutants (blocked at different stages of assembly at the restrictive temperature), and by pulse-chase kinetic studies (Edvardsson et al., 1976; D'Halluin et al.,V¥]% and reviewed by D'Halluin, 1995). Initially, the first recognisable viral assembly intermediate is a light intermediate particle (buoyant density of 1.315g/cc in a CsCl equilibrium gradient), that contains the capsid structural components pVI, pVIII, pllla, and possibly IX, which confer stability to the virion structure. Although thermolabile mutants of pVIII (H5sub304; Liu etal., 1985), and protein IX (H5dl313; Colby and Shenk, 1981), have been identified, it is as yet unclear if both have any additional functional roles during the assembly process. Also associated with light intermediate particles are the 50kd and 39kd probable scaffold proteins and the L4 lOOkd protein. The light intermediate particles mature into heavy intermediate particles (buoyant density of 1.37g/cc) with the insertion of viral DNA and the removal of the 50kd and 39kd proteins.
The left end of the viral genome including the El A region has been shown to be inserted into the virion first, with nucleotide sequences at 200 to 400bp appealing to be essential for the encapsidation of the genome (Hammerskjold and Winberg, 1980). The factors involved in binding to the ds-acting elements have not been identified, although the IVa2 protein, which is present in light intermediate particles (Edvardsson et al., 1976; D'Halluin et al., 1978) possibly in association with the Ll 52kd/55kd proteins have been implicated (Gustin et al.,
1996), as they are both known to bind DNA. The possible role of the Ll 52kd/55kd proteins in packaging was suggested by the analysis of a temperature sensitive mutant (ts369), which accumulates incomplete particles at the restrictive temperature that cany only small segments of the left end of the genome (Hasson et al., 1989 and 1992). Similarly, possible roles for DBP and pllla in DNA packaging have been suggested by the analysis of mutants, tsl9 (Roovers et al., 1990), and H2tsll2 (D'Halluin et al., 1982), both of which accumulate light intermediate particles at the restrictive temperature.
The viral DNA is condensed into a core structure by the viral proteins V, pVII and possibly pmu (Vayda and Flint, 1987). It is unclear whether the core proteins are encapsidated with the viral genome, as they are not detected in either light or heavy intermediate particles (D'Halluin 1995; Schmid and Hearing, 1995).
Young virions Hexons Pentons Fibers and others structural proteins and scaffolding proteins CX H2fs112 Light intermediates Heavy intermediates 39 kDa 33 kDa p : 1 315 p 1.37 p: 1.345 p: 1.345 100 kDa pllia 100 kDa pills pTP 100 kDa pllia pTP V Ilia TP V 50 kDa 39 kDa 33 kDa pVI pVIII pVI pVIII pVI pVIII pVII VI VIII VII X. XI. XII
Figure 1.7: Assembly pathway for Ad2/5. The different stages of virus assembly have been indicated
at the top and the polypeptides only present in some structures are indicated below. Defective mutants in the assembly pathway are indicated at the top of the arrow at the right side. CX; cyclohexamide (reproduced from D'Halluin, 1995).
After DNA packaging, young virions are matured into infectious particles by the cleavage of structural proteins pVI, pVIII, pEHa, and the core proteins pVII, pTP, and pmu (Trembley et al., 1983; reviewed by Weber, 1995). There is evidence that the LI 52kd/55kd scaffold proteins are also cleaved during virus assembly, as probable cleavage products have been identified in mature virions (Hasson et al., 1992). None of these cleavages takes place in cells infected with the Ad2tsl temperature sensitive mutant at the non-permissive temperature. The mutation has been mapped to the L3 region encoding the viral 23kd protease, with a proline substituted for lysine at residue position 137 (Yeh-Kai et al., 1983). Viral 23kd protease activity has been detected in nuclei of infected cells, and inside mature virions, but not in Ad2tsl young virions accumulated at the non-permissive temperature (Bhatti and Weber, 1979). The temperature sensitive protease is synthesised and transported to the nucleus to become associated with incomplete particles in a manner similar to the wild type protease (Anderson, 1990; Rancourt et al., 1995). The defect apparently resides in a failure of the enzyme to be activated and encapsidated. The failure to dephosphorylate protein V is another interesting consequence of Ad2tsl infection (Weber and Khitoo, 1983), the significance of which is still unclear.
1.1.4 Progressive reorganisation of host cell structure.
The eytoskeletal and nuclear morphological changes associated with adenovirus infections of cultured cells are a result of complex interactions between host and viral components which allows for efficient production of virus particles. The cyLopathic effects that are observable by light microscopy, culminate in cell death and release of progeny virions. In recent years, information provided by electron microscopy techniques such as immunocytochemistry, autoradiography,m.y/ta-hybridisation and selective staining have been complementary in determining the structure-function relationships of cellular domains at the ultrastructural level, and are reviewed in this section.