their relative merits. These include expression in E. coli, yeast, insect cells via baculovirus, and mammalian cells, listed in order of quantities of protein produced. However, while it is indeed important to produce usable amounts of protein, it is also crucial that this protein is functionally active. Ideally it would be best to purify g p l20 direct from virions released from infected cells, but this requires the use of suitable containment facilities and gives very low yields necessitating growth very large culture volumes (Pyle et aL, 1987, Robey
etal., 1986).
3.1.2.1- Correct glycosylation and conformation is important for gpl20 func tion
G p l20 has a complex tertiary structure that allows it to bind CD4 and its various coreceptors. Dénaturation of this tertiary structure by SDS or similar agents results in a loss of this binding ability (Papandreou etal., 1996). It is also heavily glycosylated having 24 potential ^-glycosylation sites all of which are used, with about 11 having high mannose- type and hybrid-type structures and 13 having complex-type oligosaccharide structures (Leonard et al., 1990). This glycosylation makes up 55% of its molecular mass.
Folding of the glycoprotein takes place in the rER, not the Golgi apparatus, at the same time as glycosylation, and has a long half-life of 30 minutes (Fennie & Lasky, 1989). That the two take place at the same time has been shown to be important (Li et al., 1993). If g p l20 is stripped of glycosylation after it has been manufactured it can still bind CD4 although at a reduced affinity (Papandreou et a l, 1996). However if the g p l20 is not glycosylated in the first instance it is incapable of binding, suggesting that the process of glycosylation is essential for the generation of the proper conformation of gp 120 to provide a CD4-binding site; but, once folded, glycosylation is not necessary to maintain a conformation that can bind CD4 (Li et al., 1993). Mutation of all 24 of the potential glycosylation sites has revealed that most are not important for infectivity/CD4 binding, save for five, all located in the amino-terminal half of g p l20, that appear vital (Lee et al.,
1992), inferring that these are important for folding.
Investigation of the V3 loop has further emphasised the importance of glycosylation. NMR analysis of V3 loop peptides show that they undergo conformational change when glycosylated, altering their ability to bind antibody (Huang et al., 1997). Furthermore abnormal glycosylation or the loss of an iV-glycan site in the loop results in increased sensitivity to neutralisation by anti-V3 mAbs and sCD4 (Back et al., 1994, Papandreou & Fenouillet, 1998), implying that the A-glycan may interfere with the binding of neutralising antibodies by limiting accessibility to neutralising sites or inducing conformational change in the V3 loop (Back et al., 1994).
If a-glucosidase, an important enzyme in glycosylation, is inhibited, this leads to production of g p l20 that has decreased CD4 binding and altered V3 immunoreactivity (Fenouillet etal., 1996, Fenouillet etal., 1997). However some cell lines such as CHO can
overcome this (Fenouillet et aL, 1996) suggesting that they have alternative pathways for glycosylation (Fenouillet et aL, 1997, Papandreou & Fenouillet, 1998). It is not clear how much these alternative pathways affect g p l20 glycosylation and whether or not they are relevant to the in vivo situation.
Work has shown that some g p l20 may bind their coreceptors in the absence of CD4. SF2 g p l20 can bind CXCR4 in this way, but it binds better if CD4 is present. However if deglycosylated, SF2 g p l20 binds CXCR4 significantly better in the absence of CD4 than does glycosylated SF2, suggesting again that glycosylation is important in conformation and function (Bandres et aL, 1998).
3.1.2.2- Selection of a suitable cell line for expression
The use of E. coli or yeast was ruled out as they do not glycosylate proteins and are unable to fold g p l20 correctly (Bandres et aL, 1998, Sohn et aL, 1996). To produce glycosylated g p l20 either a baculovirus, or mammalian-based expression system needs to be used. Production of IIIB rgp 120 in different baculovirus systems results in rgp 120 that can bind CD4 well in most but not all cases (Moore et aL, 1990). However it is neutralised less well by human antibodies than IIIB g p l20 produced in CHO cells, and many antibodies in human sera recognise glycosylation patterns only found on mammalian rgp 120 (Moore
et aL, 1990). Furthermore baculovirus-expressed g p l20 has a lower molecular weight, suggesting that it is less heavily glycosylated containing only oligosaccharides of the low mannose type (Moore et aL, 1990, Moritz et aL, 1990).
Traditionally for mammalian expression CHO cells are used, but there are several lines of evidence to suggest that a human cell line would provide a more accurate pattern of glycosylation. For example Mizuochi et al. have shown that g p l20 produced from chronically infected human H9 lymphoblastoid cells have a greater diversity of oligosaccharide structures than rgp 120 produced in CHO cells (Mizuochi et aL, 1990). Also all mammals, other than humans and Old World primates, attach an alpha-galactosyl carbohydrate structure to glycoproteins. This allows normal human serum to neutralise non-human retroviruses via naturally occurring antibodies to this structure. Thus if HIV is passaged through human cells altered to add this moiety, the HIV can be neutralised by normal human serum (Reed et aL, 1997). Hence g p l20 produced in CHO and other non primate cell lines will have a carbohydrate structure not found in vivo.
Thus, in order to minimise the compromise between similarity to g p l20 found in vivo and ease of production, a human cell line was selected. This was the 293 human kidney fibroblast cell line. This cell line has been used extensively before for protein expression (Brightwell etal., 1997, Yamagami etal., 1994), and is adherent, making easier the task of transfection and selecting clonal expressing cell lines.
The promoter from the immediate-early gene 1 of human CMV, hCMV, provides a very strong drive for protein expression (Boshart et aL, 1985, Chapman et aL, 1991, Thomsen et aL, 1984). It contains an enhancer that is active in a broad range of host cell types (Boshart et aL, 1985, Chapman et aL, 1991, Sevarino et aL, 1987), and has been used extensively as a source of transcriptional signals for the expression of heterologous proteins, including SF2 g p l20 (Chapman etal., 1991), and has been employed successfully in 293 cells (Brightwell et aL, 1997). CMV-based mammalian expression vectors are cell- cycle and serum dependent. They function best in S phase, therefore for optimum expression cells must be cultured in the presence of serum and in cycle (Brightwell et aL, 1997).
3.1.2.4- The natural signal sequence for HIV Env is very inefficient
The HIV env gene includes code for a signal sequence to direct the newly formed g p l60 to the rER where it is folded, glycosylated and cleaved into g p l20 and gp41. This signal sequence of 30 amino acids is longer than most glycoprotein signal sequences and contains an average of 5 positively charged amino acids (Li etal., 1994), that result in poor yields of g p l20 as they lead to its retention within the rER (Li et aL, 1996), with less than 40% being secreted form the cell (Murphy et aL, 1993). In addition, this signal sequence proves difficult to cleave off further reducing its effectiveness (Li et aL, 1996). If the signal sequence is removed yields of protein are greatly increased, but are non-glycosylated (Li et aL, 1994). However replacement with a non-positively charged sequence from another glycoprotein increases significantly the amount of glycosylated protein made (Li et aL,
1994).
The signal sequence from human tissue plasminogen activator, tPA, has been used before to produce both FIV env (Wang & Mullins, 1995), SIV g p l60 (Rhodes et aL, 1994), and SF2 g p l20 (Chapman etal., 1991), hence its use in the expression system described in section 3.2.1.