Picornavirus I proteinase and methods of making and using...

Details Picornavirus I proteinase and methods of making and using... Picornavirus I proteinase and methods of making and using...

1996-10-25

2001-01-30

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S006120, C435S068100, C435S252330, C435S320100, C536S023720

Reexamination Certificate

active

06180385

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is in the field of virology and molecular biology. More specifically the present invention relates to methods of making and using Picornavirus L proteinase peptides (PLPPs), including, but not limited to, expression of DNA encoding all or portions thereof, such as for Picornavirus L proteinase (PLP) and variants thereof, as well as methods for determining active sites and inhibitors of a PLP.
2. Related Background Art
Many viruses manipulate the cellular machinery of the host cell to their own advantage (Watson, J. et al. 1987
. Molecular Biology of the Gene
, Fourth Edition, Vol. II. The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.). One of the strategies of the single-stranded RNA Picornaviruses is to modify the translational machinery (Rueckert, R. R., in
Field's Virology
, Second Edition, B. N. Fields et al., eds., Raven Press, New York (1990), Vol. 1, pp. 507-548). Infection by the Picornaviruses, rhino-, entero- and aphthoviruses, leads to a reduction in the translation of capped host cell mRNAs. However, translation of viral RNAs is not affected as viral RNAs are not capped and translation of such RNAs is initiated internally (Sonenberg, N.,
Current Top. Microbiol. Immunol
. 161:23-47 (1990)).
The mechanism of this reduction is thought to occur through proteolytic cleavage of the eIF-4&ggr; polypeptide, which leads to an inability of the host cell to translate capped mRNAs (Lloyd, R. E. et al.,
J. Virol
. 61:2480-2488 (1987)). eIF-4&ggr; is a member of the eIF-4 group of translational initiation factors (the others are eIF-4A, eIF-4B, and eIF-4E) which collectively recognize the capped 5′-terminus of mRNA, unwind mRNA secondary structure, and permit the scanning by the 40S ribosomal subunit for the initiation codon (Merrick, W. C.,
Microbiol. Reviews
56:291-315 (1992); Rhoads, R. E.,
J. Biol. Chem
. 266:3017-3020 (1993)). eIF-4&ggr; is a polypeptide of calculated molecular mass 154 kDa but with an apparent mobility on SDS-PAGE corresponding to 220 kDa (eIF-4&ggr; was previously designated p220). The protein is always found as a collection of three to four bands on SDS-PAGE, but neither the heterogeneity nor aberrant mobility are understood (Yan R. et al.,
J. Biol. Chem
. 267:23226-23231 (1992)). The role of eIF-4&ggr; during initiation has not been elucidated, but recent observations on the distribution of eIF-4&ggr; polypeptides among the various initiation complexes (Joshi, B. et al.
J. Biol. Chem
. 269:2048-2055 (1994)) suggest the following model: eIF-4E first binds to the mRNA cap as a free polypeptide; it then forms a complex with eIF-4&ggr; which is already present on the 40S ribosomal subunit, thereby assembling the machinery which carries out unwinding of secondary structure. This, proteolytic cleavage of eIF-4&ggr; may separate the eIF-4E binding domain from the ribosome-binding domain and prevent the cap-dependent recruitment of mRNA to the ribosome.
The initiation of uncapped picornaviral RNAs can take place in the presence of proteolytically cleaved eIF-4&ggr; as it occurs internally on a 450 nucleotide segment of the 5′ UTR, known as the internal ribosome entry segment (IRES). This event takes place in all picornaviruses, although there may be differences in the mechanisms as there is little similarity between the IRES elements of rhino- and enteroviruses and those of cardio- and aphthovirus and that of Hepatitis A virus (Jackson, R. et al.,
Trends in Biochem. Sci
. 15:477-483 (1990)).
However, the relevance of the eIF-4&ggr; cleavage to the host cell shut-off is still controversial. Firstly, several reports claim that cleavage of eIF-4&ggr; alone is not sufficient to elicit the host cell shut-off (e.g., Bonneau & Sonenberg,
J. Virol
. 61:986-991 (1987); Perez & Carrasco,
Virology
189:178-186 (1992)). Secondly, the proteolytical activity carrying out the eIF-4&ggr; cleavage has been the subject of dispute. For rhino- and enteroviruses, a mechanism involving the activation of a cellular proteinase by the 2A proteinase had been proposed; cleavage of the eIF-4&ggr; molecule was then performed by the former (Kr{umlaut over (a)}usslich, H.-G. et al.,
J. Virol
. 61:2711-2718 (1987); Wyckoff, E. E. et al.,
Proc. Natl. Acad. Sci. USA
. 87:9529-9533 (1990); Wyckoff, E. E. et al.,
J. Virol
. 66:2943-2951 (1992)). However, recent experiments with purified recombinant 2A proteinases have contradicted this hypothesis. The findings that the 2A proteinase cleavage sequence on eIF-4&ggr; is similar to the preferred cleavage specificity of the proteinases and the ability of these 2A proteinases to cleave a peptide of this sequence show that the 2A proteinases do indeed cleave eIF-4&ggr; directly (Lamphear, B. J. et al.,
J. Biol. Chem
. 268:19200-19203 (1993); Sommergruber, W. H., et al.,
Virology
198:741-745 (1994)).
The situation in the Picornavirus aphthoviruses (e.g., Foot-and-Mouth Disease Virus, FMDV) is different; eIF-4&ggr; cleavage is mediated by the viral-encoded leader L proteinase and not by the 2A proteinase (Devaney, M. A. et al.,
J. Virol
. 62:4407-4409 (1988); Lloyd, R. E. et al.,
J. Virol
. 62:4216-4223 (1988)). However, the nature of the in vivo cleavage products is not clear; one report (Medina, M. E. et al.,
Virology
194:355-359 (1993)) states that they are identical to those found during poliovirus infection, whereas two reports describe different products (Lloyd, R. E. et al.,
J. Virol
. 62:4216-4223 (1988); Kleina & Grubman,
J. Virol
. 66:7168-7175 (1992)). The site of cleavage of the FMDV L proteinase on eIF-4&ggr; has not yet been identified. It is also not known whether cleavage is a direct event.
The nature of the L proteinase itself is also poorly understood. Amino acid sequence comparisons have indicated a similarity to papain-like thiol-proteinases (Gorbalenya, A. E. et al.,
FEBS Lett
. 288:201-205 (1991)); the inhibition of the enzyme by E64, a specific inhibitor of this class of proteinases supports this suggestion (Kleina & Grubman,
J. Virol
. 66:7168-7175 (1992)). Although the 2A proteinases of rhino- and enteroviruses are also thiol-proteinases, they are not related to papain; instead, they have a high similarity to serine proteinases, such as chymotrypsin and &agr;-lytic proteinase (Argos, P. et al.,
Nucleic Acids Res
. 12:7251-7267 (1984); Bazan & Fletterick,
Proc. Natl. Acad. Sci. USA
85:7872-7876 (1988); Gorbalenya, A. E. et al.,
FEBS Lett
. 194:253-257 (1986)). In addition, the FMDV L proteinase and the 2A proteinases are located at different positions on the viral polyprotein. The L proteinase is encoded at the extreme N-terminus of the polyprotein, with cleavage taking place between the C-terrninus of the L proteinase and the N-terminus of VP4, whilst rhino- and enterovirus 2A proteinases cleave between the C-terminus of VP1 and their own N-terminus. Furthermore, two forms of the L proteinase (Lab and Lb; see
FIG. 1
) are found in the infected cell, as translation of the FMDV RNA can begin at one of two AUG codons (Sangar, D. V. et al.,
Nucleic Acids Res
. 15:3305-3315 (1987)). Both forms exhibit the same enzymatic activities (Medina, M. E. et al.,
Virology
194:355-359 (1993)).
The expression of viral proteinases has generally proved to be difficult for two reasons. Firstly, most viral proteinases (including the HIV proteinase, rhinovirus 2A proteinase and enteroviral 2A proteinase) are toxic for the
E. coli cell
. Secondly, these proteins are insoluble at high levels of expression. Both problems were encountered with the Lb proteinase. Previously the modification of the bacterial T7 RNA polymerase expression system has been used with some success for HRV2 and CVB4 2A proteinase expression (Liebig, H.-D. et al.,
Biochemistry
32:7581-7588 (1993)).
The general importance of inhibiting virally coded proteinase has been moved back into the spotlight of possible anti viral therapeutic approaches not least by studies with the proteinase of human immunodeficiency virus 1 (HIV I). By deletion a

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