Modified small RNA viruses

Details Modified small RNA viruses Modified small RNA viruses

1999-07-02

2001-06-26

Park, Hankyel T. (Department: 1648)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

C435S236000, C435S237000, C424S185100, C424S189100

Reexamination Certificate

active

06251654

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to small RNA viruses and virus-like particles (VLPs) having altered or substituted Ig-like domains so as to modify host cell tropism or, in other words, the specificity of host cell binding and infection. The invention also relates to the use of such small RNA viruses and VLPs in insecticidal and medicinal applications.
BACKGROUND TO THE INVENTION
Formally recognised small RNA viruses include members of Picornaviradae, the Nodaviradae and the Tetraviradae. However, there are many unrecognised insect viruses that also fall into this category. The Tetraviradae are a family of small isometric insect viruses with unenveloped, icosahedral capsids 35-41 nm in diameter and single-stranded positive-sense RNA (ss+RNA) genomes. They have not received wide attention from virologists. Their known host range is confirmed to only a few families of moths in a single insect order, the Lepidoptera (moths, butterflies), making them the only small RNA virus family restricted to insect hosts. While they appear to be effective at controlling several of their hosts that are important insect pests, they have been little used in this regard. The lack of a cell culture system or, until recently, a reliable means to obtain the virus from laboratory reared insects made it necessary to rely on sporadically available field-collected material of uncertain quality. Such was the difficulty that only recently did it emerge that there are actually two groups of tetraviruses, Nudaurelia &bgr;-like viruses having a mono-partite genome of ca. 6 kb and Nudaurelia &ohgr;-like viruses having a bi-partite genome comprising ss RNAs of 5.3 and 2.5 kb. There are only two known Nudaurelia &ohgr;-like viruses. The complete genome of one member (
Helicoverpa armigera
stunt virus—HaSV) has been previously sequenced by the present inventors. The other member is Nudaurelia &ohgr; virus (N&ohgr;V) which has been partially sequenced.
One of the most intriguing aspects of infections by tetraviruses is that they appear only to infect a single tissue type, which in the case of HaSV is the midgut. In a definitive experiment that highlights the specificity of HaSV, the present inventors showed that its midgut specificity prevailed even when virus was injected into the haemocoel of larvae, thereby exposing host non-midgut cell types not normally exposed to HaSV. The presence of virus was examined by using cloned cDNA probes on Northern blots of RNA extracted from midguts and from the rest of the carcasses from three groups of larvae, one injected with HaSV, one fed HaSV and uninfected controls. They observed a positive signal only in the midgut RNA of both groups of larvae treated with HaSV.
Further evidence for specific binding of HaSV particles to a particular cell type comes from a rigorous examination of larvae of
H. armigera
infected with HaSV. The sensitive immuno-histochemistry technique of immuno-gold staining with silver enhancement was employed on a series of cross- and sagittal-sections of infected larvae. Sections in this series were also examined with electron microscopy. Staining appeared only in midgut cells despite close attention to tissues from the foregut, fat, body, salivary gland, and brain. Both types of differentiated cells of the midgut, the columnar and goblet cells, were found to be infected, as were the much smaller undifferentiated regenerative cells at the basal membrane. Although all these midgut cell types were found to be infected, analysis of virus binding to cells in sections of wax-embedded midgut showed that only goblet cells, and not columnar cells, were the primary target of HaSV binding.
The two known &ohgr;-like viruses show a high degree of sequence identity. That is, the amino acid sequences of the coat proteins of the two &ohgr;-like viruses show an overall 67% identity (76% similarity). This comparison defined four domains in the coat (capsid) protein, with two regions of high homology (ca. 80% identity and containing extensive stretches of sequence reaching over 95% identity) (Hanzlik et al., 1995). A 49 residue amino-terminal domain shows lower homology, as does a 165 residue sequence located towards the middle of the sequence and showing 33% identity. Surprisingly, the high overall sequence identity is not reflected in a detectable serological relationship suggesting that the central domain of low sequence homology is exposed on the capsid surface as the sole immunogenic portion of the intact virion. As first suggested by Hanzlik et al. (1995), this region is responsible for the differing host specificities of the two viruses.
The present inventors have now surprisingly realised that the central domain (corresponding to residues 287 to 416) of HaSV forms a structure belonging to the Immunoglobulin (Ig) superfamily. Other protein domains whose structures show an Ig-like fold include the variable (V) and constant (C) domains found on antibodies (e.g. the Fab fragment of IgGs), the HLA surface antigens of the MHC complex and cell adhesion proteins and receptors (e.g. the CD4 receptor recognised by HIV gp 120). Mediation of cell adhesion to other cells or the extracellular matrix by these proteins is central to development, differentiation, the immune response and tissue structure and healing. Many of these proteins are also used as receptors by viruses (Lentz (1990).
Recent studies based on cell adhesion assays and analysis of artificial lipid bilayers attached to plates have elucidated the basis of cell adhesion promoted by binding of surface proteins. These studies are exemplified by work on the binding between the MHC class II and CD4 proteins, which mediate adhesion of antigen presenting cells (APCs) and CD4
+
T cells in the immune response. Soluble (monomeric) CD4 (sCD4) fails to inhibit the MHC class II-specific proliferative response of T-cell clones (Hussey et al., 1988) or the binding of MHC class II
+
B cells to CD4-transfected COS-7 cells in cell adhesion assays, even at a concentration of 100 &mgr;M (Sakihama et al., 1995a). This implies that the affinity of the monomeric sCD4 for the MHC class II proteins is >10
−4
M. It has now been shown that oligomerization of CD4 molecules on the surface of CD4
+
cells is required for stable binding to MHC class II proteins, by increasing the avidity of the interaction between these cell adhesion protein molecules (Sakihama et al., 1995 a,b). This oligomerization follows an initial interaction between 1 or 2 CD4 molecules and MHC class II dimers. Characterization of chimaeric CD4 molecules has shown that the membrane proximal domains 3 and/or 4 appear to be involved in oligomerization.
The present inventors have now recognised that the lack of sequence similarity between the Ig-like domain of HaSV and the corresponding domain of N&ohgr;V may allow tetravirus particles to be used as icosahedral platforms capable of carrying altered Ig-like domains or substituted tertiary structures and thereby show modified host cell binding specificities.
The Ig-like domain forms a prominent protrusion which interacts with either quasi 3-fold or icosahedral 3-fold related subunits on the surface of the tetravirus capsid. The icosahedral particles therefore present a defined oligomeric form of the Ig-like domain which is likely to allow stable binding of the complete capsid to the cell-surface receptor, analogous to the binding between CD4 and MHC class II oligomers. Support for this notion comes from the findings of Weber and Karjalainen (1993), who reported that a soluble, pentameric immunofusion construct of mouse CD4 and human C&mgr; could inhibit the interaction between polymer-bound mouse sCD4 and B cells, whereas a soluble monomeric immunofusion construct of mouse CD4 and mouse C&kgr; could not.
DISCLOSURE OF THE INVENTION
Thus, in a first aspect, the present invention provides an isolated small RNA virus of a kind which includes an Ig-like domain within the wild-type coat protein(s), wherein said Ig-like domain has been altered or substituted so as to modify host cell t

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