Mapping of antigenic determinants and regions of RNA - protein interactions in physalis mottle virus

Abstract

PhMV belongs to the tymo group of plant viruses, whose particles are stabilized predominantly by protein–protein interactions. The genome consists of a ssRNA of Mr 2.0 × 10^6 encapsidated in an icosahedral shell of 180 identical subunits of Mr 20,000. As the present thesis is concerned with the study of the architecture of PhMV, the architecture of plant viruses in general is discussed in some detail in Chapter 1.

All the icosahedral capsids are constructed from a large number of either identical or only a few distinct classes of protein subunits. This leads to a high degree of symmetry in the capsid structure. Caspar and Klug (1962) proposed the theory of quasi-equivalence to explain the presence of more than 60 subunits in viral capsids. This theory leads to the possible values of a so-called triangulation number (T), which represents the mean number of triangles into which each icosahedral face might be divided. The number of quasi-equivalent protein subunits on the capsid is 60T.

Broadly, four kinds of structures can be distinguished among the spherical plant viruses whose architecture is known. Tymo-, bromo-, and cucumoviruses have pentamer and hexamer clusters, which can be seen as distinct morphological units. Tombus-, sobemo-, and STNV groups of viruses are stabilized by protein–RNA as well as protein–protein interactions. High-resolution structures are available for these groups of viruses. AMV has bacilliform particles of varying size. The comoviruses, on the other hand, have two different protein subunits, with the shell occupying different symmetry-related positions. These four groups are discussed in some detail with respect to the structural organization of protein and RNA.

All the icosahedral viruses studied so far have been shown to contain an eight-stranded ?-barrel motif. This motif is also maintained in other animal and insect icosahedral viruses studied so far. This barrel consists mainly of two twisted antiparallel 4-stranded sheets, leading to a "jelly-roll" topology.

Among viruses with helical arrangement of protein subunits, TMV is the most well-studied. The study of structure and assembly of TMV has provided insights into the mode of interactions between protein and RNA in rod-shaped viruses.

Monoclonal antibodies are used as tools in elucidating the architecture of plant viruses. The principles involved in the production of MAbs and their use in general are presented along with a description of the architecture of TMV and SBMV as probed with MAbs. By using MAbs which could recognize the intact TMV as well as the coat protein subunits, it was shown that these MAbs specifically recognized the extremity of the virus particle containing the 5’ end of the RNA. Further, these studies established that short stacks of disks do not nucleate TMV assembly. With the use of MAbs specific to the N-terminal region of SBMV, it was demonstrated that the N-terminal region, which is buried in the native particle, becomes exposed upon swelling of the particles due to the removal of metal ions.

Tymoviruses are unique in their architecture in that the capsid shell is stabilized predominantly by strong protein–protein interactions. The RNA can be released without disruption of the capsid structure by a variety of chemical and physical methods. The detailed X-ray structural information on any tymovirus is not yet available. It has also not been possible to reassemble the virus from isolated components due to the extreme stability of the capsid structure. The nature of RNA–protein, protein–protein, and RNA–RNA interactions in TYMV has been discussed in some detail.

BDMV, a member of the tymo group, infects Solanaceous plants causing systemic mottling symptoms. Studies on the in vitro disassembly of BDMV(I) at alkaline pH and high ionic strength demonstrated the presence of polyamines within the virus particles and their role in maintaining the integrity of the nucleocapsid. Comparison of the primary structure of BDMV(I) determined earlier in this laboratory with other viral coat proteins suggested that polyamines in tymoviruses might replace the role of the basic amino terminal arm in maintaining particle stability. A phylogenetic tree constructed after pairwise alignment of the tymoviral coat protein sequence showed that BDMV(I) was not a strain of BDMV(E), but a separate tymovirus, and it was therefore renamed PhMV.

From the review of the literature, it was clear that spherical plant viruses have contributed substantially to our understanding of the molecular structure, function, and reassembly of viruses. While TYMV has been the subject of many detailed studies, other members of this group have been studied in less detail. The present study was undertaken with a view to understand the architecture of PhMV in particular and tymoviruses in general.

The main objectives of the present investigations were: (a) To map the viral epitopes using polyclonal antibodies; (b) To extend these studies to epitope mapping using monoclonal antibodies; (c) To locate regions of RNA–protein interactions through cross-linking studies; (d) To express the PCR-amplified coat protein gene of PhMV in E. coli to explore the possibility of using the expressed protein in epitope mapping and assembly studies.

The materials and methods used in this study, such as isolation and characterization of PhMV from infected N. glutinosa plants (Figs. 2.1–2.5); proteolytic and chemical cleavage of PhMV-P; purification of peptides; electrotransfer of peptides onto PVDF and nitrocellulose; preparation of polyclonal antibodies and different protocols for obtaining monoclonal antibodies; antibody screening procedures like ELISA, RIA; methods for UV and NaHSO? cross-linking; and procedures for polymerase chain reaction, are described in Chapter 2.

In order to map the epitopes on PhMV, initially polyclonal antibodies to PhMV and PhMV-P were characterized in terms of antigen dilution curves, antibody dilution curves, and standard inhibition curves (Figs. 3.1–3.3). Both AbV and AbP reacted with PhMV and PhMV-P, indicating that both antibodies reacted with the surface epitopes of the viral capsids.

Reactivity of AbV and AbP with the tryptic peptides showed common antigenic determinants (Figs. 3.4 and 3.5), indicating that the exposed regions are common in both PhMV and PhMV-P.

Only IgM MAbs were obtained using in vitro sensitized immunization protocols (Figs. 3.6A, 3.6B). These could not be used in epitope mapping because of their low affinity.

In order to obtain IgG MAbs, fusion was performed with splenocytes obtained from in vivo immunization of PhMV and PhMV-P. High-affinity IgG MAbs were obtained: 3 against PhMV-P and 5 against PhMV, which cross-reacted with both PhMV and PhMV-P (Figs. 4.1–4.6).

There were 3 and 2 unique epitopes recognized by the virus and protein MAbs, respectively (Tables 4.2 and 4.3). However, the results were ambiguous. Hence, further mapping of the epitopes using peptides obtained from cleavage of the protein was undertaken.

The virus MAbs probably recognized epitopes within residues 59–141. These MAbs did not recognize shorter fragments.

Using CNBr and tryptic digestion products of PhMV-P and a truncated coat protein lacking the N-terminal 21 amino acid residues, the epitope for MAb PA3B2 was identified to be within amino acid residues 22–36 (Figs. 4.9, 4.10A, 4.10B, 4.11–4.13 and Table 4.5). The epitope for MAb PA3B2 corresponded to the predicted epitope between amino acid residues 22–30.

Using CNBr-digested peptides, the epitope for MAbs PB5G9 and PF12C9 could be located within residues 59–141. The epitope for these MAbs was further narrowed down using clostripain and V8-cleavage products and was found to reside within residues 75–110 (Figs. 4.14–4.17). A predicted epitope (98–107) within this region may correspond to the recognition site of these two MAbs.

To locate the specific region of protein that interacts with RNA in situ, UV and NaHSO? cross-linking were performed.

Tryptic digestion of UV/NaHSO? cross-linked PhMV-P yielded many peptides (peak 1 and many peaks numbered together as 4) (Figs. 5.2 and 5.3). Peak 1 was further subjected to rechromatography (Fig. 5.4). The peak eluting at 4.4 min, with absorbance at 225 nm as well as 260 nm, was sequenced (Table 5.1). This peptide corresponded to Val(9)–Lys(10). Rechromatography of peak 4 yielded many peptides linked to RNA, which might correspond to the N-terminal blocked peptide.

Isolation of peptides from NaHSO? cross-linked PhMV-P yielded a number of peptides (numbered 4, Fig. 5.7, panels A–D), all of which might correspond to the N-terminal blocked peptide carrying different lengths of oligonucleotide chains (Figs. 4.8–4.11).

PhMV lacks the N-terminal basic amino acid residues, like other tymoviruses. It was found to be structurally similar to SBMV, a virus stabilized by RNA–protein interactions. The size of the coat protein of SBMV was 28K. Hence, it was proposed that PhMV lacks the first 60 residues that were disordered in SBMV (Fig. 1.7). The epitope 22–30 in PhMV could correspond to the ?3C strand of SBMV, and Lys-10, which was buried, could correspond to ?A and ?3B strands of SBMV. Hence, the immunological and cross-linking studies on PhMV are consistent with the canonical ?-barrel structure observed for other viruses.

In order to obtain native PhMV-P for in vitro assembly studies and site-directed mutagenesis, the coat protein gene of PhMV-P was amplified by PCR using a cDNA clone Tc20 as the template (Figs. 6.2 and 6.3) and two primers: one sense primer containing an EcoRI site and the other antisense primer containing a HindIII site.

The PCR product (PCR-CP) was cloned into the EcoRI/HindIII site of pKK223-3 (Fig. 6.4) after internalization of the restriction sites by kinase, Klenow, and ligase reactions, followed by EcoRI/HindIII digestion of PCR-CP.

Since no expressing clones were obtained by this procedure, blunt-end ligation of PCR-CP was performed in the SmaI site of pKK223-3. The clones were screened by colony hybridization (Fig. 6.5). Two clones, 6-5 and 10-31, were found to contain the insert (Figs. 6.6 and 6.7), but both did not express PhMV-P (Fig. 6.8).

Since in these clones the distance between the SD sequence and the initiation codon of PhMV-P was 18 nt (Fig. 6.9), EcoRI digestion of 10-31 was performed to remove the intervening 8 extra nucleotides.

One of the clones, 10-31-11, obtained after EcoRI digestion followed by ligation, was found to express the protein upon induction with IPTG (Fig. 6.10).

IPTG (0.5 mM) gave optimum expression when induced for 10 h (Figs. 6.11 and 6.13).

The results described in this thesis show that in tymoviruses, one of the sites of RNA–protein interactions is at the N-terminus, even though there is no preponderance of basic residues in this region. The decreased basicity in the N-terminus of tymoviruses could account for the weak RNA–protein interaction. The region 22–36 following this buried N-terminal region is a major epitope, and these results are consistent with the canonical ?-barrel structure of viruses. The availability of an expressing clone for the coat protein now enables further studies on the involvement of specific regions of the CP in viral stability through site-directed mutagenesis.