Structure-function relationships of physalis mottle virus (PhMV) replicase

Abstract

Physalis mottle virus (PhMV) belongs to the tymovirus group of (+)strand plant viruses that infect solanaceous plants. Its icosahedral protein shell is made up of 180 identical protein subunits, each of Mr 20 kDa. The intact virus particles encapsidate a genomic singlestranded RNA of approximately 6 kb (Mr ~2 × 10). The 3terminal 1255 nucleotides of the PhMV RNA were characterized in an earlier study (Jacob et al., 1992). This region included a 149nucleotide 3terminal noncoding region capable of folding into a tRNAlike structure, the complete coatprotein coding region, and the Cterminal 178 amino acid residues of the replicase protein. Purification and characterization of viral replicases have received great attention in recent years because of their central role in the life cycle of (+)strand RNA viruses. Extensive studies exist for segmented viral replicases, whereas replicases of monopartite viruses have been far less explored (De Graaf and Jaspars, 1994). The present work was undertaken to elucidate the nucleotide sequence of the 5terminal region of the PhMV genome and to determine the structure-function relationships of the PhMV replicase, a monopartite virus.

(1) Chapter I presents a selected review of literature on the organization and expression of (+)strand viral genomes, with emphasis on replication strategies. It also reviews structure-function relationships of replicases from various systems, particularly tymoviruses, and cisacting elements in (+)strand RNA viruses involved in replicase interaction. (2) The objectives of this thesis were: (a) to generate a cDNA library representing the 5terminal region of the PhMV genome, determine its nucleotide sequence, analyse the sequence, and subclone the 5noncoding region; (b) to isolate and characterize PhMV RdRp and establish an invitro replication system to understand (+)strand viral replication; (c) to identify cisacting elements within the 5 and 3 noncoding regions capable of interacting with the RdRp; (d) to identify viral and hostencoded factors in PhMV RdRp preparations that specifically recognize the 5 and 3 noncoding regions. (3) Chapter II describes materials and methods. A novel northwestern blotting technique for identifying RNAbinding proteins in crude membrane preparations of PhMV RdRp is reported. In this method, [³²P]labelled transcripts are used to probe nitrocellulose membranes directly, bypassing UV crosslinking (Hayes and Buck, 1993). (4) Chapter III reports the nucleotide sequence of the 5terminal 1273 bases of PhMV obtained from overlapping clones. This region includes a 147nucleotide 5noncoding sequence followed by two extensively overlapping ORFs spaced seven nucleotides apart. The first ORF encodes a putative 69 kDa overlapping protein (OP), in the -1 reading frame relative to the downstream 206 kDa replicase protein (RP) ORF. The sequence determined includes the Nterminal 375 amino acids of OP and the Nterminal 373 amino acids of RP. The RP sequence is highly conserved and contains an RNA methyltransferase motif typical of sindbislike (+)strand viral replicases (Rozanov et al., 1992). (5) The 5noncoding region contains a 31nucleotide pyrimidinerich stretch and several shorter pyrimidine runs. It can fold into a stem-loop structure, with pyrimidinerich stretches located in loops, as seen in other tymoviruses. It also contains tandem repeats of a 26nucleotide sequence (positions 54-80 and 95-124), capable of forming stem-loops with the repeats in the stems. Analysis also showed that sequences resembling internal control region 2 (ICR2) motifs of tRNA genes occur at nucleotides 37-46. (6) Chapter IV describes isolation and characterization of PhMV RdRp. Crude membrane fractions from PhMVinfected Nicotiana glutinosa leaves supported PhMVspecific RNA synthesis in vitro. Activity peaked at three weeks postinfection and produced predominantly ssRNA. Reaction products corresponded to genomic (6 kb) and subgenomic (0.75 kb) RNAs. Optimal reaction conditions included 20 mM MgCl, 2 mM each of GTP/CTP/ATP, 5 Ci [³²P]UTP, 3 pmol PhMV RNA, and 1 h incubation. Solubilized RdRp showed strict template specificity for PhMV RNA. (7) Chapter V reports the identification of cisacting regions important for RdRp binding. Sense and antisense transcripts representing the 5 and 3terminal noncoding regions were generated using invitro transcription. These transcripts specifically inhibited PhMV RNA synthesis in vitro, whereas yeast tRNA did not, demonstrating specificity. (8) Inhibition by the 3sense transcript likely reflects the tRNAlike structure’s role as the promoter for (-)strand initiation. Yeast tRNA failed to inhibit, suggesting additional sequence/structural features unique to PhMV. The antisense 3 transcript may inhibit via its ICRlike sequence, as seen in TYMV (Boyer et al., 1991). (9) Inhibition by the 5sense transcript suggests cisacting elements at the 5 end. Inhibition by the 5antisense transcript (3 end of the (-)strand) suggests promoter activity for (+)strand synthesis. A sequence resembling a tRNA gene promoter (5GGCUGAAACA3) occurs at nucleotides 133-141. Binding of RdRp to both ends suggests structural similarity of the 5 and 3 termini. (10) Chapter VI describes attempts to identify viralencoded polypeptides in the replicase complex. Antipeptide antibodies (Bransom et al., 1991) against TYMV replicase were used. The Cterminal antiserum recognized PhMV proteins, including 70 kDa and 110 kDa polypeptides, suggesting an additional cleavage upstream of the known site. Western blots were inconclusive, so northwestern analysis was used. (11) Northwestern analysis identified three RNAbinding proteins-45 kDa, 30 kDa, and 20 kDa-unique to infected tissue and capable of binding specifically to 5 and 3 terminal transcripts. Transcripts from other viral regions or other viruses showed no binding. This is the first direct evidence for specific ciselementrecognizing proteins in a tymovirus replicase preparation. The 45 kDa and 30 kDa proteins may be hostencoded; the 20 kDa RNAbinding protein is distinct from PhMV coat protein. Purification is required for full characterization.

Summary This study characterizes: • the 5terminal region of the PhMV genome, including pyrimidinerich motifs, stem-loop structures, and an ICR2like element; • an invitro PhMVspecific RdRp system producing ssRNA; • cisacting elements at both genome termini that interact with RdRp; • three RNAbinding proteins (45 kDa, 30 kDa, 20 kDa) that bind specifically to these elements. The work lays the foundation for future identification of minimal cisacting sequences, complete invitro replication systems, and characterization of RNAbinding proteins.