Plant pararetroviruses are classified as members of the family Caulimoviridae which comprises 6 genera 1. Like retroviruses, members of this group of viruses use reverse transcriptase for replication of the genome 23. However, they differ in two major points: retroviruses have an RNA genome whereas pararetroviruses have a DNA genome; and, a proviral form of retroviruses is integrated into the host chromosome whereas the DNA of pararetroviruses accumulates within the nucleus as multiple copies of a circular chromosome 4.

Retroviruses and pararetroviruses show similarities in their molecular organization and replication process and are phylogenetically related. These groups of viruses direct the production of a terminally redundant RNA which is used as a replicative intermediate and as mRNA. Many of the genes encoded by pararetroviruses are homologous in sequence and/or analogous in function to those of retroviruses. The genome of all replication-competent retroviruses consists of three major genetic elements that are arranged in the order gag-pol-env (structural-replication-envelope proteins). Each protein is produced as a result of frameshifting during translation, or suppression of stop codons in the polyproteins. Products of the gag ORF represent the structural components of the viral matrix, i.e. capsid and nucleocapsidproteins; the pol domains generally comprise the protease, reverse transcriptase, ribonuclease and an endonuclease/integrase. Pararetroviruses encode the gag-pol core, but lack an integrase, as viral DNA integration into the host chromosome is not required 5.

Retrovirus and pararetrovirus polyproteins are believed to be processed by their own aspartate proteases 367. These proteases contain several conserved regions when compared with each other and share consensus sequences in the active site; however, they show no homology with other viral proteases. Protein cleavages by these and other proteases are dependant on amino acid sequence and conformation near the cleavage site 6.

Rice tungro is a major rice disease in southeast asia and is caused by a double infection by two viruses 89: Rice tungro bacilliform virus (RTBV) 1011, a member of the genus Badnavirus in the family Caulimoviridae 1, and Rice tungro spherical virus (RTSV), a single-stranded RNA virus and member of the genus Waikivirus in the family Sequiviridae 1213. RTBV is responsible for symptoms of the disease 8 and RTSV is required for the transmission of the two viruses by the leafhopper vector Nephotettix virescens 1415.

RTBV genome is a double stranded DNA of 8.0 kbp with two site-specific discontinuities resulting from replication by reverse transcriptase 316. The RTBV genome has four open reading frames (ORF) 17. ORF3 has similarities with the gag-pol core of retroviruses. The polyprotein (P3) contains a putative movement protein (MP), the capsid protein (CP), the aspartate protease (PR), and the reverse transcriptase (RT) with a ribonuclease H activity. The N- and C- terminal amino acid (aa) sequences of the CP were deduced from MALDI-TOF mass spectral analysis 18. The location of the CP domain, which is encoded by aa 477–791 in P3, was confirmed by immunodetection reactions using antibodies raised against different segments of ORF3 18. P3 sequence aa 985–995 shows homologies to the active site of aspartate proteases encoded by retroviruses and pararetroviruses, with the sequence DSGS believed to be the RTBV protease active site. Changing of D to A (aspartic acid to an alanine) in this sequence affects the proteolytic processing of the RT 19. Detection of RTBV gene products in vivo revealed a major protein of 13.5 kDa in virus preparations, by using an antibody raised against the domain aa 881–1098 1020. The antibody was used in immunogold labelling reactions and showed binding on the surface of the virus particle, suggesting that the PR is located at or near the surface of the virus particle 20.

In this paper, we used mass spectrometry analysis to characterize the RTBV protease domain in a protein associated with purified virions, and confirm the hypothesis by in silico modelling and in vitro expression studies. Analysis of products of in vitro processing of P3 suggests that RTBV aspartate protease is involved in the release of the CP from the polyprotein P3.

Retroviral proteases have been studied for many years as they are essential in the control of the replication of retroviruses. Retroviral proteases contain about 100 amino acids residues in length, and contain one copy of the active site Asp-Thr-Gly or Asp-Ser-Gly 22. It was proposed that they are active as homo-dimers 23. Crystallographic structures of Rous sarcoma virus (RSV) and Human immunodeficiency virus (HIV) showed that the PR of the viruses are dimeric, with two copies of the active site being brought into close proximity at the junction between the dimer partners 2425. To date, numerous retroviral proteases have been investigated, and their structures determined 26. The retroviral protease is formed by duplication of four structural elements: a hairpin, a wide loop, an α-helix and a second hairpin. Active site sequences are placed in the extended loop of the structural model, implying that the active site is a number of amino acids away from the N-terminal of retroviral proteins.

The CP domain was previously characterized by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry analysis 18. A single CP domain was identified, with positions of the amino- and carboxy- termini of the CP at aa 477 and 791, respectively. The molecular mass of the CP was determined to be 37,303 Da with an estimated pI of 9.43. A basic pI can explain the absence of peaks related to the CP in our mass spectral analysis. In this paper, virions of RTBV were subjected to in-line HPLC electrospray ionization mass spectrometry analysis. The HPLC column is expected to have retained the CP prior to mass spectrometry analysis. The molecular mass of the protein found by these analyses was determined to be 13,794 ± 4 Da, with a putative isoelectric point of 6.3. We suspected that this protein was the viral protease that was encoded in P3 protein.

Five peptide domains, with the predicted mass of the protein identified in virions, were found to contain the active site DSGS, the active site of an aspartate protease, and the conserved region IIG (Figure 2). We compared the derived sequences of the five putative proteins with sequences of retroviral proteases, a process that led us to predict that the peptide comprising aa 965–1085 represents the protease, based on the position of the active site within the predicted sequence. In the predicted protein the Aspartic acid residue of active site would be 23 amino acid away from N-terminus; the active site is 25 and 37 aa from the N-terminus for HIV and RSV, respectively.

To confirm if the target sequence is or is not a protease, we used a combination of sequence and structural predictions procedures to build a structural model for the RTBV protease (Figure 4-A) with RSV protease as a template (Figure 4-B). The consistency in fold recognition was given by reliable scores against different templates for the same SCOP classification (SCOP classification b.50.1). The proteins of this fold show a closed beta barrel. In the model, some of the beta strands elements are missing, however the remaining beta sheets can be arranged in a predicted conformation as they are product of sequence duplications. Keeping in mind that the protease domain is part of a multi-domain protein, additional interactions among the domains may influence the folding of individual subunits. The conservation in the predicted active site, and the predicted overall folding of sequences led us to predict that the domain posseses a protease activity. Subsequent experiment evidence that confirmed that the predicted active site can be inactive by mutation of D to A, the predicted secondary structure and the fold recognition analyses with model building led us to conclude that the protein comprises an aspartate protease.

Plasmids that encode peptides PR and mPR were introduced for expression in E. coli: however, PR did not accumulate in E.coli while mPR was expressed normally and reacted with Ab-RTBV and with Ab-PR antibodies. In other studies, the mutation of D to A in the active site of the RTBV protease was shown to affect its activity 19. We suggest that PR did not accumulate in E. coli because the peptide was an active protease that was not tolerated in the host. Previous attempts to express proteases in E. coli have had similar outcomes 2728.

An antiserum against the region aa 881–1098 of the P3 was produced in previous studies 20; this peptide includes the protease domain. Using this antibody a protein of approximately 13.5 kDa was detected by western blots assays in virus preparations and in infected tissues, suggesting that the protein that was detected represented the viral protease 20. Furthermore, the antiserum was used to label virus particles, and revealed that the label was attached to virions. The characterization of PR domain, and the immunodetection reactions performed in the present studies are in agreement with the previous results, and also with studies performed by Marmey et al., 18, which investigated a peptide comprising aa 806–961 of P3, that was referred to as IR. IR did not react with Ab-RTBV serum, suggesting that the IR region did not contain sequences related to the CP. Our results support the hypothesis that peptide IR corresponds to the intervening region between CP and PR, and that it may be involved in the processing of P3 18. With the present study the N- and C- terminal amino acid sequences are now characterized for CP, PR and RT-Rnase H 1819. However, we did not identify apparent sequence similarities between the cleavage sites that would be used by the PR (Figure 8). Such a lack of sequence similarity is usual for viral aspartate proteases 2930. Other details that remain to beclarified in the organization of P3 include: characterization of the movement domain, and the order and rates in which the various sites on P3 are cleaved.

A previous work conducted in insect cells using baculovirus based constructs, including constructs in which the active site of the protease was mutated revealed that RT was processed by PR 19. In the present work, in vitro processing of CP by PR was demonstrated in E.coli. If immunoprecipitations with antibodies were not achieved for technical reasons, presence of the 37 kDa peptide was associated with co-existence of CP and active PR in the construct. It is the first time that such a processing is demonstrated for pararetroviruses (e.g. Commelina yellow mottle virus, Banana streak virus, Cacao swollen shoot virus), where CP and PR are components of the same polyprotein.

Our results clarify the organization of P3, and its processing by its own protease and lead to a more complete understanding of the replication process and possible points of control of pararetroviruses.

The author(s) declare that they have no competing interests.

PM carried out the designing of primers, the construction of various plasmids, the in vitro expression analysis and pulse chase labelling experiments; ARM performed the structural modelling analysis; AdK analyzed various sequences by computer; RNB and CMF were principal investigators. All authors read and approved the final manuscript.