Cooperation in Viral Movement: The Geminivirus BL1 Movement Protein Interacts with BR1 and Redirects It from the Nucleus to the Cell Periphery

For plant viruses to systemically infect a host requires the active participation of viral-encoded movement proteins. It has been suggested that BL1 and BR1, the two movement proteins encoded by the bipartite geminivirus squash leaf curl virus (SqLCV), act cooperatively to facilitate movement of the viral single-stranded DNA genome from its site of replication in the nucleus to the cell periphery and across the cell wall to adjacent uninfected cells. To better understand the mechanism of SqLCV movement, we investigated the ability of BL1 and BR1 to interact specifically with each other using transient expression assays in insect cells and Nicotiana tabacum cv Xanthi protoplasts. In this study, we showed that when individually expressed, BL1 is localized to the periphery and BR1 to nuclei in both cell systems. However, when coexpressed in either cell type, BL1 relocalized BR1 from the nucleus to the cell periphery. This interaction was found to be specific for BL1 and BR1, because BL1 did not relocalize the SqLCV nuclear-localized AL2 or coat protein. In addition, mutations in BL1 known to affect viral infectivity and pathogenicity were found to be defective in either their subcellular localization or their ability to relocalize BR1, and, thus, identified regions of BL1 required for correct subcellular targeting or interaction with BR1. These findings extend our model for SqLCV movement, demonstrating that BL1 and BR1 appear to interact directly with each other to facilitate movement cooperatively and that BL1 is responsible for providing directionality to movement of the viral genome.     

The geminivirus BR1 movement protein binds single-stranded DNA and localizes to the cell nucleus.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.     

A Phage Single-Stranded DNA (ssDNA) Binding Protein Complements ssDNA Accumulation of a Geminivirus and Interferes with Viral Movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.     

Transgenic tobacco plants expressing the geminivirus BL1 protein exhibit symptoms of viral disease.

Bipartite geminiviruses, such as squash leaf curl virus (SqLCV), encode two movement proteins (MPs), BR1 and BL1, that are essential for viral movement in and subsequent infection of the host plant. To elucidate the biochemical functions of these MPs and define their respective contributions to viral infection, we have generated transgenic Nicotiana benthamiana plants expressing SqLCV BR1 and BL1. Transgenic plants expressing BR1 or a truncated BL1 were phenotypically indistinguishable from wild-type N. benthamiana. In contrast, transgenic plants expressing full-length BL1, alone or in combination with BR1, were strikingly abnormal both in their growth properties and phenotypic appearance, with leaves that were mosaic and curled under, thus mimicking typical SqLCV disease symptoms in this host. BL1 was localized to the cell wall and plasma membrane fractions, whereas BR1 was predominantly in the microsomal membrane fraction. These findings demonstrate that expression of BL1 in transgenic plants is sufficient to produce viral disease symptoms, and they further suggest that BL1 and BR1 carry out distinct and independent functions in viral movement.     

Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.

The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and squash leaf curl virus (SqLCV) have two single-stranded circular genomic DNAs, the A and B components, thought to be replicated from double-stranded circular DNA intermediates. Although it has been presumed that the origin sequences for viral replication are located in the highly conserved 200-nucleotide common region (CR) present in both genomic components and that the viral-encoded AL1 protein interacts with these sequences to effect replication, there has been no evidence that this is in fact so. We have investigated these questions, demonstrating selectivity and sequence specificity in this protein-DNA interaction. Simple component switching between the DNAs of TGMV and SqLCV and analysis of replication in leaf discs showed that whereas the A components of both TGMV and SqLCV promote their own replication and that of their cognate B component, neither replicates the noncognate B component. Furthermore, using an in vivo functional replication assay, we found that cloned viral CR sequences function as a replication origin and direct the replication of nonviral sequences in the presence of AL1, with both circular single-stranded and double-stranded DNA being synthesized. Finally, by the creation of chimeric viral CRs and specific subfragments of the viral CR, we demonstrated sequence-specific recognition of the replication origin by the AL1 protein, thereby localizing the origin to an approximately 90-nucleotide segment in the AL1 proximal side of the CR that includes the conserved geminiviral stem-loop structure and approximately 60 nucleotides of 5' upstream sequence. By deletional analysis, we further demonstrated that the conserved stem-loop structure is essential for replication. These studies identify the functional viral origin of replication within the CR, demonstrating that sequence-specific recognition of this origin by the AL1 protein is required for replication.     

Identification of domains in brome mosaic virus RNA-1 and coat protein necessary for specific interaction and encapsidation.

Even though many single-stranded RNAs are present in the cytoplasm of infected cells, encapsidation by brome mosaic virus (BMV) coat protein is specific for BMV RNA. Although the highly conserved 3' region of each of the three BMV genomic RNAs is an attractive candidate for the site of recognition by the coat protein, band shift and UV cross-linking assays in the presence of specific and nonspecific competitors revealed only nonspecific interactions. However, BMV RNA-1 formed a retarded complex (complex I) with the coat protein in the absence of competitors, and two domains of RNA-1 that specifically bound coat protein in a small complex (complex II), presumably early in the encapsidation process, were identified. Strong nonspecific, cooperative binding was observed in the presence of high concentrations of coat protein, suggesting that this provides the mechanism leading to rapid encapsidation seen in vivo. In contrast, no binding to a coat protein mutant lacking the N-terminal 25 amino acids that has been shown to be incapable of encapsidation in vivo (R. Sacher and P. Ahlquist, J. Virol. 63:4545-4552, 1989) was detected in vitro. The use of deletion mutants of RNA-1 revealed the presence of domains within the coding region of protein 1a that formed complexes with purified coat protein. One deletion mutant (B1SX) lacking these domains was only slightly more effective in dissociating RNA-1-coat protein complexes than were nonspecific competitors, further suggesting that regions other than the 3' end can participate in the selective encapsidation of BMV RNAs.     

A viral movement protein as a nuclear shuttle. The geminivirus BR1 movement protein contains domains essential for interaction with BL1 and nuclear localization.

For the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, BR1 and BL1. These act in a cooperative manner to transport the viral single-stranded DNA genome from its site of replication in the nucleus to the cell periphery (A.A. Sanderfoot, S.G. Lazarowitz [1995] Plant Cell 7: 1185-1194). We have proposed that BR1 functions as a nuclear shuttle protein, transporting the viral single-stranded DNA to and from the nucleus as a complex that is recognized by BL1 for movement to adjacent cells. To further investigate this, we expressed BR1 mutants known to affect viral infectivity in Spodoptera frugiperda insect cells and Nicotiana tabacum L. cv Xanthi protoplasts and found these to be defective in either their nuclear targeting or their ability to be redirected to the cell periphery when co-expressed with BL1. Translational fusions to beta-glucuronidase and alanine-scanning mutagenesis further demonstrated that the C-terminal 86 amino acids of BR1 contains a domain(s) essential for its interaction with BL1 and identified two nuclear localization signals within the N-terminal 113 residues of BR1. These nuclear localization signals were precisely located within distinct 16- and 22-peptide segments of BR1. These studies support and extend our model for squash leaf curl virus movement, showing that BR1 has a domain structure, with an N-terminal region required for nuclear targeting and a C-terminal region required for its interaction with BL1.     

The C terminus of brome mosaic virus coat protein controls viral cell-to-cell and long-distance movement

To investigate the functional domains of the coat protein (CP; 189 amino acids) of Brome mosaic virus, a plant RNA virus, 19 alanine-scanning mutants were constructed and tested for their infectivity in barley and Nicotiana benthamiana. Despite its apparent normal replicative competence and CP production, the C-terminal mutant F184A produced no virions. Furthermore, virion-forming C-terminal mutants P178A and D182A failed to move from cell to cell in both plant species, and mutants D181A and V187A showed host-specific movement. These results indicate that the C-terminal region of CP plays some important roles in virus movement and encapsidation. The specificity of certain mutations for viral movement in two different plant species is evidence for the involvement of host-specific factors.     

A single amino acid change in the coat protein of Maize streak virus abolishes systemic infection, but not interaction with viral DNA or movement protein

Functional coat protein (CP) is important for host plant infection by monopartite geminiviruses. We identified a proline-cysteine-lysine (PCK) motif at amino acids 180–182 of the maize streak virus (MSV) CP that is conserved in most of the cereal–infecting Mastreviruses. Substitution of the lysine (K) with a valine (V) in the CP of MSV to produce mutant MSVCP182V abolished systemic infection in maize plants, although the mutant replicated around the inoculation site and, unlike other MSV CP mutants, enabled single-stranded (ss) DNA accumulation in suspension cells. The stability of the mutant protein, CP182V, in infected cells was confirmed by immunoblotting, but virions could not be detected. Like the wild-type (wt) CP, CP182V localized to the nucleus when expressed in insect and tobacco cells, and the Escherichia coli-expressed protein bound both ss and double-stranded DNA and interacted with movement protein in vitro. Taken together, these data suggest that mutation of amino acid 182 affects virion formation of MSV, either by affecting encapsidation per se or by affecting particle stability, and that virions are necessary for the long-distance movement of MSV in maize plants.     

The 60-residue C-terminal region of the single-stranded DNA binding protein of herpes simplex virus type 1 is required for cooperative DNA binding

ICP8 is the major single-stranded DNA (ssDNA) binding protein of the herpes simplex virus type 1 and is required for the onset and maintenance of viral genomic replication. To identify regions responsible for the cooperative binding to ssDNA, several mutants of ICP8 have been characterized. Total reflection X-ray fluorescence experiments on the constructs confirmed the presence of one zinc atom per molecule. Comparative analysis of the mutants by electrophoretic mobility shift assays was done with oligonucleotides for which the number of bases is approximately that occluded by one protein molecule. The analysis indicated that neither removal of the 60-amino-acid C-terminal region nor Cys254Ser and Cys455Ser mutations qualitatively affect the intrinsic DNA binding ability of ICP8. The C-terminal deletion mutants, however, exhibit a total loss of cooperativity on longer ssDNA stretches. This behavior is only slightly modulated by the two-cysteine substitution. Circular dichroism experiments suggest a role for this C-terminal tail in protein stabilization as well as in intermolecular interactions. The results show that the cooperative nature of the ssDNA binding of ICP8 is localized in the 60-residue C-terminal region. Since the anchoring of a C- or N-terminal arm of one protein onto the adjacent one on the DNA strand has been reported for other ssDNA binding proteins, this appears to be the general structural mechanism responsible for the cooperative ssDNA binding by this class of protein.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

Replication of wheat dwarf virus DNA in protoplasts and analysis of coat protein mutants in protoplasts and plants.

The replication of wheat dwarf virus (WDV) in protoplasts derived from a Triticum monococcum suspension cell system was investigated. The production of circular viral double-stranded DNA (dsDNA) forms consistent with the replication of the viral genome was observed. In comparison to whole plants, the production of viral single-stranded DNA (ssDNA) was reduced, possibly due to only low levels of viral coat protein being produced in the protoplasts. Mutations introduced into the viral coat protein open reading frame (ORF) did not affect the ability of the viral DNA to replicate, and a deletion of ca. 400 bp was tolerated. However, these mutations abolished the infectivity of the viral genome when agroinoculated onto wheat plants, providing evidence that, contrary to the case for the bipartite geminiviruses, the coat protein is essential for infection by WDV.     

Encapsidation of heterologous RNAs by bacteriophage MS2 coat protein.

The RNA bacteriophages of E. coli specifically encapsidate a single copy of the viral genome in a protein shell composed mainly of 180 molecules of coat protein. Coat protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon. We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with coat protein. To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from coat protein expressed from a plasmid. The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the coat protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA. When coat protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well. However, elevation of the level of operator-lacZ RNA relative to coat protein results in its selective encapsidation at the expense of cellular RNAs. Our results are consistent with the proposition that this single protein-RNA interaction accounts both for translational repression and viral genome encapsidation.     

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Role of the coat Protein-RNA interaction in the life cycle of bacteriophage MS2

The coat protein of the RNA bacteriophage MS2 interacts with viral RNA to translationally repress replicase synthesis. This protein-RNA interaction is also thought to play a role in genome encapsidation. In this study the strength of the interaction was perturbed by constructing a recombinant genome containing a super-repressing coat mutation. Because replicase synthesis is prematurely repressed, the mutant produces plaques about five orders of magnitude less efficiently than wild-type. The few plaques obtained are second-site revertants of the original coat mutation and fall into two categories. Those of the first type contain nucleotide substitutions within the translational operator that reduce or destroy its ability to bind coat protein, showing that this interaction is not necessary for genome encapsidation. Revertants of the second type are double mutants in which one substitution converts the coat initiator AUG to AUA and the other substitutes an A for the G normally present two nucleotides upstream of the coat start codon. The mutation of the coat protein gene AUG to AUA, by itself, reduces coat protein synthesis to a few percent of the wild-type level. The second substitution destabilizes the coat initiator stem-loop and restores coat protein synthesis to within a few fold of wild-type levels. Received: 17 June 1996 / Accepted: 5 December 1996     

Mutagenesis in ORF AV2 affects viral replication in <Emphasis Type="BoldItalic">Mungbean yellow mosaic India virus</Emphasis>

Mungbean yellow mosaic India virus (MYMIV) is a whitefly-transmitted begomovirus with a bipartite genome. We investigate the functions of the MYMIV-AV2 protein, the open reading frame present upstream of the coat protein gene in DNA A component. The ability of MYMIV-AV2 mutants to replicate, spread and cause symptoms in legume hosts, blackgram, cowpea and French bean was analysed. Plants agroinoculated with mutants K73R, C86S and the double mutant C84S,C86S showed increase in severity of symptoms compared with the wild type. However, mutants W2S and H14Q,G15E caused marked attenuation of symptoms. While the double mutants C84S,C86S caused a 50-fold increase in double-stranded supercoiled and single-stranded DNA accumulation, the mutations W2S and H14Q,G15E showed a decrease in double-stranded supercoiled and single-stranded viral DNA accumulation. Because AV2 mutants affect the ratio between open circular and supercoiled DNA forms, we hypothesize that these mutations may modulate the functions of the replication initiation protein.     

Dissecting the multifunctional role of the N‐terminal domain of the Melon necrotic spot virus coat protein in RNA packaging,viral movement and interference with antiviral plant defence

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S‐domain builds a robust protein shell around the viral genome, whereas the C‐terminal protruding domain, or P‐domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N‐terminal domain, or R‐domain, and the arm region, which connects the R‐domain and S‐domain, are involved in different key steps of the viral cycle, such as cell‐to‐cell movement and the suppression of RNA silencing and pathogenesis through their RNA‐binding capabilities. Deletion mutants revealed that the CP RNA‐binding ability was abolished only after complete, but not partial, deletion of the R‐domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA‐binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R‐domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full‐length CPs were required to encapsidate full‐length genomes. We also found that the R‐domain carboxyl portion and the arm region were essential for efficient cell‐to‐cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R‐domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.     

Portable encapsidation signal of the L-A double-stranded RNA virus of S. cerevisiae

The (+) single-stranded RNA (ssRNA) of the L-A virus is the species packaged to form new viral particles. Empty L-A viral particles specifically bind viral (+) ssRNA, and a sequence 400 bases from the 3' end is necessary for this activity. We show that its stem-loop structure, the A residue protruding from the stem, and the loop sequence are all important for the binding, and that this 34 base region is sufficient for the binding. M1, a satellite virus of L-A, has a similar structure on its (+) strand that is likewise sufficient for the binding. Heterologous RNA with the binding sequence from L-A or M1, when expressed in vivo, was packaged in L-A viral particles. Thus, the sites necessary to bind to empty particles are encapsidation signals for the L-A virus. Since the pol domain of the 180 kd minor coat protein appears to be responsible for the binding, this result suggests that the RNA polymerase molecule recognizes the viral genome for packaging.