Biomedical subjects

S G Lazarowitz

Publications and source records attributed to S G Lazarowitz.

At least 19 recordsLinked to original sources

PubMed1999

Probing plant cell structure and function with viral movement proteins.

Virus-encoded movement proteins are the principal strategy by which all plant viruses counter the primary physical defense of the plant to infection - the cell wall - to produce systemic infection and disease. Our understanding of how these proteins act at the molecular and cellular level has increased enormously in the past decade and ushered in an exciting new era of plant virology as an approach to investigating plant cell structure and function. The earliest studies focused on how movement proteins interacted with plasmodesmata, and were an important element in demonstrating the dynamic nature of these intercellular channels. Current efforts are focused on the role of movement proteins in coordinating the replication of viral genomes and the vectorial movement of the progeny genomes through the infected cell, as well as into adjacent cells. Movement proteins are thus providing unique approaches to unravel the fundamental mechanisms by which macromolecular transport is directed and integrated within and between plant cells.

Cell Wall↗

PubMed1999

Nuclear export in plants. Use of geminivirus movement proteins for a cell-based export assay.

The nuclear export of proteins and RNAs has been studied in heterokaryons or by microinjecting test substrates into nuclei of HeLa cells or Xenopus oocytes. We have previously shown that the two movement proteins BR1 and BL1 encoded by the plant pathogenic squash leaf curl virus act in a coordinated manner to facilitate virus cell-to-cell movement and that one of these (BR1) is a nuclear shuttle protein. By using a novel in vivo cell-based assay for nuclear export in which nuclear-localized BR1 is trapped by BL1 and redirected to the cortical cytoplasm, we demonstrate that residues 177 to 198 of BR1 contain a leucine-rich nuclear export signal (NES) of the type found in the Rev protein encoded by the human immunodeficiency virus and in Xenopus TFIIIA. We further show that the TFIIIA NES can functionally replace the NES of BR1 in both nuclear export and viral infectivity. These findings suggest that this basic pathway for nuclear export is highly conserved among plant and animal cells and in yeast.

Amino Acid Sequence↗

PubMed1998

Asymmetric infectivity of pseudorecombinants of cabbage leaf curl virus and squash leaf curl virus: implications for bipartite geminivirus evolution and movement.

The bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) have distinct host ranges. SqLCV infects a broad range of plants within the Cucurbitaceae, including pumpkin and squash, and CLCV has a broad host range within Brassicaceae that includes cabbage and Arabidopsis thaliana. Despite this, the genomic A components of these viruses share a high degree of sequence identity, particularly in the gene encoding the replication protein AL1, and their common regions are 77% identical. However, there is unexpected sequence diversity in the common regions of the two CLCV genomic A and B components, these being only 80% identical. Based on these sequence similarities, we investigated the host range properties of pseudorecombinants of SqLCV and CLCV. We found that in a pseudorecombinant virus consisting of the A component of CLCV and the B component of SqLCV, both components replicated in tobacco protoplasts, and this pseudorecombinant was infectious and caused systemic disease in Nicotiana benthamiana, a common host to all bipartite geminiviruses. However, this pseudorecombinant did not move systemically in pumpkin or Arabidopsis, despite the demonstrated replication compatibility of the genome components. As a result of the greater sequence differences between the common regions, the pseudorecombinant of SqLCV A and CLCV B components neither replicated the CLCV B component nor systemically infected any of the hosts tested. These findings demonstrate that for different geminiviruses with distinct host ranges, the replication origins and AL1 proteins can be sufficiently similar to permit infectious pseudorecombinants, but replication alone is not sufficient to cause systemic disease, and host range may ultimately be limited at the level of movement. The results of this study further suggest that CLCV is an evolving virus that can provide insights into how new bipartite geminiviruses arise from mixed infections.

Base Sequence↗

PubMed1998

The bipartite geminivirus coat protein aids BR1 function in viral movement by affecting the accumulation of viral single-stranded DNA.

The movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.

Amino Acid Sequence↗

PubMed1997

The geminivirus BL1 movement protein is associated with endoplasmic reticulum-derived tubules in developing phloem cells.

Plant viruses encode movement proteins that are essential for systemic infection of their host but dispensable for replication and encapsidation. BL1, one of the two movement proteins encoded by the bipartite geminivirus squash leaf curl virus, was immunolocalized to unique approximately 40-nm tubules that extended up to and across the walls of procambial cells in systemically infected pumpkin leaves. These tubules were not found in procambial cells from pumpkin seedlings inoculated with BL1 mutants that are defective in movement. The tubules also specifically stained with antisera to binding protein (BiP), indicating that they were derived from the endoplasmic reticulum. Independent confirmation of this endoplasmic reticulum association was obtained by subcellular fractionation studies in which BL1 was localized to fractions that contained both endoplasmic reticulum membranes and BiP. Thus, squash leaf curl virus appears to recruit the endoplasmic reticulum as a conduit for cell-to-cell movement of the viral genome.

Animals↗

PubMed1996

Getting it together in plant virus movement: cooperative interactions between bipartite geminivirus movement proteins.

To move cell-to-cell and systemically infect the host, plant viruses must cross the barrier posed by the plant cell wall. Plant viruses accomplish this through strategies that alter the architecture of the infected cell, eliminating this barrier through the action of viral-encoded 'movement proteins'. Detailed studies of a number of cytoplasmically replicating viruses suggest that movement proteins interact with components of the cytoskeleton and transport systems of the plant cell to allow passage of progeny into adjacent cells. Recent work on the two movement proteins encoded by the phloem-restricted geminivirus squash leaf curl virus has defined unique aspects of nuclear transport and protein protein interaction in the movement of this nuclear-replicating virus, and suggests that post-translational phosphorylation may be important in the regulation of movement protein function.

Journal Article↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1995

Both bipartite geminivirus movement proteins define viral host range, but only BL1 determines viral pathogenicity.

Bipartite geminiviruses such as squash leaf curl virus (SqLCV) encode two movement proteins (MPs), BR1 and BL1, that are essential for virus movement and systemic infection of host plants. BR1 has been implicated in the host range properties of the virus, and BL1 in viral pathogenic properties. To more precisely examine the roles of each MP, we have introduced missense and deletion mutations into the coding sequence of both BR1 and BL1, and examined the effects of these mutations on viral infectivity and the production of disease symptoms in pumpkin, squash, and Nicotiana benthamiana. For each MP, a range of mutant phenotypes from partially to fully defective was observed that affected the overall level and rate of infectivity. However, only mutations in BL1, and not BR1, affected the severity of disease symptoms, confirming our earlier finding that BL1 is responsible for the production of disease symptoms. For all mutants, the cucurbit hosts were found to be more permissive for viral movement than was N. benthamiana, and several mutations in both BL1 and BR1 produced host-specific phenotypes, retaining high levels of infectivity in pumpkin and squash, but abolishing infectivity for N. benthamiana. Unexpectedly, functional SqLCV coat protein (AR1) was found to specifically mask the phenotypes of certain BR1 mutations, suggesting some redundancy of function between coat protein and the BR1 MP and an interaction of AR1 with the viral movement pathway. AR1 and BR1 have similar nucleic acid binding affinities, suggesting a possible mechanism for the observed effects of the viral coat protein on viral movement.

Amino Acid Sequence↗

PubMed1994

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.

Animals↗

PubMed1993

A single missense mutation in the BR1 movement protein alters the host range of the squash leaf curl geminivirus.

The bipartite geminiviruses causing squash leaf curl disease, SqLCV-E and SqLCV-R, have a broad host range that includes Nicotiana benthamiana, pumpkin, squash, and beans. We have characterized a naturally occurring mutant of the SqLCV-R B component (BRtd1) which has lost the ability to infect N. benthamiana, but still retains full infectivity for pumpkin and squash. The mutation responsible for this host range alteration was localized to the coding region of BR1, a gene essential for viral movement. Sequence analysis identified three missense mutations in the BR1 gene encoded by BRtd1. By site-directed mutagenesis, one of these missense mutations, a substitution of Cys for Arg at position 98 in the BR1 protein, has been identified as responsible for the altered host range properties of BRtd1. These results establish a role for the BR1 movement protein in determining the host range properties of SqLCV.

Agrobacterium tumefaciens↗

PubMed1993

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.

Base Sequence↗

PubMed1992

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.

Base Sequence↗

PubMed1991

Infectivity and complete nucleotide sequence of the cloned genomic components of a bipartite squash leaf curl geminivirus with a broad host range phenotype.

Through cloning and molecular analysis we have identified two highly homologous bipartite geminiviruses as causing squash leaf curl disease. Mechanical and Agrobacterium-mediated inoculation of plants with cloned viral DNA components identified the two genomic components of SqLCV-E, a squash leaf curl virus with an unexpectedly broad host range for a whitefly-transmitted geminivirus. Nucleotide sequence analysis of the genome of this virus showed it to have the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses. Sequence comparison with the genomic components of tomato golden mosaic virus and bean golden mosaic virus revealed a close evolutionary relationship with these two bipartite geminiviruses, with which SqLCV-E shares common hosts. These studies provide clear molecular evidence for the assignment of SqLCV to the subfamily of bipartite geminiviruses.

Base Sequence↗

PubMed1991

Molecular characterization of two bipartite geminiviruses causing squash leaf curl disease: role of viral replication and movement functions in determining host range.

The genomes of two distinct, but highly homologous, bipartite geminiviruses have been identified in and cloned from extracts of squash leaf curl diseased field squash. These two squash leaf curl viruses (SqLCVs) have covalently closed, circular single-stranded DNA genomes with the same bipartite component organization characteristic of other whitefly-transmitted geminiviruses. Infectivity studies using virus preparations or cloned viral genomic components on different potential host plants demonstrated that these two SqLCVs have different host range phenotypes which can be explained by specific interactions among the different viral genomic components that act to influence viral replication and systemic movement in the plant. Analysis of Agrobacterium-inoculated leaf discs demonstrated that replication of the restricted virus was rescued in trans by the nonrestricted virus, providing an explanation for the mixtures of viral DNA components often found in particular hosts in the field. Sequence analysis of the common regions of these two SqLCVs identified a 13-base deletion in the restricted virus as compared to the nonrestricted virus, suggesting a potential sequence alteration likely to be involved in their host range phenotypic differences and strengthening the conclusion based on hybridization studies of their close evolutionary relationship. Also identified in the original field squash was a defective viral component which appeared to interfere with movement of the restricted SqLCV in its normally permissive hosts and accounted for another aspect of host range variation observed for this virus.

Base Sequence↗

PubMed1989

Maize streak virus genes essential for systemic spread and symptom development.

The entire genome of single component geminiviruses such as maize streak virus (MSV) consists of a single-stranded circular DNA of ~2.7 kb. Although this size is sufficient to encode only three average sized proteins, the virus is capable of causing severe disease of many monocots with symptoms of chlorosis and stunting. We have identified viral gene functions essential for systemic spread and symptom development during MSV infection. Deletions and gene replacement mutants were created by site-directed mutagenesis and insertion between flanking MSV or reporter gene sequences contained in Agrobacterium T-DNA derived vectors. Following Agrobacterium-mediated inoculation of maize seedlings, the mutated MSV DNAs were excised from these binary vectors by homologous recombination within the flanking sequences. Our analyses show that the capsid gene of MSV, while not required for replication, is essential for systemic spread and subsequent disease development. The ;+' strand open reading frame (ORF) located immediately upstream from the capsid ORF and predicted to encode a 10.9 kd protein was also found to be dispensable for replication but essential for systemic spread. By this analysis, MSV sequences that support autonomous replication were localized to a 1.7 kb segment containing the two viral intergenic regions and two overlapping complementary ;-' strand ORFs. Despite the inability of the gene replacement mutants to spread systemically, both inoculated and newly developed leaves displayed chlorotic patterns similar to the phenotype observed in certain developmental mutants of maize. The similarity of the MSV mutant phenotype to these developmental mutants is discussed.

Journal Article↗

PubMed1988

Infectivity and complete nucleotide sequence of the genome of a South African isolate of maize streak virus.

The complete infectious genome of a South African isolate of the geminivirus maize streak (MSV-S) has been cloned, characterized, and sequenced. Using an A. tumefaciens Ti plasmid delivery system, the cloned -2.7 kb single circular MSV component was shown to be necessary and sufficient for infection of maize. Based on sequence analysis of the infectious clone, MSV-S is highly homologous to the previously characterized Kenyan and Nigerian isolates. While the genomic organization of MSV-S has elements in common with each of these previously characterized isolates, it is identical to neither and its analysis addresses the discrepancies between them. The result is a somewhat simplified and unified picture of the viral genome, the structural organization of which is essentially identical to that of wheat dwarf virus.

Base Sequence↗

PubMed1982

Simian virus 40 mutant with transposed T-antigen and VP1 genes.

A simian virus 40 mutant with the T-antigen gene transposed to the late region of the viral genome has been constructed. This transposed molecule directed the synthesis of a full-sized, 92,000-dalton large T antigen in both permissive and nonpermissive cells. This large T antigen functioned in the initiation of viral DNA replication and in the transformation of nonpermissive cells. T-antigen synthesis by this transposed genome had the characteristics of late transcription, thus indicating that functional large T antigen of simian virus 40 is not required for the initiation of late transcription.

Animals↗

PubMed1982

Organization of the Epstein-Barr virus DNA molecule. II. Fine mapping of the boundaries of the internal repeat cluster of B95-8 and identification of additional small tandem repeats adjacent to the HR-1 deletion.

We used cloned BamHI fragments from Epstein-Barr virus strain B95-8 [EBV(B95-8)]DNA to obtain detailed restriction maps of the region of the genome adjacent to the large internal repeat cluster. These maps together with the results of hybridization experiments using a 3.1-kilobase repeat probe defined more precisely the location of the injection between the internal repeat cluster and the flanking unique-sequence DNA. On one side (UL), the repeat sequences extended 600 +/- 80 base pairs (bp) into BamHI-Y; on the other side (US), they extended 1,300 +/- 200 bp into BamHI-C. Therefore, EBV(B95-8) DNA contained a nonintegral number of 3.1-kilobase repeat units, namely, 12.6 copies. The mapping studies also revealed a second series of internal tandem repetitions in EBV(B95-8) DNA located within the BamHI-H fragment. This cluster comprised 11 copies of a 135-bp repeat unit which contained a single site for the NotI restriction endonuclease. Hybridization to these cloned EBV(B95-8) fragments using total EBV(HR-1) DNA as probe indicated that the deletion in EBV(HR-1) removed all 3,000 bp of unique-sequence DNA which lay between the large 3.1-kilobase and the small 135-bp repeat clusters. Thus, the deletion which destroyed the transforming ability in the EBV(HR-1) virus was bounded on either side by tandem repetitions.

Base Sequence↗