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1.
Arch Virol ; 160(2): 375-87, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25449306

RESUMO

Geminiviruses are DNA viruses that infect several economically important crops, resulting in a reduction in their overall yield. These plant viruses have circular, single-stranded DNA genomes that replicate mainly by a rolling-circle mechanism. Geminivirus infection results in crosstalk between viral and cellular factors to complete the viral life cycle or counteract the infection as part of defense mechanisms of host plants. The geminiviral replication initiator protein Rep is the only essential viral factor required for replication. It is multifunctional and is known to interact with a number of host factors to modulate the cellular environment or to function as a part of the replication machinery. This review provides a holistic view of the research related to the viral Rep protein and various host factors involved in geminiviral DNA replication. Studies on the promiscuous nature of geminiviral satellite DNAs are also reviewed.


Assuntos
Replicação do DNA/genética , DNA Circular/genética , DNA Satélite/genética , Proteínas de Ligação a DNA/genética , Geminiviridae/genética , DNA Circular/biossíntese , DNA Viral/biossíntese , DNA Viral/genética , Doenças das Plantas/virologia , Ligação Proteica , Proteínas Virais/genética , Replicação Viral/genética
2.
Arch Virol ; 160(4): 959-67, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25643815

RESUMO

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.


Assuntos
DNA Helicases/metabolismo , Tospovirus/enzimologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , DNA Helicases/química , DNA Helicases/genética , DNA Viral/genética , Inativação Gênica , Mutação de Sentido Incorreto , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Nicotiana/genética , Nicotiana/virologia , Tospovirus/química , Tospovirus/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genética
3.
Virus Genes ; 48(3): 486-93, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24442717

RESUMO

Geminiviral replication initiator protein (Rep) is a key player in geminiviral rolling circle mode of replication. However, the virus exploits various host cellular machineries for its replication. Study of these host factors is important to understand the geminiviral DNA replication in greater details. With this view, we screened for the peptides interacting with the Rep protein of a representative of geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), employing phage display technique. Through this screen, we have identified a host transcription factor, NAC083, as a potential MYMIV-Rep-binding partner. In silico docking studies also suggested possible binding of NAC083 peptide to MYMIV-Rep. We validated the interaction between MYMIV-Rep and Arabidopsis thaliana full-length NAC083 protein using in vitro pull-down assay and yeast two-hybrid analysis. NAC proteins are well-known transcription factors belonging to the largest gene families in plants. This study demonstrates for the first time the interaction of NAC083, a member of NAC transcription factor family, with MYMIV-Rep protein thereby indicating its possible role in MYMIV DNA replication.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/virologia , Begomovirus/metabolismo , DNA Helicases/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Begomovirus/genética , DNA Helicases/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Virais/genética
4.
FASEB J ; 26(3): 1142-60, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22171001

RESUMO

Geminiviruses primarily encode only few factors, such as replication initiator protein (Rep), and need various host cellular machineries for rolling-circle replication (RCR) and/or recombination-dependent replication (RDR). We have identified a host factor, RAD54, in a screen for Rep-interacting partners and observed its role in DNA replication of the geminivirus mungbean yellow mosaic India virus (MYMIV). We identified the interacting domains ScRAD54 and MYMIV-Rep and observed that ScRAD54 enhanced MYMIV-Rep nicking, ATPase, and helicase activities. An in vitro replication assay demonstrated that the geminiviral DNA replication reaction depends on the viral Rep protein, viral origin of replication sequences, and host cell-cycle proteins. Rad54-deficient yeast nuclear extract did not support in vitro viral DNA replication, while exogenous addition of the purified ScRAD54 protein enhanced replication. The role of RAD54 in in planta replication was confirmed by the transient replication assay; i.e., agroinoculation studies. RAD54 is a well-known recombination/repair protein that uses its DNA-dependent ATPase activity in conjunction with several other host factors. However, this study demonstrates for the first time that the eukaryotic rolling-circle replicon depends on the RAD54 protein.


Assuntos
Begomovirus/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Begomovirus/genética , Begomovirus/fisiologia , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Interações Hospedeiro-Patógeno , Microscopia Eletrônica , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Replicação Viral
5.
Nucleic Acids Res ; 38(20): 7037-53, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20571080

RESUMO

Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a ∼35 kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single-stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Antiprotozoários/farmacologia , Núcleo Celular/metabolismo , Ciprofloxacina/farmacologia , DNA de Protozoário/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eritrócitos/parasitologia , Teste de Complementação Genética , Organelas/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia Estrutural de Proteína
6.
Nucleic Acids Res ; 35(3): 755-70, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17182628

RESUMO

Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.


Assuntos
DNA Helicases/metabolismo , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Geminiviridae/genética , Proteína de Replicação A/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Bioensaio , Replicação do DNA , Teste de Complementação Genética , Mutação , Pisum sativum/genética , Pisum sativum/crescimento & desenvolvimento , Pisum sativum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteína de Replicação A/genética , Nicotiana/virologia , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genética
7.
Nucleic Acids Res ; 35(9): 2861-74, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17430964

RESUMO

Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.


Assuntos
Proteínas de Bactérias/química , DnaB Helicases/química , Helicobacter pylori/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Teste de Complementação Genética , Estrutura Terciária de Proteína , Deleção de Sequência , Homologia Estrutural de Proteína
8.
Nucleic Acids Res ; 34(21): 6362-77, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17142233

RESUMO

Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).


Assuntos
Begomovirus/enzimologia , Begomovirus/genética , DNA Helicases/metabolismo , Replicação do DNA , Proteínas Virais/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/genética , DNA Viral/biossíntese , Deleção de Sequência , Proteínas Virais/genética
9.
Nucleic Acids Res ; 31(23): 6828-40, 2003 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-14627816

RESUMO

Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5' to 3' helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.


Assuntos
Proteínas de Bactérias , DNA Helicases/metabolismo , Helicobacter pylori/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Clonagem Molecular , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/isolamento & purificação , DnaB Helicases , Escherichia coli/enzimologia , Escherichia coli/genética , Teste de Complementação Genética , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Dados de Sequência Molecular , Nucleotídeos/farmacologia , Cloreto de Potássio/farmacologia , Temperatura , Uridina Trifosfato/metabolismo
10.
Protoplasma ; 253(2): 467-75, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25944245

RESUMO

Minichromosome maintenance 2-7 (MCM2-7) proteins are conserved eukaryotic replicative factors essential for the DNA replication at its initiation and elongation step, and act as a licensing factor. The MCM2-7 and MCM4/6/7subcomplex exhibit DNA helicase activity, and are therefore regarded as the replicative helicase. The MCM proteins have not been studied in detail in plant system. Here, we present the biochemical characterization of Arabidopsis thaliana MCM3 single subunit and show that it exhibits in vitro unwinding and ATPase activities. AtMCM3 shows a greater unwinding activity with 5' forked partial DNA duplex substrate as compared to 3' forked and non-forked substrates. ATP and magnesium ion are indispensable for its DNA helicase activity. Specifically, ATP and dATP are the preferred nucleotides for its unwinding activity. The directionality of the AtMCM3 has been determined to be in 3' to 5' direction. The oligomerization status of AtMCM3 single subunit protein indicates that it is present in different multimeric forms. The unraveling of the helicase activity of AtMCM3 will provide better insights into the plant DNA replication.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/fisiologia , Biocatálise , DNA de Plantas/química , DNA de Cadeia Simples/química , Hidrólise , Componente 3 do Complexo de Manutenção de Minicromossomo/fisiologia , Ligação Proteica , Estrutura Quaternária de Proteína
11.
Virus Res ; 172(1-2): 35-45, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23276684

RESUMO

Designing artificial microRNAs (amiRs) targeting the genes responsible for viral replication, transmission and symptom development after viral infection offers a promising strategy to contain the multiplication and spread of geminiviruses in host plants. Here, we report the design of two amiRs targeting the middle region of the AV1 (coat protein) transcript (amiR-AV1-3) and the overlapping region of the AV1 and AV2 (pre-coat protein) transcripts (amiR-AV1-1) of a model geminivirus, Tomato leaf curl virus (ToLCV). Our analyses demonstrate that transgenic tomato plants expressing amiR-AV1-1, propagated until the T2 generation and were highly tolerant to Tomato leaf curl New Delhi virus (ToLCNDV), whereas those harboring amiR-AV1-3 exhibited only moderate tolerance. Biochemical analyses revealed that in these cases, the amiRs acted through the slicing mechanism, cleaving their respective targets. Although ToLCVs are generally difficult targets for manipulations related to virus resistance, our data reveal that an amiR strategy could be employed to protect plants in an effective manner.


Assuntos
Begomovirus/crescimento & desenvolvimento , Proteínas do Capsídeo/antagonistas & inibidores , MicroRNAs/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/virologia , Begomovirus/genética , Proteínas do Capsídeo/genética , Solanum lycopersicum , MicroRNAs/genética
12.
Plant Physiol Biochem ; 49(1): 13-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21075002

RESUMO

Tomato leaf curl virus (ToLCV) is a member of family geminiviridae that constitute rapidly emerging group of phytopathogens posing threat to a large number of vegetable crops worldwide. Three different genomes are found to be associated with ToLCV viz., DNA-A, DNA-B and beta satellite DNA. MicroRNAs (miRs) are known to govern several fundamental processes in eukaryotes, including basal defense mechanisms. In animals, it has been demonstrated that certain host miRs prevent viral establishment by directly interfering with pathogen replication or by binding to viral transcripts. However, in spite of the existence of huge families of phytopathogenic viruses, no such mechanism has been observed in plants. In the present study, we performed in silico analysis to investigate whether tomato encoded miR/miR* sequences possess any potential to bind to viral genome and/or encoded ORFs. We observed that different sequences can bind to ToLCNDV DNA-A, ToLCNDV DNA-B and ToLCNDV associated DNA beta genomes and most of the encoded ORFs. Interestingly, our analysis revealed that several miR* species could similarly target genome and ORFs of ToLCNDV suggesting novel role of miR* in host defense response. This observation holds much importance as miR* molecules are presently thought to follow degradation pathway and are not assigned with any function. Moreover, we could predict targets for these miR* sequences that are generally involved in plant metabolism. Overall, these results shed light on new paradigm of intricate host-pathogen interactions via miRNA pathway.


Assuntos
Begomovirus/genética , DNA Viral , Genoma de Planta , Genoma Viral , Interações Hospedeiro-Patógeno/genética , MicroRNAs/metabolismo , Solanum lycopersicum/genética , Sequência de Bases , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Fases de Leitura Aberta , Doenças das Plantas/genética , Doenças das Plantas/virologia , Imunidade Vegetal/genética
13.
Virus Res ; 160(1-2): 395-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21600246

RESUMO

Tomato leaf curl viruses (ToLCV) infect tomato plants and eventually cause several phenotypic defects, notably in the leaves in the form of upward curling. The entry of virus triggers plants' basal defense responses which eventually introduce temporal changes in the transcriptome to evade the pathogen attack. In this study, we have identified about 20 tomato ESTs using subtractive hybridization that were induced in tomato leaves upon agro-infection with the constructs bearing the dimers of Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and DNA-B components. The induced ESTs belonged to the class of genes that play crucial roles in innate immunity, plants metabolism and ethylene signaling. The expression of few of these ESTs was validated by northern blot analysis and two out of six selected genes expressed exclusively in the infected leaf tissues. Besides leaves, the expression status of selected genes was checked in a wide variety of tissues (flower, fruit, stem and root) of both healthy and infected plants by RT-PCR. These results suggest that the flower and fruit tissues, similar to leaves, exhibited induction of most of the genes while the stem and root tissues suffered from down-regulation. Overall, these results indicate that the hosts' transcriptome undergoes considerable changes in response to viral infection.


Assuntos
Begomovirus/patogenicidade , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Solanum lycopersicum/virologia , Northern Blotting , Etiquetas de Sequências Expressas , Hibridização de Ácido Nucleico , Doenças das Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Int J Biol Sci ; 5(2): 97-117, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19173032

RESUMO

Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways.


Assuntos
Interferência de RNA/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Chlamydomonas reinhardtii/metabolismo , MicroRNAs/genética , MicroRNAs/fisiologia , Modelos Biológicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia
15.
Virology ; 380(1): 75-83, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18703212

RESUMO

In order to explore the mechanism of geminivirus DNA replication, we show that the Replication initiator (Rep) protein encoded by Mungbean yellow mosaic India virus (MYMIV), a member of the family Geminiviridae, binds specifically to the iterons present in the viral DNA replication origin (CR-A) in a highly ordered manner that might be a prerequisite for the initiation of replication. MYMIV Rep also acts as a helicase during the post-initiation stage and is upregulated in presence of the RPA32 subunit of Replication Protein A. The implication of these findings on the initiation and elongation stages of MYMIV DNA replication has been discussed.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Geminiviridae/fisiologia , Origem de Replicação/fisiologia , Proteína de Replicação A/metabolismo , Transativadores/metabolismo , DNA Helicases/genética , DNA Viral/biossíntese , Geminiviridae/genética , Proteína de Replicação A/genética , Transativadores/genética , Transcrição Gênica , Replicação Viral/genética
16.
Nucleic Acids Symp Ser (Oxf) ; (52): 507-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776476

RESUMO

MicroRNAs are approximately 21- 25 nt long RNA species that are critical regulators of transcriptome across the eukaryotes. Growing number of evidences clearly supports their involvement in plant leaf development. ToLCV infection severely affects the morphology of mature tomato leaves. To investigate the mechanism underlying the virus- host interaction, we focussed our studies on expression of microRNAs and the irrespective targets under normal and ToLCV infection. We have cloned Myb33, ARF4 homolog, Argonaute1, Apetala2, SBP transcription factor and RBOH from tomato and checked their expression by RT-PCR. Our work suggests that miR159 is upregulated while miR164 and miR171 are downregulated under viral infection. Our studies shed light on the impact of ToLCV infection on host transcriptome.


Assuntos
Begomovirus , MicroRNAs/metabolismo , Doenças das Plantas/virologia , Solanum lycopersicum/genética , Solanum lycopersicum/virologia , Biomarcadores/metabolismo , Solanum lycopersicum/metabolismo , Doenças das Plantas/genética
17.
J Virol ; 78(21): 11890-903, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15479830

RESUMO

Proliferative cell nuclear antigen (PCNA), a conserved plant protein as well as an important replication factor, is induced in response to geminivirus infection in the resting cells of the phloem tissues. The biochemical role of PCNA in rolling circle replication (RCR) of geminivirus DNA has not been explored in detail. The initiation of RCR of the bipartite genome of a geminivirus, Indian mung bean yellow mosaic virus (IMYMV), is mainly controlled by viral protein Rep (or AL1 or AC1). The role of host PCNA in RCR of IMYMV was revealed by studying the physical and functional interactions between recombinant PCNA and recombinant IMYMV Rep. Pea nuclear PCNA as well as recombinant pea PCNA showed binding to recombinant Rep in experiments involving both affinity chromatography and yeast two-hybrid approaches. The contacting amino acid residues of PCNA seemed to be present throughout a wide region of the trimeric protein, while those of Rep appeared to be localized only in the middle part of the protein. The site-specific nicking-closing activity and the ATPase function of IMYMV Rep were impaired by PCNA. These observations lead to interesting speculations about the control of viral RCR and dynamic profiles of protein-protein interactions at the RCR origin of the geminiviruses.


Assuntos
Fabaceae/virologia , Geminiviridae/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Sítios de Ligação , Replicação do DNA , Regulação para Baixo , Antígeno Nuclear de Célula em Proliferação/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química
18.
J Virol ; 78(5): 2405-13, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14963136

RESUMO

Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.


Assuntos
Geminiviridae/genética , Geminiviridae/fisiologia , Plantas/virologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/virologia , Replicação Viral , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Genes Virais/genética , Teste de Complementação Genética , Genoma Viral , Modelos Biológicos , Doenças das Plantas/virologia , Plasmídeos/genética , Reação em Cadeia da Polimerase
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