Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator

Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.     

High-affinity Rep-binding is not required for the replication of a geminivirus DNA and its satellite

The 682-nt satellite DNA (sat-DNA) of Tomato leaf curl virus (TLCV) depends on the helper virus for its replication. In contrast to the strict specificity that exists in each geminivirus for its cognate replication associated protein (Rep), TLCV sat-DNA can utilize Rep encoded by distinct geminiviruses. We have used a combination of protein-binding assays and mutagenesis to show that repeat motifs in TLCV and sat-DNA are essential for Rep-binding in vitro. Surprisingly, mutants of TLCV and sat-DNA impaired in their ability to bind TLCV Rep in vitro were infectious in tomato. Thus, in contrast to other geminiviruses reported, TLCV and sat-DNA replication is independent of the high-affinity in vitro Rep binding. These results prompt a reassessment of the current model of geminivirus replication where Rep/DNA interaction is a highly specific step in the initiation of rolling circle replication.     

Self-interaction of Abutilon mosaic virus replication initiator protein (Rep) in plant cell nuclei

Geminiviruses replicate their circular single-stranded DNA genome in nuclei of infected plant cells. Their replication initiator proteins (Reps) possess interaction domains for homo- and hetero-oligomerization as shown previously by in vitro studies and yeast two hybrid assays. Here, homo-oligomerization and cellular localization of the Abutilon mosaic virus (AbMV) Rep was analysed with bimolecular fluorescence complementation (BiFC) in epidermal tissues of Nicotiana benthamiana. BiFC revealed that Rep oligomers accumulated within the nucleoplasm, but were excluded from nucleoli as indicated by a nucleoli/cajal body marker. A similar subcellular distribution was observed for Rep fused to full-length cyan fluorescent protein. To examine whether tagged Reps were functionally active, N. benthamiana plants transgenic for a dimeric AbMV DNA B were inoculated with the BiFC expression constructs and nucleic acids were analysed by rolling circle amplification/restriction fragment length polymorphism as well as Southern blot hybridization. The results confirmed that the modified AbMV Rep was able to transreplicate DNA B.     

MYMIV replication initiator protein (Rep): roles at the initiation and elongation steps of MYMIV DNA replication

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.     

Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed.     

Interaction between coat protein and replication initiation protein of Mung bean yellow mosaic India virus might lead to control of viral DNA replication

In addition to their encapsidation function, viral coat proteins (CP) contribute to viral life cycle in many different ways. The CPs of the geminiviruses are responsible for intra- as well as inter-plant virus transmission and might determine the yield of viral DNA inside the infected tissues by either packaging the viral DNA or interfering with the viral replicative machinery. Since the cognate Rep largely controls the rolling circle replication of geminiviral DNA, the interaction between Rep and CP might be worthwhile to examine for elucidation of CP-mediated control of the viral DNA copy number. Here a reasonably strong interaction between Rep and CP of the geminivirus Mung bean yellow mosaic India virus is reported. The domain of interaction has been mapped to a central region of Rep. The replication initiation activity of Rep, i.e., its nicking and closing function, is down regulated by CP. This report highlights how CP could be important in controlling geminiviral DNA replication.     

Expression of full-length and truncated Rep genes from Mungbean yellow mosaic virus-Vigna inhibits viral replication in transgenic tobacco

Mungbean yellow mosaic virus-Vigna (MYMV-Vig) is a bipartite geminivirus that causes a severe yellow mosaic disease in blackgram. An assay was developed to study MYMV-Vig replication by agroinoculation of tobacco leaf discs with partial dimers of the virus. This assay, in a non-host model plant, was used to evaluate pathogen-derived resistance contributed by MYMV-Vig genes in transgenic plants. Viral DNA accumulation was optimum in tobacco leaf discs cultured for 10 days after infection with Agrobacterium tumefaciens strain Ach5 containing partial dimers of both DNA A and DNA B of MYMV-Vig. Transgenic tobacco plants with MYMV-Vig genes for coat protein (CP), replication-associated protein (Rep)-sense, Rep-antisense, truncated Rep (T-Rep), nuclear shuttle protein (NSP) and movement protein (MP) were generated. Leaf discs from transgenic tobacco plants, harbouring MYMV-Vig genes, were agroinoculated with partial dimers of MYMV-Vig and analyzed for viral DNA accumulation. The leaf discs from transgenic tobacco plants harbouring CP and MP genes supported the accumulation of higher levels of MYMV-Vig DNA. However, MYMV-Vig accumulation was inhibited in one transgenic plant harbouring the Rep-sense gene and in two plants harbouring the T-Rep gene. Northern analysis of these plants revealed a good correlation between expression of Rep or T-Rep genes and inhibition of MYMV-Vig accumulation.     

Interaction of geminivirus Rep protein with replication factor C and its potential role during geminivirus DNA replication

Geminivirus DNA replication during the rolling-circle stage depends on the use of a DNA primer, a strategy poorly understood as compared with other eukaryotic viral systems that rely on RNA or protein as primers. Here we have used wheat dwarf virus (WDV) with the aim of elucidating the events leading to recruitment of cell factors at the replication origin. We have identified a novel interaction of WDV Rep, the replication initiation protein, with the large subunit of the wheat replication factor C complex (TmRFC-1). In other systems, the heteropentameric RFC clamp loader complex stimulates loading of DNA polymerase delta to the primer-template. Expression of TmRFC-1 is subjected to cell-cycle regulation, with a peak in early S-phase. We show that WDV Rep stimulates binding of recombinant TmRFC-1 to a model substrate containing a 3'-OH terminus and a WDV Rep-binding site. This was confirmed using cellular fractions enriched for wheat RFC complex, supporting the idea that, in addition to generating a 3'-OH terminus during initiation of DNA replication, WDV Rep could participate in the recruitment of RFC to the newly formed primer. We propose that this pathway may represent an initial event to facilitate the assembly of other replication factors, e.g., PCNA and/or DNA polymerase delta, a model that could also apply to other eukaryotic replicons, such as nanoviruses, circoviruses, and parvoviruses with a similar DNA replication strategy.     

Characterisation of the interaction between WRN,the helicase/exonuclease defective in progeroid Werner's syndrome,and an essential replication factor,PCNA

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.     

Insights into the functional characteristics of geminivirus rolling-circle replication initiator protein and its interaction with host factors affecting viral DNA replication

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.     

Cpr1 cyclophilin and Ess1 parvulin prolyl isomerases interact with the tombusvirus replication protein and inhibit viral replication in yeast model host

To identify host proteins interacting with the membrane-bound replication proteins of tombusviruses, we performed membrane yeast two-hybrid (MYTH) screens based on yeast cDNA libraries. The screens led to the identification of 57 yeast proteins interacting with replication proteins of two tombusviruses. Results from a split ubiquitin assay with 12 full-length yeast proteins and the viral replication proteins suggested that the replication proteins of two tombusviruses interact with a similar set of host proteins. Follow-up experiments with the yeast Cpr1p cyclophilin, which has prolyl isomerase activity that catalyzes cis-trans isomerization of peptidyl-prolyl bonds, confirmed that Cpr1p interacted with the viral p33 replication protein in yeast and in vitro. Replication of Tomato bushy stunt virus replicon RNA increased in cpr1Δ yeast, while over-expression of Cpr1p decreased viral replication. We also show that the Ess1p parvulin prolyl isomerase partly complements Cpr1p function as an inhibitor of tombusvirus replication.     

Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM)

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA₃₂, which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins.     

Identification of an octanucleotide motif sequence essential for viral protein, DNA, and progeny virus biosynthesis at the origin of DNA replication of porcine circovirus type 2

A plasmid-based transfection system capable of generating infectious porcine circovirus type 2 (PCV2) was established. This system was then used in mutagenesis studies to investigate the involvement of a "conserved" nonanucleotide (which constitutes a portion of the loop sequence) located at the origin of DNA replication of PCV2 with respect to viral protein synthesis, DNA self-replication, and progeny virus production. The results demonstrated that an octanucleotide (AGTATTAC) embedded in the loop sequence is essential for virus replication. This octanucleotide can be further condensed to an essential core element (ECE) represented by AxTAxTAC. The positions specified by the indicated nucleotides are critical for viral DNA replication and stable infectious virus production, and they cannot be substituted by other bases, while the positions indicated by x can accept variable bases and yield stable progeny viruses.     

Interactions of the cellular CCAAT displacement protein and human papillomavirus E2 protein with the viral origin of replication can regulate DNA replication

Previously, we and others have shown that CCAAT displacement protein (CDP) negatively regulates the papillomavirus promoters. Overexpression of CDP has been shown to inhibit high-risk human papillomavirus virus (HPV) and bovine papillomavirus DNA replication in vivo presumably through reduction in expression of viral replication proteins, E1 and E2. Sequence analysis of the HPV origin indicates several potential CDP-binding sites with one site overlapping the E1-binding site. Therefore, CDP could also negatively regulate papillomavirus replication directly by preventing the loading of the initiation complex. We show here that purified CDP inhibits in vitro HPV DNA replication. Footprint analysis demonstrated that CDP binds the E1-binding site and the TATA box, and that the binding of purified CDP to the E1-binding site is decreased by the addition of purified E2 protein. Consistent with this, E2-independent in vitro HPV replication is inhibited by CDP to a greater extent than E2-dependent replication. These results suggest that binding of E2 at the E2-binding site may play an important role in overcoming the inhibition of E1 initiation complex formation caused by the binding of negative regulators like CDP to the origin of replication.     

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

Replication protein A (RPA) is the main eukaryotic ssDNA-binding protein with essential roles in DNA replication, recombination, and repair. RPA maintains the DNA as single-stranded and also interacts with other DNA-processing proteins, coordinating their assembly and disassembly on DNA. RPA binds to ssDNA in two conformational states with opposing affinities for DNA and proteins. The RPA–protein interactions are compatible with a low DNA affinity state that involves DNA-binding domain A (DBD-A) and DBD-B but not with the high DNA affinity state that additionally engages DBD-C and DBD-D. The structure of the high-affinity RPA–ssDNA complex reported here shows a compact quaternary structure held together by a four-way interface between DBD-B, DBD-C, the intervening linker (BC linker), and ssDNA. The BC linker binds into the DNA-binding groove of DBD-B, mimicking DNA. The associated conformational change and partial occlusion of the DBD-A–DBA-B protein–protein interaction site establish a mechanism for the allosteric coupling of RPA–DNA and RPA–protein interactions.     

Host transcription factor Rpb11p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins

Previous genome-wide screens identified over 100 host genes whose deletion/down-regulation affected tombusvirus replication and 32 host genes that affected tombusvirus RNA recombination in yeast, a model host for replication of Tomato bushy stunt virus (TBSV). Down-regulation of several of the identified host genes affected the accumulation levels of p33 and p92(pol) replication proteins, raising the possibility that these host factors could be involved in the regulation of the amount of viral replication proteins and, thus, they are indirectly involved in TBSV replication and recombination. To test this model, we developed a tightly regulated expression system for recombinant p33 and p92(pol) replication proteins in yeast. We demonstrate that high accumulation level of p33 facilitated efficient viral RNA replication, while the effect of p33 level on RNA recombination was less pronounced. On the other hand, high level of p92(pol) accumulation promoted TBSV RNA recombination more efficiently than RNA replication. As predicted, Rpb11p, which is part of the polII complex, affected the accumulation levels of p33 and p92(pol) as well as altered RNA replication and recombination. An in vitro assay with the tombusvirus replicase further supported that Rpb11p affects TBSV replication and recombination only indirectly, via regulating p33 and p92(pol) levels. In contrast, the mechanism by which Rpt4p endopeptidase/ATPase and Mps1p threonine/tyrosine kinase affect TBSV recombination is different from that proposed for Rpb11p. We propose a model that the concentration (molecular crowding) of replication proteins within the viral replicase is a factor affecting viral replication and recombination.     

Rep68 protein of adeno-associated virus type 2 interacts with 14-3-3 proteins depending on phosphorylation at serine 535

Rep78/68 proteins of adeno-associated virus type 2 (AAV-2) are involved in many aspects of the viral life cycle, including replication, gene expression, and site-specific integration. To understand the molecular mechanisms of the actions of Rep proteins, we searched for Rep68-interacting cellular proteins by utilizing a one-step affinity purification technique and identified two members of 14-3-3 proteins (14-3-3 epsilon and gamma). We found that phosphorylation of 535Ser at the carboxy terminus of Rep68 was critical for its association with 14-3-3. The association of 14-3-3 proteins to Rep68 resulted in reduction of the affinity of Rep68 for DNA. Furthermore, genome DNA replication of a recombinant mutant virus carrying a phosphorylation-deficient Rep68 (Ser535Ala) was more efficient than that of the wild-type virus. These results suggest that phosphorylation of Rep68 and subsequent association with 14-3-3 proteins regulates Rep-mediated functions during the AAV life cycle.     

血清乙肝病毒外膜大蛋白检测及其与病毒复制的关系

目的探讨血清乙型肝炎病毒外膜大蛋白（LHBs）检测对于判定乙型肝炎病毒（HBV）复制的临床意义。方法分别采用ELISA法、时间分辨免疫荧光分析法和实时荧光定量PCR法检测340份慢性乙型肝炎患者血清中LHBs、Pre-S1蛋白、HBV-M和HBVDNA，并进行相关性分析。结果340份慢性乙型肝炎患者血清中LHBs水平与HBVDNA拷贝数变化相一致，两者呈正相关（r=0．899。P=0．038）；在不同模式的HBeAg血清中，LHBs与HBVDNA的阳性率差异均无统计学意义（P均〉0．05）；HBV DNA阳性血清中LHBs阳性率（83．15％）明显高于Pre-S1蛋白和HBeAg的阳性率（50．54％和54．48％），差异有统计学意义（P均〈0．05）。结论血清LHBs水平能反映HBV感染者体内HBV复制程度，其灵敏度高于Pre-S1蛋白和HBeAg，可作为判断HBV复制新的血清学指标。     

Local and global changes in the morphology and distribution of replication centres in rapidly expanding nuclei

Sperm pronuclei assembled inXenopus egg extracts undergo a 10-fold increase in volume over a typical 150-min incubation. Double immunofluorescence with anti-proliferating cell nuclear antigen (PCNA) antibodies and biotinylated anologues of thymidine can be used to distinguish different phases of S-phase. The mean diameter of pronuclei at initiation was 12.42 µm. During the elongation phase of S-phase the mean diameter increased to 17.44 µm, equal to a sevenfold increase in nuclear volume. Confocal microscopy was used to investigate the morphology and distribution of replication centres through S-phase. In early S-phase nuclei, the mean centre-to-centre distance between any replication centre and its 10 nearest neighbours was 1.24 µm; this decreased significantly to 0.90 µm in mid S-phase nuclei and 0.99 µm in late S-phase nuclei. In addition, the morphology of replication foci also changed. In early S-phase nuclei, each replication focus was a discrete entity. However, as S-phase progressed neighbouring replication foci appeared to fuse giving rise to ring-like structures or string-like structures. Three-dimensional reconstructed images of z-series collected through early and late S-phase nuclei indicated that as nuclei enlarge replication centres appear to accumulate at the nuclear periphery. A model is proposed to accommodate the results described below.