A Host RNA Helicase-Like Protein,AtRH8, Interacts with the Potyviral Genome-Linked Protein,VPg, Associates with the Virus Accumulation Complex,and Is Essential for Infection

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.Plant viruses are obligate intracellular parasites that infect many agriculturally important crops and cause severe losses each year. One of the common characteristics of plant viruses is their relatively small genome that encodes a limited number of viral proteins, making them dependent on host factors to fulfill their infection cycles (Maule et al., 2002; Whitham and Wang, 2004; Nelson and Citovsky, 2005; Decroocq et al., 2006). In order to establish a successful infection, the invading virus must recruit an array of host proteins (host factors) to translate and replicate its genome and to move locally from cell to cell via the plasmodesmata and systemically via the vascular system. It has been suggested that down-regulation or mutation of some of the required host factors may result in recessively inherited resistance to viruses (Kang et al., 2005b).Potyviruses, belonging to the genus Potyvirus in the family Potyviradae, constitute the largest group of plant viruses (Rajamäki et al., 2004). Potyviruses have a single positive-strand RNA genome approximately 10 kb in length, with a viral genome-linked protein (VPg) covalently attached to the 5′ end and a poly(A) tail at the 3′ end (Urcuqui-Inchima et al., 2001; Rajamäki et al., 2004). The viral genome contains a single open reading frame (ORF) that translates into a polypeptide with a molecular mass of approximately 350 kD, which is cleaved into 10 mature proteins by viral proteases (Urcuqui-Inchima et al., 2001). Recently, a novel viral protein resulting from a frameshift in the P3 cistron has been reported (Chung et al., 2008). Of the 11 viral proteins, VPg is a multifunctional protein and the only other viral protein present in the viral particles (virions) besides the coat protein and the cylindrical inclusion protein (CI; Oruetxebarria et al., 2001; Puustinen et al., 2002; Gabrenaite-Verkhovskaya et al., 2008). The nonstructural protein is linked to the viral RNA by a phosphodiester bond between the 5′ terminal uridine residue of the RNA and the O⁴-hydroxyl group of amino acid Tyr (Murphy et al., 1996; Oruetxebarria et al., 2001; Puustinen et al., 2002). Mutation of the Tyr residue that links VPg to the viral RNA abolishes virus infectivity completely (Murphy et al., 1996). In infected cells, VPg and its precursor NIa are present in the nucleus and in the membrane-associated virus replication vesicles in the cytoplasm (Carrington et al., 1993; Rajamäki and Valkonen, 2003; Cotton et al., 2009). As a component of the replication complex, VPg may serve as a primer for viral RNA replication (Puustinen and Mäkinen, 2004) and as an analog of the m⁷G cap of mRNAs for the viral genome to recruit the translation complex for translation (Michon et al., 2006; Beauchemin et al., 2007; Khan et al., 2008). Furthermore, VPg has been suggested to be an avirulence factor for recessive resistance genes in diverse plant species (Moury et al., 2004; Kang et al., 2005b; Bruun-Rasmussen et al., 2007). Thus, VPg plays a pivotal role in the virus infection process. The molecular identification of VPg-interacting host proteins and the subsequent functional characterization of such interactions may advance knowledge of the intricate virus replication mechanisms and help develop novel antiviral strategies.Previous studies have shown that VPg and its precursor NIa interact with several host proteins, including three essential components of the host protein translation apparatus (Thivierge et al., 2008). The first protein is the cellular translation initiation factor eIF4E or its isoform eIF(iso)4E, identified through a yeast two-hybrid screen using VPg as a bait (Wittmann et al., 1997; Schaad et al., 2000). The protein complex of VPg and eIF4E is an essential component for virus infectivity (Robaglia and Caranta, 2006). Mutations and knockout of eIF4E or eIF(iso)4E confer resistance to infection (Lellis et al., 2002; Ruffel et al., 2002; Nicaise et al., 2003; Gao et al., 2004; Kang et al., 2005a; Ruffel et al., 2005; Decroocq et al., 2006; Bruun-Rasmussen et al., 2007). It is well known that potyviruses recruit selectively one of the eIF4E isoforms, depending on specific virus-host combinations (German-Retana et al., 2008). For instance, in Arabidopsis (Arabidopsis thaliana), eIF(iso)4E is required for infection by Turnip mosaic virus (TuMV), Plum pox virus (PPV), and Lettuce mosaic virus, while eIF4E is indispensable for infection by Clover yellow vein virus (Duprat et al., 2002; Lellis et al., 2002; Sato et al., 2005; Decroocq et al., 2006). The second cellular protein interacting with VPg is another translation initiation factor, eIF4G. Analysis of Arabidopsis knockout mutants for eIF4G or its isomers eIF(iso)4G1 and eIF(iso)4G2 has yielded results supporting the idea that the recruitment of eIF4G for potyvirus infection is also isoform dependent (Nicaise et al., 2007). Recently, poly(A)-binding protein (PABP), the translation initiation factor that bridges the 5′ and 3′ termini of the mRNA into proximity, has been proposed to be essential for efficient multiplication of TuMV (Dufresne et al., 2008). PABP was previously documented to interact with NIa, a VPg precursor containing both VPg and the proteinase NIa-Pro (Léonard et al., 2004). As the translation factors eIF(iso)4E and PABP have been found to be internalized in virus-induced vesicles, it has been suggested that the interactions between VPg and these translation factors are crucial for viral RNA translation and/or replication (Beauchemin and Laliberté, 2007; Beauchemin et al., 2007; Cotton et al., 2009). Besides these three translation factors, a Cys-rich plant protein, potyvirus VPg-interaction protein, was also found to associate with VPg (Dunoyer et al., 2004). This plant-specific VPg-interacting host protein contains a PHD finger domain and acts as an ancillary factor to support potyvirus infection and movement (Dunoyer et al., 2004).In this study, we describe the identification of an Arabidopsis DEAD-box RNA helicase (DDX), AtRH8, and a peach (Prunus persica) DDX-like protein, PpDDXL, both interacting with the potyviral VPg protein. Using the atrh8 mutant, we demonstrate that AtRH8 is not required for plant growth and development in Arabidopsis but is necessary for infection by two plant potyviruses, PPV and TuMV. Furthermore, we present evidence that AtRH8 colocalizes with the virus accumulation complex in potyvirus-infected leaf tissues, which reveals a possible role of AtRH8 in virus infection. Finally, we have identified the VPg-binding region (VPg-BR) of AtRH8 and PpDDX and show that overexpression of the VPg-BR either from AtRH8 or PpDDXL suppresses virus accumulation.     

Density-Dependent Benefits in Ant-Hemipteran Mutualism? The Case of the Ghost Ant Tapinoma melanocephalum (Hymenoptera: Formicidae) and the Invasive Mealybug Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

Although density-dependent benefits to hemipterans from ant tending have been measured many times, few studies have focused on integrated effects such as interactions between ant tending, natural enemy density, and hemipteran density. In this study, we tested whether the invasive mealybug Phenacoccus solenopsis is affected by tending by ghost ants (Tapinoma melanocephalum), the presence of parasitoids, mealybug density, parasitoid density and interactions among these factors. Our results showed that mealybug colony growth rate and percentage parasitism were significantly affected by ant tending, parasitoid presence, and initial mealybug density separately. However, there were no interactions among the independent factors. There were also no significant interactions between ant tending and parasitoid density on either mealybug colony growth rate or percentage parasitism. Mealybug colony growth rate showed a negative linear relationship with initial mealybug density but a positive linear relationship with the level of ant tending. These results suggest that benefits to mealybugs are density-independent and are affected by ant tending level.     

Two domains that control prefusion stability and transport competence of the measles virus fusion protein

Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability.     

Identification of chemokine receptor CCR4 antagonist

The present study reports the identification and hits to leads optimization of chemokine receptor CCR4 antagonists. Compound 12 is a high affinity, non-cytotoxic antagonist of CCR4 that blocks the functional activity mediated by the receptor.     

高山红景天愈伤颗粒组织悬浮培养动力学及工艺的研究

研究了高山红景天愈伤组织颗粒悬浮培养过程的生长及红景天甙合成的规律,发现红景天甙合成与细胞生长偶联,两者之间的关系表示为q=5．38×10^-3μ。其产率系数Yx/s及维持系数m分别为0．66g·g^-1和0．033g·(g·d)^-1。在3.5L气升式反应器中研究了培养过程的操作特性,发现“发泡”现象几乎不存在,氧传递系数K1a的变化不同常规植物细胞的悬浮培养．愈伤组织颗粒在培养过程中经历一个由小变大,后叉变小的过程。     

Preservation of fertility in females treated for cancer

Advancements of diagnosis and treatment have substantially improved cancer survival rates in the last few decades. The increasing number of survivors focuses attention on long-term effects caused by cancer treatment and its impact on quality of life. Ovarian failure is one of the major sequelae of cytotoxic chemotherapy and/or radiotherapy in female children and reproductive-age women. Oncologists should address the patients about fertility preservation options before therapy. Embryo cryopreservation is the only well-established method for females in preserving fertility; however other strategies including ovarian suppression, ovarian transposition and cryopreservation of oocytes and ovarian tissue are still experimental. Patients need advice and to know which are the most practical options for them. This article reviews the available fertility preservation methods in women, and the related issues including normal physiology of the ovary, effect of anticancer therapy on fertility, role of the oncologist and ethics. We performed a MEDLINE search from 1971 to 2011 in a similar way as Jensen et al. 2011, using the following MeSH terms: antineoplastic agents; ovarian failure; premature; infertility, female; fertility preservation; child and cancer; reproductive technologies, assisted.     

MicroRNA–Mediated Repression of the Seed Maturation Program during Vegetative Development in Arabidopsis

The seed maturation program only occurs during late embryogenesis, and repression of the program is pivotal for seedling development. However, the mechanism through which this repression is achieved in vegetative tissues is poorly understood. Here we report a microRNA (miRNA)–mediated repression mechanism operating in leaves. To understand the repression of the embryonic program in seedlings, we have conducted a genetic screen using a seed maturation gene reporter transgenic line in Arabidopsis (Arabidopsis thaliana) for the isolation of mutants that ectopically express seed maturation genes in leaves. One of the mutants identified from the screen is a weak allele of ARGONAUTE1 (AGO1) that encodes an effector protein for small RNAs. We first show that it is the defect in the accumulation of miRNAs rather than other small RNAs that causes the ectopic seed gene expression in ago1. We then demonstrate that overexpression of miR166 suppresses the derepression of the seed gene reporter in ago1 and that, conversely, the specific loss of miR166 causes ectopic expression of seed maturation genes. Further, we show that ectopic expression of miR166 targets, type III homeodomain-leucine zipper (HD-ZIPIII) genes PHABULOSA (PHB) and PHAVOLUTA (PHV), is sufficient to activate seed maturation genes in vegetative tissues. Lastly, we show that PHB binds the promoter of LEAFY COTYLEDON2 (LEC2), which encodes a master regulator of seed maturation. Therefore, this study establishes a core module composed of a miRNA, its target genes (PHB and PHV), and the direct target of PHB (LEC2) as an underlying mechanism that keeps the seed maturation program off during vegetative development.