Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55GAG) of the virion nucleocapsid proteins

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55GAG) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1–22) and central (aa 70–100) regions of Vif to be essential for its interaction with Pr55GAG, but deletion of the carboxy-terminal (aa 158–192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55GAG showed that a 35-amino-acid region of the protein bridging the MA(p17)–CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp21) near the amino terminus of Vif showed it to be important for the interaction with Pr55GAG. By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif–Pr55GAG interaction

European stone fruit Yellows phytoplasma in Japanese plum and Myrobalan plum in Bosnia and Herzegovina

Stone fruits from commercial as well as abandoned orchards were evaluated for European Stone Fruit Yellows phytoplasma (ESFY) presence during 2004-2007 years. Orchards were monitored in western and southern districts of Bosnia and Herzegovina. In the first survey conducted in period of 2004 till 2005 the causal agent of ESFY was identified on peach (Prunus persica) and apricot (Prunus armeniaca) plants in both surveyed districts. During 2007, a new survey was performed and samples were taken from symptomatic and symptomless plants of European plum (Prunus domestica), Japanese plum (Prunus salicina), Myrobalan plum (Prunus cerasifera) and cherry (Prunus avium). Samples were analyzed using real-time PCR and nested PCR approaches. In this extended survey, the presence of ESFY phytoplasma was additionally identified in Japanese plum and myrobalan plum trees

European stone fruit Yellows phytoplasma in Japanese plum and Myrobalan plum in Bosnia and Herzegovina

Stone fruits from commercial as well as abandoned orchards were evaluated for European Stone Fruit Yellows phytoplasma (ESFY) presence during 2004-2007 years. Orchards were monitored in western and southern districts of Bosnia and Herzegovina. In the first survey conducted in period of 2004 till 2005 the causal agent of ESFY was identified on peach (Prunus persica) and apricot (Prunus armeniaca) plants in both surveyed districts. During 2007, a new survey was performed and samples were taken from symptomatic and symptomless plants of European plum (Prunus domestica), Japanese plum (Prunus salicina), Myrobalan plum (Prunus cerasifera) and cherry (Prunus avium). Samples were analyzed using real-time PCR and nested PCR approaches. In this extended survey, the presence of ESFY phytoplasma was additionally identified in Japanese plum and myrobalan plum trees.Keywords: Bosnia and Herzegovina, myrobalan plum, Japanese plum, phytoplasma, ESFY, PC

Diagnostics of fruit trees phytoplasmas – the importance of latent infections

In the period 2000-2008 more than 1300 fruit trees from different regions of Slovenia were tested for the quarantine phytoplasmas Apple proliferation (AP), Pear decline (PD), and European stone fruit yellows (ESFY). The majority of samples were collected within systematic official surveys, which was conducted for assessing the presence of these phytoplasma in Slovenia in production and mother plant orchards. Samples were taken from trees with and without expressed symptoms. DNA was extracted from the symptomatic shoots. In addition some roots from asymptomatic trees were sampled for the evaluation of latent infections. The presence of phytoplasmas was analyzed with a nested PCR, RFLP and a real time PCR (Hren et al., 2007). AP, PD and ESFY were confirmed as being present in several areas in Slovenia where fruit trees are cultivated. AP was found not only in apple, but also in stone fruit trees such as cherry, apricot and plum (Mehle et al., 2007). By using highly sensitive diagnostic methods, such as real time PCR, some latent infections were detected and they were confirmed next year also by less sensitive methods

Diagnostics of fruit trees phytoplasmas – the importance of latent infections

In the period 2000-2008 more than 1300 fruit trees from different regions of Slovenia were tested for the quarantine phytoplasmas Apple proliferation (AP), Pear decline (PD), and European stone fruit yellows (ESFY). The majority of samples were collected within systematic official surveys, which was conducted for assessing the presence of these phytoplasma in Slovenia in production and mother plant orchards. Samples were taken from trees with and without expressed symptoms. DNA was extracted from the symptomatic shoots. In addition some roots from asymptomatic trees were sampled for the evaluation of latent infections. The presence of phytoplasmas was analyzed with a nested PCR, RFLP and a real time PCR (Hren et al., 2007). AP, PD and ESFY were confirmed as being present in several areas in Slovenia where fruit trees are cultivated. AP was found not only in apple, but also in stone fruit trees such as cherry, apricot and plum (Mehle et al., 2007). By using highly sensitive diagnostic methods, such as real time PCR, some latent infections were detected and they were confirmed next year also by less sensitive methods.Keywords: Phytoplasma, AP, PD, ESFY, latent infection, fruit tree

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive

The RNA Polymerase PB2 Subunit of Influenza A/HongKong/156/1997 (H5N1) Restrict the Replication of Reassortant Ribonucleoprotein Complexes

BACKGROUND: Genetic reassortment plays a critical role in the generation of pandemic strains of influenza virus. The influenza virus RNA polymerase, composed of PB1, PB2 and PA subunits, has been suggested to influence the efficiency of genetic reassortment. However, the role of the RNA polymerase in the genetic reassortment is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reconstituted reassortant ribonucleoprotein (RNP) complexes, and demonstrated that the PB2 subunit of A/HongKong/156/1997 (H5N1) [HK PB2] dramatically reduced the synthesis of mRNA, cRNA and vRNA when introduced into the polymerase of other influenza strains of H1N1 or H3N2. The HK PB2 had no significant effect on the assembly of the polymerase trimeric complex, or on promoter binding activity or replication initiation activity in vitro. However, the HK PB2 was found to remarkably impair the accumulation of RNP. This impaired accumulation and activity of RNP was fully restored when four amino acids at position 108, 508, 524 and 627 of the HK PB2 were mutated. CONCLUSIONS/SIGNIFICANCE: Overall, we suggest that the PB2 subunit of influenza polymerase might play an important role for the replication of reassortant ribonucleoprotein complexes

Identifying Determinants of Cullin Binding Specificity Among the Three Functionally Different Drosophila melanogaster Roc Proteins via Domain Swapping

BACKGROUND: Cullin-dependent E3 ubiquitin ligases (CDL) are key regulators of protein destruction that participate in a wide range of cell biological processes. The Roc subunit of CDL contains an evolutionarily conserved RING domain that binds ubiquitin charged E2 and is essential for ubiquitylation. Drosophila melanogaster contains three highly related Roc proteins: Roc1a and Roc2, which are conserved in vertebrates, and Roc1b, which is specific to Drosophila. Our previous genetic data analyzing Roc1a and Roc1b mutants suggested that Roc proteins are functionally distinct, but the molecular basis for this distinction is not known. METHODOLOGY/PRINCIPAL FINDINGS: Using co-immunoprecipitation studies we show that Drosophila Roc proteins bind specific Cullins: Roc1a binds Cul1-4, Roc1b binds Cul3, and Roc2 binds Cul5. Through domain swapping experiments, we demonstrate that Cullin binding specificity is strongly influenced by the Roc NH(2)-terminal domain, which forms an inter-molecular beta sheet with the Cullin. Substitution of the Roc1a RING domain with that of Roc1b results in a protein with similar Cullin binding properties to Roc1a that is active as an E3 ligase but cannot complement Roc1a mutant lethality, indicating that the identity of the RING domain can be an important determinant of CDL function. In contrast, the converse chimeric protein with a substitution of the Roc1b RING domain with that of Roc1a can rescue the male sterility of Roc1b mutants, but only when expressed from the endogenous Roc1b promoter. We also identified mutations of Roc2 and Cul5 and show that they cause no overt developmental phenotype, consistent with our finding that Roc2 and Cul5 proteins are exclusive binding partners, which others have observed in human cells as well. CONCLUSIONS: The Drosophila Roc proteins are highly similar, but have diverged during evolution to bind a distinct set of Cullins and to utilize RING domains that have overlapping, but not identical, function in vivo

Species difference in ANP32A underlies influenza A virus polymerase host restriction.

Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals