Sequential packaging of RNA genomic segments during the assembly of Bluetongue virus

Bluetongue virus (BTV), a member of the Orbivirus genus within the Reoviridae family, has a genome of 10 double-stranded RNA segments, with three distinct size classes. Although the packaging of the viral genome is evidently highly specific such that every virus particle contains a set of 10 RNA segments, the order and mechanism of packaging are not understood. In this study we have combined the use of a cell-free in vitro assembly system with a novel RNA–RNA interaction assay to investigate the mechanism of single-stranded (ss) RNAs packaging during nascent capsid assembly. Exclusion of single or multiple ssRNA segments in the packaging reaction or their addition in different order significantly altered the outcome and suggested a particular role for the smallest segment, S10. Our data suggests that genome packaging probably initiates with the smallest segment which triggers RNA–RNA interaction with other smaller segments forming a complex network. Subsequently, the medium to larger size ssRNAs are recruited until the complete genome is packaging into the capsid. The untranslated regions of the smallest RNA segment, S10, is critical for the instigation of this process. We suggest that the selective packaging observed in BTV may also apply to other members of the Reoviridae family.     

Host factor Rab11a is critical for efficient assembly of influenza A virus genomic segments

It is well documented that influenza A viruses selectively package 8 distinct viral ribonucleoprotein complexes (vRNPs) into each virion; however, the role of host factors in genome assembly is not completely understood. To evaluate the significance of cellular factors in genome assembly, we generated a reporter virus carrying a tetracysteine tag in the NP gene (NP-Tc virus) and assessed the dynamics of vRNP localization with cellular components by fluorescence microscopy. At early time points, vRNP complexes were preferentially exported to the MTOC; subsequently, vRNPs associated on vesicles positive for cellular factor Rab11a and formed distinct vRNP bundles that trafficked to the plasma membrane on microtubule networks. In Rab11a deficient cells, however, vRNP bundles were smaller in the cytoplasm with less co-localization between different vRNP segments. Furthermore, Rab11a deficiency increased the production of non-infectious particles with higher RNA copy number to PFU ratios, indicative of defects in specific genome assembly. These results indicate that Rab11a+ vesicles serve as hubs for the congregation of vRNP complexes and enable specific genome assembly through vRNP:vRNP interactions, revealing the importance of Rab11a as a critical host factor for influenza A virus genome assembly.     

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.     

Mutational analyses of packaging signals in influenza virus PA, PB1, and PB2 genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3′ and 5′ termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5′ sites in the PA or PB1 coding regions but extended across the entire (80-base) 5′ coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.     

Analysis of ribonucleoproteins of influenza virus containing genomic and complementary RNA

The density and sedimentation characteristics of ribonucleoproteins (RNP) containing genomic RNA from influenza virus and RNA complementary have been studied. Radioactive RNA from infected cells has been used for analysis. RNA classes of interest were isolated by reannealing with abundant nonradioactive genomic and complementary RNA and separation of resulting duplexes in electrophoresis. The RNP containing antigenomic virus-specific RNA are practically identical to "genomic" RNP for their sedimentation and density characteristics. The "plus" RNP is characterized by the stoichiometric mode of RNA protein interaction.     

Features of mutated changes of genomic RNA of cold-adapted and hr-variants of influenza group A virus, detected by RNA:RNA hybridization]

The presence of mutations in the majority of the genes of cold-adapted strains A/Leningrad/134/17/57 (H2N2), A/Leningrad/134/47/57 (H2N2) and A/PR/8/59/1 (H1N1) of influenza A virus has been demonstrated by the RNA-RNA hybridization with the subsequent electrophoresis of double-stranded RNA in 7.5% polyacrylamide gel. The strains were cultivated 17, 47 and 59 passages in the chicken embryos at 25 degrees C. In the genomes of variants passaged in chicken embryos at optimal temperature of incubation 36 degrees C (hr-variants) the used technique permits identification of a single mutant gene. The obtained data suppose the attenuation of cold-adapted vaccine strains of influenza A virus and their high genetic stability to be a result of selection of the variants obtaining multiple mutations in the genome during passaging of the virions at cold temperature. The attenuation of hr-variants is defined by 1-2 mutations (first of all in HA-gene) that makes understandable their inability to serve as donors for recombinant live influenza vaccines construction.     

流感病毒基因组进化研究进展

流感病毒先后造成了1918、1957、1968和2009年等多次全球性大流感,对人类的生命健康和社会生活形成了巨大的威胁。流感病毒的基因组进化研究为揭示病毒致病机理、疫情监测、准备疫苗和研发抗病毒药物提供了巨大的帮助。文章以流感病毒基因组进化机制为核心,结合与基因组进化密切相关的抗原性和抗药性等表型进化,对流感病毒基因组进化研究的相关进展予以介绍。     

Apo alpha-lactalbumin and lysozyme are colocalized in their subsequently formed spherical supramolecular assembly

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.     

Simplified polyacrylamide gel electrophoresis and silver staining for analysis and preparation of influenza virus RNA segments.

The influenza virus has a genome consisting of eight RNA segments. A simplified technique to study the RNA segmental pattern by silver staining after gel electrophoresis has been developed. In addition, individual RNA segments could be isolated by a combination of polyacrylamide gel electrophoresis and isotachophoresis.     

Role of sindbis virus capsid protein region II in nucleocapsid core assembly and encapsidation of genomic RNA

Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.     

Analysis of the reassorted influenza virus clone genome: data for use of ordered selection of RNA segments

Chicken embryonated eggs were coinfected with influenza A/FPV/Rostock and A/FPV/Weybridge strains. 25 plaque isolates were obtained from the mixed yield and their genetic content was analysed by polyacrylamide gel electrophoresis of H3-uridine-labelled vRNA in a modified gel system. At least 18 clones out of 25 plaque isolates proved to be reassortants; however, only one among them contained the homologous RNA-segments belonging to both parents. The results are in agreement with the concept of an ordered recruitment of vRNA-segments into virions.     

The genomic rate of molecular adaptation of the human influenza A virus

Quantifying adaptive evolution at the genomic scale is an essential yet challenging aspect of evolutionary biology. Here, we develop a method that extends and generalizes previous approaches to estimate the rate of genomic adaptation in rapidly evolving populations and apply it to a large data set of complete human influenza A virus genome sequences. In accord with previous studies, we observe particularly high rates of adaptive evolution in domain 1 of the viral hemagglutinin (HA1). However, our novel approach also reveals previously unseen adaptation in other viral genes. Notably, we find that the rate of adaptation (per codon per year) is higher in surface residues of the viral neuraminidase than in HA1, indicating strong antibody-mediated selection on the former. We also observed high rates of adaptive evolution in several nonstructural proteins, which may relate to viral evasion of T-cell and innate immune responses. Furthermore, our analysis provides strong quantitative support for the hypothesis that human H1N1 influenza experiences weaker antigenic selection than H3N2. As well as shedding new light on the dynamics and determinants of positive Darwinian selection in influenza viruses, the approach introduced here is applicable to other pathogens for which densely sampled genome sequences are available, and hence is ideally suited to the interpretation of next-generation genome sequencing data.