Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.     

Mapping the inter-RNA interaction of bacterial virus phi29 packaging RNA by site-specific photoaffinity cross-linking

During replication, the lengthy genome of double-stranded DNA viruses is translocated with remarkable velocity into a limited space within the procapsid. The question of how this fascinating task is accomplished has long been a puzzle. Our recent investigation suggests that phi29 DNA packaging is accomplished by a mechanism similar to the driving of a bolt with a hex nut and that six packaging RNAs (pRNAs) form a hexagonal complex to gear the DNA-translocating machine (Chen, C., and Guo, P. (1997) J. Virol. 71, 3864-3871; Zhang, F., Lemieux, S., Wu, X., St.-Arnaud, S., McMurray, C. T., Major, F., and Anderson, D. (1998) Mol. Cell 2, 141-147; Guo, P., Zhang, C., Chen, C., Garver, K., and Trottier, M., (1998) Mol. Cell 2, 149-155). In the current study, circularly permuted pRNAs were used to position an azidophenacyl photoreactive cross-linking agent specifically at a strategic site that was predicted to be involved in pRNA-pRNA interaction. Cross-linked pRNA dimers were isolated, and the sites of cross-link were mapped by primer extension. The cross-linked pRNA dimer retained full activity in phi29 procapsid binding and genomic DNA translocation, indicating that the cross-link distance constraints identified in dimer formation reflect the native pRNA complex. Both cross-linked dimers either containing or not containing the interlocking loops for programmed hexamer formation bound procapsid equally well; however, only the one containing the interlocking loops programmed for hexamer formation was active in phi29 DNA packaging. The cross-linked pRNA dimers were also identified as the minimum binding unit necessary for procapsid binding. Primer extension of the purified cross-linked pRNA dimers revealed that base G(82) was cross-linked to bases G(39), G(40), A(41), C(49), G(62), C(63), and C(64), which contribute to the formation of the three-way junction, suggesting that these bases are proximate in the formation of pRNA tertiary structure. Interestingly, the photoaffinity agent in the left interacting loop did not cross-link directly to the right loop as expected but cross-linked to bases adjacent to the right loop. These data provide a background for future modeling of pRNA tertiary structure.     

噬菌体phi29 pRNA载体在RNA干扰中的初步应用研究

RNA干扰是在细胞胞质中双链RNA(dsR-NA)介导的序列特异性mRNA的降解[1]。这个过程是由21~25个被称为小干扰RNA(si RNA)形成的dsRNA完成[2]。目前,这一技术已经广泛应用于研究基因的功能,病毒感染治疗等方面。但是,si RNA在体内容易降解,干扰作用持续的时间不长。新的研究表明枯     

Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29 DNA-protein p3 replication.

The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off.     

Evidence that the neck appendages are adsorption organelles in Bacillus subtilis bacteriophage phi29.

A mutant of Bacillus subtilis unable to adsorb phage phi29 efficiently has been isolated. This mutant can be infected by host range mutants of the phage. Since the host range mutations map in cistron 12, which codes for neck appendage protein, this would tend to confirm that these organelles are involved in viral adsorption.     

Sex and the evolution of intrahost competition in RNA virus phi6.

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model''s predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of phi6, and suggest that genetic conflicts can evolve in RNA viruses.     

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.     

An additional dimer linkage structure in Moloney murine leukemia virus RNA.

We have identified an additional dimerization linkage structure in the genome of Moloney murine leukemia virus (MoMLV). Retroviral genomes have long been known to be linked at their 5' ends to form dimers. In MoMLV, a hairpin loop functioning as a dimer linkage structure (DLS) has previously been identified at nucleotides 278-303. Here, we describe RNA dimers formed from sections of the MoMLV 5' untranslated region that do not contain the previously described MoMLV DLS. These dimers exhibit the distinctive characteristics previously described for whole genome dimers. We have mapped this novel region to nucleotides 199-243. This sequence contains a stem-loop structure (nucleotides 204-227) much like the 278-303 region. We describe the chemical and thermal stability of dimers containing the 204-227 stem-loop as well as kinetics and salt-dependence of dimer formation. Our results show that dimerization of MoMLV RNA can be nucleated at multiple sites and suggest that the 5' untranslated region may contain separately folding and dimerizing domains.     

A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree of amino acid sequence similarity. Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis. Although structural analyses indicate that chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd for receptor binding, active dimer formation may require high local concentrations of MCP-1. Our data further suggest that the dimer interface can be a target for MCP-1 inhibitory drugs.     

Human immunodeficiency virus type 1 RNA outside the primary encapsidation and dimer linkage region affects RNA dimer stability in vivo.

To characterize the cis-acting determinants that function in RNA dimer formation and maintenance, we examined the stability of RNA dimers isolated from virus particles containing mutations in the encapsidation region of human immunodeficiency virus type 1 (HIV-1). The genomic RNAs of all mutants containing lesions in elements required for in vitro dimerization exhibited thermal stability similar to that of wild-type (WT) HIV-1. These data indicate that the eventual formation of stable dimeric RNA in vivo is not absolutely dependent on the elements that promote dimer formation in vitro. Surprisingly, mutants that lacked a large segment of the middle portion of the genome, outside the likely primary dimer linkage region, formed RNA dimers that were measurably more stable than WT. In addition, the insertion of one or multiple copies of a foreign gene, which resulted in a series of vectors that approached RNA length similar to that of WT RNA, still exhibited augmented dimer stability. These results suggest that there are regions in the HIV-1 genome outside the primary dimer initiation and dimer linkage regions that can negatively affect dimer stability.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

A splice hepadnavirus RNA that is essential for virus replication.

According to the current model of hepadnavirus gene expression, the viral envelope proteins are produced from unspliced subgenomic RNAs, in contrast to the retroviral mechanism, where the subgenomic env RNA is generated by RNA splicing. We now describe and characterize a novel duck hepatitis B virus RNA species which is derived from the RNA pregenome by loss of a 1.15 kb intron. This RNA (termed spliced L RNA) codes for the large surface protein (L protein), as does the previously described unspliced mRNA (the preS RNA); however, it differs in 5' leader sequence and promoter control. Mutational analysis indicates that the spliced L RNA is functionally important for virus replication in infected hepatocytes and ducks, but not for virus formation from transfected DNA genomes. This suggests that the newly discovered second pathway for L protein synthesis plays a distinct role in an early step in the viral life cycle.     

The procapsid binding domain of phi29 packaging RNA has a modular architecture and requires 2'-hydroxyl groups in packaging RNA interaction

The phi29 packaging RNA (pRNA) is an essential component in the phi29 bacteriophage DNA packaging motor, the strongest biomolecular motor known today. Utilizing Mg2+-dependent intermolecular base pairing interactions between two 4-nucleotide loops within the pRNA procapsid binding domain, multiple copies of pRNA form a ring-shaped complex that is indispensable for packaging motor function. To understand pRNA structural organization and pRNA/pRNA interaction, studies were carried out on pRNA closed dimers, the simplest functional pRNA complex believed to be the building blocks for assembling the oligomeric ring. Tertiary folding and interactions in various pRNA mutants were evaluated based on measured closed dimer affinity that is directly linked to the proper positioning of the interacting loops. The data revealed that the procapsid binding domain contains two autonomous modules that are capable of interacting noncovalently to form a fully active species in pRNA/pRNA interaction. Deleting the 2'-hydroxyl groups in one of the interacting loops weakens the dimer affinity by 125-fold, suggesting potential tertiary interactions involving these 2'-hydroxyl groups. The results provide evidence that nonbase functional groups are involved in pRNA folding and interaction and lead to a simple model that describes the pRNA monomer configuration in terms of three arms spanning a hinge. The functional constructs developed here will aid biophysical and biochemical investigations of pRNA structure and function, as well as developments of pRNA-based technology for nanoscience and gene therapy.     

S-Layers as a basic building block in a molecular construction kit

Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.     

A defective interfering RNA that contains a mosaic of a plant virus genome

A symptom-modulating RNA associated with tomato bushy stunt virus (TBSV) was investigated with respect to physical and biological properties. Linear RNA of approximately 396 nucleotides was packaged in viral coat protein and was dependent on TBSV for replication. Coinoculation of the small RNA with TBSV resulted in the attenuation of TBSV-induced symptoms and depression of virus synthesis in whole plants. Nucleotide sequence analysis revealed that the symptom-modulating RNA was derived from 5', 3', and internal segments of the TBSV genome. The identification of this symptom-modulating RNA as a co-linear deletion mutant of the helper virus genome establishes it as the first definitive defective interfering RNA (DI RNA) to be identified in association with a plant virus.