A MicroRNA Encoded by Autographa californica Nucleopolyhedrovirus Regulates Expression of Viral Gene ODV-E25

Baculovirus-encoded microRNAs (miRNAs) have been described in Bombyx mori nucleopolyhedrovirus; however, most of their functions remain unclear. Here we report the identification and characterization of an miRNA encoded by Autographa californica nucleopolyhedrovirus. The identified miRNA, AcMNPV-miR-1, perfectly matched a segment in the coding sequence of the viral gene ODV-E25 and downregulated ODV-E25 mRNA expression, which likely resulted in a reduction of infectious budded virions and accelerated the formation of occlusion-derived virions.     

杆状病毒ac68基因的克隆、表达与抗体制备

目的：对苜蓿丫纹夜蛾核多角体病毒（Autographa califorica multicapsid nucleopoly hedrovirus,AcMNPV）开放阅读框68（open reading frame,ORF68,ac68）进行原核表达,制备该蛋白的多克隆抗体,为深入研究其功能提供基础。方法：将ac68基因克隆至原核表达载体pET28a上,在大肠杆菌BL21（DE3）中表达Ac68蛋白,通过His抗体检测进一步验证所表达的蛋白为带有组氨酸的融合蛋白。以纯化的Ac68蛋白作为抗原,免疫昆明小鼠制备多克隆抗体。结果：实现了ac68基因的原核表达,获得了该蛋白的多克隆抗体并在AcMNPV感染的Sf-9细胞中检测到一条大小为25kD左右的特异杂交带。结论：获得的抗体可用于Ac68蛋白功能的进一步研究。     

杆状病毒AcMNPV p35基因的克隆表达及多抗制备

用PCR方法扩增得到苜蓿丫纹夜蛾核多角体病毒(Autographa californic anucleopolyhedrovirus,AcM N-PV)p35基因,将其克隆至质粒pET-32a( )上,构建得到重组质粒pET-p35,转化大肠杆菌BL21(DE3),经IPTG诱导,表达了1条约为55 ku的蛋白带。以Ni2 -NTA偶连抗体检测证明所表达的蛋白为带有组氨酸的融合蛋白。采用割胶回收的方法纯化融合蛋白,以纯化的融合蛋白制备多克隆抗体,效价为1/1 024。免疫印迹分析表明,该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。     

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.     

Functional Analysis of the Autographa californica Multiple Nucleopolyhedrovirus GP64 Terminal Fusion Loops and Interactions with Membranes

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) glycoprotein GP64 is the major envelope protein of the budded virus (BV). GP64 is a class III fusion protein that mediates BV attachment to the cell surface and low-pH-triggered membrane fusion between the BV envelope and the endosome membrane during entry. Class III fusion proteins contain terminal looped structures that are believed to interact with membranes. To examine the functions of 3 loops found at the apex of the GP64 postfusion structure, we generated 2-alanine substitutions that scanned the two so-called fusion loops (loop 1 and loop 2) plus an adjacent loop structure (loop 3) that is closely attached to loop 2 and is also found at the apex of the GP64 postfusion structure. We identified essential residues from Y75 to T86 (loop 1) and N149 to H156 (loop 2) that are required for fusion activity, but no essential residues in loop 3. Further analysis revealed that critical fusion loop residues fall within two groups that are associated with either membrane merger (hemifusion) or fusion pore expansion. We next examined the interactions of soluble GP64 proteins and BV with membranes composed of various phospholipids. BV interacted directly with small unilamellar vesicles (SUVs) comprised of phospholipids phosphatidylcholine and phosphatidic acid (PC/PA) or phosphatidylcholine and phosphatidylserine (PC/PS) under neutral and acidic pH. We also examined the interactions of soluble GP64 constructs containing substitutions of the most hydrophobic residues within each of the two fusion loops. We found that a 2-residue substitution in either single loop (loop 1 [positions 81 and 82] or loop 2 [positions 153 and 154]) was not sufficient to substantially reduce the GP64-liposome interaction, but the same substitutions in both fusion loops severely reduced the GP64-liposome association at neutral pH. These results suggest that critical hydrophobic residues in both fusion loops may be involved in the interaction of GP64 with host cellular membranes and direct GP64-membrane interactions may represent a receptor-binding step prior to a low-pH-triggered conformational change.     

杆状病毒AcMNPV vp39基因的克隆、表达与抗体制备

目的:构建苜蓿丫纹夜蛾核多角体病毒(Autographa californica nucleopolyhedro virus,AcMNPV)VP39的原核表达载体,表达、纯化蛋白并制备多克隆抗体。方法:用PCR方法扩增vp39基因,并将其克隆至pET-21a( )上,转化到大肠杆菌BL21(DE3)中进行诱导表达,采用割胶回收的方法纯化融合蛋白,纯化的融合蛋白作为抗原,免疫新西兰大白兔,Western blot检测抗体活性。结果:构建了pET-VP39原核表达质粒,含有该质粒的大肠杆菌经IPTG诱导超量表达了一个与预期理论值相符的约为40kDa的融合蛋白。对制备的抗体进行免疫印迹分析表明该抗血清能与感染苜蓿丫纹夜蛾核多角体病毒的细胞蛋白样品发生特异性反应。结论:获得了兔抗AcMNPV-VP39多克隆抗体,为进一步深入研究VP39在病毒侵染过程中与宿主因子的相互作用提供了检测工具。     

Mutations in the M4 Domain of the Torpedo californica Nicotinic Acetylcholine Receptor Alter Channel Opening and Closing

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time. Received: 27 September 1996/Revised: 28 January 1997     

Mutations That Alter the Timing and Pattern of Cubitus Interruptus Gene Expression in Drosophila Melanogaster

The cubitus interruptus (ci) gene is a member of the Drosophila segment polarity gene family and encodes a protein with a zinc finger domain homologous to the vertebrate Gli genes and the nematode tra-1 gene. Three classes of existing mutations in the ci locus alter the regulation of ci expression and can be used to examine ci function during development. The first class of ci mutations causes interruptions in wing veins four and five due to inappropriate expression of the ci product in the posterior compartment of imaginal discs. The second class of mutations eliminates ci protein early in embryogenesis and causes the deletion of structures that are derived from the region including and adjacent to the engrailed expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks the formation of the ventral naked cuticle. The loss of ci expression at these two different stages in embryonic development correlates with the subsequent elimination of wingless expression. Adults heterozygous for the unique ci(Ce) mutation have deletions between wing veins three and four. A similar wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative serine/threonine kinase. In ci(Ce)/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both the ci mRNA and protein appears to be critical for normal Drosophila development.     

Segregation of Induced Frameshift Mutations and the Sequence of Gene Replication in ESCHERICHIA COLI K12

We have constructed a strain of E. coli K12 carrying six mutations induced by the acridine half-mustard ICR-191. The mutations are widely spaced on the E. coli linkage map and are all easily reverted by ICR-191. Mapping of ten independent revertants for each of five markers indicated that the reversions induced by ICR-191 occurred near the original mutations. Exponentially and nonsynchronously growing cultures of this strain were exposed to ICR-191 for 0.85 generation, quickly washed free of mutagen, and resuspended in the original medium minus mutagen. Total viable cell number maintained its exponential increase both during and immediately after exposure to mutagen, whereas the number of revertants of any particular type remained constant for a characteristic period after removal of mutagen before finally assuming an exponential increase. Theoretically, the length of such a segregation lag should depend on the position of the particular reverted gene in the sequence of gene replication: the earlier a gene is replicated in the chromosome replication cycle, the longer its segregation lag should be. Our results are consistent with this prediction and fit a unidirectional, clockwise replication scheme with an origin between 55 and 74 min on the E. coli linkage map. The results also fit a very asymmetric bidirectional replication scheme.     

Phenotypic and genetic analysis of Lymantria dispar nucleopolyhedrovirus few polyhedra mutants: mutations in the 25K FP gene may be caused by DNA replication errors.

We previously demonstrated that polyhedron formation (PF) mutants arise at a high frequency during serial passage of the Lymantria dispar nucleopolyhedrovirus (LdMNPV) in the L. dispar 652Y cell line (J. M. Slavicek, N. Hayes-Plazolles, and M. E. Kelly, Biol. Control 5:251-261, 1995). Most of these PF mutants exhibited the traits of few polyhedra (FP) mutants; however, no large DNA insertions or deletions that correlated with the appearance of the FP phenotype were found. In this study, we have characterized several of the PF mutants at the phenotypic and genetic levels. Genetic techniques were used to group the mutations in the LdMNPV PF mutants to the same or closely linked genes. Wild-type viruses were recovered after coinfection of L. dispar 652Y cells with certain combinations of PF mutants. These viruses were analyzed by restriction endonuclease analysis and found to be chimeras of the original PF mutants used in the coinfections. Marker rescue experiments localized the mutations in one group of PF isolates to the region containing the LdMNPV 25K FP gene. The mutations in these PF mutants were identified. Four of five of the LdMNPV FP mutants contain small insertions or deletions within the 25K FP gene. The fifth LdMNPV FP mutant analyzed contained a large deletion that truncated the C terminus of the 25K FP gene product. All of the deletions occurred within the same potential hairpin loop structure, which had the lowest free energy value (most stable hairpin) of the five potential hairpin loop structures present in the 25K FP gene. One of the insertion mutants contained an extra base within a repetitive sequence. These types of mutations are likely caused by errors that occur during DNA replication. The relationship between the types of mutations found within the LdMNPV 25K FP gene and DNA replication-based mutagenesis is discussed.     

Mutations within the mepA Operator Affect Binding of the MepR Regulatory Protein and Its Induction by MepA Substrates in Staphylococcus aureus

The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates.     

棉铃虫病毒HaSNPV fp25k基因的克隆表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)fp25k基因的序列,设计引物,引入适当的酶切位点,利用PCR扩增出基因片段.将该基因片段克隆至原核表达镲载体pProEXHTb,经IPTG诱导,在大肠杆菌DH5α中获得了高效表达,表达产物的大小为32kDa.纯化蛋白产物免疫家兔制备抗血清.该抗血清可与原核表达的GST-FP25K融合蛋白及在感染的昆虫细胞中表达的FP25K蛋白发生特异性免疫反应.该抗体的获得为深入研究FP25K蛋白的功能提供了基础.