Mutations that confer resistance to mycophenolic acid and ribavirin on Sindbis virus map to the nonstructural protein nsP1.

SVMPA, a mutant of Sindbis virus derived by serial passage on Aedes albopictus mosquito cells maintained after infection in the presence of mycophenolic acid (MPA), is resistant not only to MPA but also to ribavirin. Both of these compounds inhibit the synthesis of GMP and thereby reduce the level of GTP. We had suggested earlier that SVMPA had become resistant to MPA because it coded for an altered RNA guanylyltransferase enzyme with an increased affinity for GTP, enabling it to replicate in cells with reduced levels of GTP. We now report that the MPA-resistant phenotype of SVMPA has been mapped to the coding region for the nonstructural viral protein, nsP1. By replacing the nucleotide sequence between 88 and 1404 of the infectious clone of Sindbis virus (i.e., the Toto 1101 plasmid) with the corresponding sequence from SVMPA cDNA, we were able to generate recombinant Sindbis virus expressing the drug-resistant phenoptype. SVMPA has three base substitutions in the region between nucleotides 88 and 1404 which lead to predicted amino acid changes in the Sindbis virus nsP1 protein: the replacement of Gln at residue 21 by Lys, Ser at residue 23 by Asn, and Val at residue 302 by Met. These results, taken together with previous data from our laboratory associating the RNA methyltransferase with nsP1, (1) are consistent with the idea that an alteration of the RNA guanylyltransferase is responsible for the MPA-resistant phenotype and (2) support the idea that an important function of nsP1 relates to the modification of the 5' terminus of the Sindbis virus mRNAs.     

Cleavage between nsP1 and nsP2 initiates the processing pathway of Sindbis virus nonstructural polyprotein P123

The cleavage between nsP1 and nsP2 and that between nsP2 and nsP3 in the Sindbis virus nonstructural polyproteins was studied with respect to order of processing and enzyme-substrate relationships, using site-specific mutants in which the cleavage sites had been altered. The penultimate Gly in nsP1 or nsP2 or both was substituted by Ala, Val, or Glu, and processing was studied in vitro. Substitution with Ala resulted in partial cleavage whereas substitution with Val or Glu totally abolished cleavage at the mutagenized site. Abolishment of cleavage at the nsP2/nsP3 site did not affect processing at the nsP1/nsP2 site in the precursor polyprotein P123, and nsP1 and P23 were produced. When cleavage at the nsP1/nsP2 site was abolished, however, processing at the nsP2/nsP3 site was also prevented and P123 accumulated. To investigate why cleavage at the nsP1/nsP2 site should be required for cleavage at the nsP2/nsP3 site, the mutagenized polypeptides were used as enzymes in trans-cleavage experiments. We found that P123 can cleave the nsP1/nsP2 site but not the nsP2/nsP3 site, whereas P23 can cleave the nsP2/nsP3 site very efficiently. Thus, cleavage at the nsP1/nsP2 site by P123 is required to produce an enzyme capable of cleaving the nsP2/nsP3 site. Release of nsP4 from P1234 appears to be independent of the other cleavages and occurs primarily immediately after translation. These mutations were also transferred into a full-length cDNA clone of Sindbis virus and virus was recovered. Mutants defective in the cleavage of the nsP2/nsP3 site were temperature sensitive, growing at a slightly reduced rate compared to wild-type virus at 30 degrees but growing poorly at 40 degrees. Mutants defective in the cleavage of both the nsP1/nsP2 site and the nsP2/nsP3 site were viable but grew poorly compared with wild-type at any temperature.     

Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli.

We have constructed two plasmids, pSR5-42 and pSR5-Toto, which under lac control expressed the SVLM21 and the SVToto forms, respectively, of the Sindbis virus nonstructural protein, nsP1. The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60,000 and an amino terminal sequence in agreement with that expected for nsP1. Following induction with IPTG, cells carrying pSR5-42 (which contains the SVLM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5-Toto (which contains the SVToto gene sequence). This result is in agreement with what is observed when methyltransferase is measured in cells infected with SVLM21 and SVSTD (or SVToto), respectively. These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.     

The first 28 amino acids of mature LamB are required for rapid and efficient export from the cytoplasm

Our laboratory has been utilizing the Escherichia coli outer membrane protein LamB to study the mechanism of protein localization. Various lines of evidence suggest that, in addition to a signal sequence, regions within the mature protein are required for efficient localization. In particular, studies using LamB-LacZ hybrid proteins have identified regions between amino acids 27 and 49 of mature LamB, which may play an important role in localization. To elucidate further the function of these regions, a series of in-frame deletions that remove varying lengths of early lamB sequences was constructed. The effects of these deletions on export of a large LamB-LacZ hybrid protein, 42-1, and on export of an otherwise wild-type LamB protein were determined. We find a strong correlation between the sequences deleted and the export phenotypes these deletions impart to both LamB and the LamB-LacZ42-1 hybrid protein. On the basis of these findings, the deletions can be divided into several distinct classes that define a region within mature LamB that participates in localization. This region extends amino terminally from amino acid 28 of the mature protein and functions in the rapid and efficient localization of LamB from the cytoplasm.     

Site-specific single amino acid changes to Lys or Arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost

A hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser(118), Glu(132), Glu(138), Gln(178), and Ser(180)). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants.     

Low pH-dependent Sindbis virus-induced fusion of BHK cells: differences between strains correlate with amino acid changes in the E1 glycoprotein

Expression of alphavirus glycoproteins on the surface of infected cells leads to cell fusion after exposure to acidic pH. Two strains of Sindbis virus, AR339 (SV) and neuroadapted Sindbis virus (NSV), which differ in virulence for weanling mice, were found to differ in pH-dependent fusion. BHK-21 cells infected with SV fused maximally after shifting to pH 5.4, whereas cells infected with NSV required a lower pH, pH 4.8, for maximal fusion. No difference was noted in the optimal pH for agglutination of goose erythrocytes (5.75 for both viruses). To determine the molecular basis for the difference in fusion a series of recombinant viruses was constructed using a cDNA clone of Sindbis virus from which infectious RNA can be transcribed in vitro. Cells infected with a recombinant virus that had the SV E1 and NSV E2 genes had a fusion response curve as a function of pH like SV, while cells infected with recombinant virus with the NSV E1 and SV E2 genes fused like NSV. The E1 glycoproteins of SV and NSV differ at two positions: Val-72 in SV is Ala in NSV (a change near the putative fusion site), and Gly-313 in SV is Asp in NSV. Recombinant viruses which had Val-72 (SV) and Asp-313 (NSV) or Ala-72 (NSV) and Gly-313 (SV) had a lowered pH of fusion like NSV suggesting that both positions participate in determining some aspect of the conformational change in the E1-E2 heterodimer associated with pH-dependent fusion.     

表皮葡萄球菌生物被膜形成相关icaC敲除突变株的构建及其表型的改变

目的构建表皮葡萄球菌脚阴性突变株，以期获得仅icaC基因不同而其他遗传背景与表皮葡萄球菌1457株相同的突变株，对脚基因产物在细菌形成生物被膜中的作用进行研究。方法构建同源重组质粒pBT2-△icaC，经金黄色葡萄球菌RN4220株修饰后电转化入表皮葡萄球菌1457株。将含重组质粒的表皮葡萄球菌1457株在40℃多次传代，筛选出icaC敲除突变株（△icaC株）。检测△icaC以株生长曲线，采用微量板半定量法检测细菌生物被膜的形成能力，并采用斑点免疫印迹法检测细菌多糖黏附因子的合成。结果使用同源重组法敲除表皮葡萄球菌1457株基因组中的脚基因，△icaC株的生长曲线与表皮葡萄球菌1457株相似，但生物被膜形成能力及细胞外多糖黏附因子含量显著降低。结论表皮葡萄球菌1457株中脚基因的缺失对细菌生长无明显影响，但显著降低细胞外多糖黏附因子含量及生物被膜形成能力，为进一步研究表皮葡萄球菌脚基因在生物被膜形成中的作用奠定基础。     

Both phthiocerol dimycocerosates and phenolic glycolipids are required for virulence of Mycobacterium marinum

Phthiocerol dimycocerosates (PDIMs) and structurally related phenolic glycolipids (PGLs) are complex cell wall lipids unique to pathogenic mycobacteria. While these lipids have been extensively studied in recent years, there are conflicting reports on some aspects of their biosynthesis and on the role of PDIMs and especially PGLs in virulence of Mycobacterium tuberculosis. This has been complicated by the natural deficiency of PGLs in many clinical strains of M. tuberculosis and the frequent loss of PDIMs in laboratory M. tuberculosis strains. In this study, we isolated seven mutants of Mycobacterium marinum deficient in PDIMs and/or PGLs in which multiple genes of the PDIM/PGL biosynthetic locus were disrupted by transposon insertion. Zebrafish infection experiments showed that M. marinum strains lacking one or both of these lipids were avirulent, suggesting that both PDIMs and PGLs are required for virulence. We also found that these strains were hypersensitive to antibiotics and exhibited increased cell wall permeability. Our studies provide new insights into the biosynthesis of PDIMs/PGLs and may help us to understand the role of PDIMs and PGLs in M. tuberculosis virulence.     

Both VH and VL chains of polyreactive IgM antibody are required for polyreactivity: expression of Fab in Escherichia coli.

Monoclonal polyreactive antibodies can bind to many structurally dissimilar self and non-self antigens. Neither the precise antigen-binding site on the polyreactive antibody molecule nor the molecular basis of polyreactivity has been elucidated. The present study was initiated to see whether antibody genes encoding the Fab fragment of a human monoclonal polyreactive IgM antibody (MoAb 67) could be efficiently expressed in Escherichia coli, and whether the bacterially expressed Fab fragments possessed biological activity. cDNA encoding the variable domains of the heavy and light chains of MoAb 67 were cloned, amplified by polymerase chain reaction (PCR) and expressed in E. coli. Neither the recombinant heavy nor light chain showed antigen-binding activity. In contrast, the recombinant Fab 67 fragment showed the same antigen-binding reactivity profile as the native IgM antibody. It is concluded that the antigen-binding activity of polyreactive antibodies resides in the Fab fragment, and that both the heavy and light chains are required for activity.     

Cellular inhibitors of apoptosis proteins cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome

Pathogen and danger recognition by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. The cellular inhibitor of apoptosis proteins (cIAPs) function in apoptosis and innate immunity, but their role in modulating the inflammasome and the inflammatory caspases is unknown. Here we report that the cIAPs are critical effectors of the inflammasome and are required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1. Deficiency in cIAP1 (encoded by Birc2) or cIAP2 (Birc3) impaired caspase-1 activation after spontaneous or agonist-induced inflammasome assembly, and Birc2(-/-) or Birc3(-/-) mice or mice administered with an IAP antagonist had a dampened response to inflammasome agonists and were resistant to peritonitis. Our results describe a role for the cIAPs in innate immunity and further demonstrate the evolutionary conservation between cell death and inflammation mechanisms.     

Suppressor mutations that allow sindbis virus RNA polymerase to function with nonaromatic amino acids at the N-terminus: evidence for interaction between nsP1 and nsP4 in minus-strand RNA synthesis

The alphavirus RNA polymerase, nsP4, invariably has a Tyr residue at the N-terminus. Previously we reported that the N-terminal Tyr residue of nsP4 of Sindbis virus, the type species of the genus Alphavirus, can be substituted with Phe, Trp, or His without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for Met, were lethal or quasilethal. Here we report the identification of two suppressor mutations in nsP4 (Glu-191 to Leu and Glu-315 to Gly, Val, or Lys) and one in nsP1 (Thr-349 to Lys) that allow nsP4 with nonaromatic amino acids at the N-terminus to function at 30 degrees C. The suppressor mutation at nsP4 Glu-315 occurred most frequently. All three suppressor mutations suppressed the effects of Ala, Arg, or Leu at the N-terminus of nsP4 with almost equal efficiency and thus the effect of the suppressing mutation is independent of the nsP4 N-terminal residue. Reconstructed mutants containing nsP1-T349K or nsP4-E315G combined with Ala-nsP4 had a defect in minus-strand RNA synthesis at 40 degrees C. A double mutant containing nsP4-Q191L combined with Ala-nsP4 was unstable and could not be tested for RNA synthesis because it reverted to temperature-independence too rapidly. Combinations of nsP1-T349K or nsP4-E315G with Leu, Arg, His, or any aromatic amino acid at the N-terminus of nsP4 also made the mutant viruses temperature sensitive. The results from this study and from a previous report on the shutoff of minus-strand RNA synthesis at 40 degrees C with the nsP1-A348T mutation in ts11 suggests that the N-terminus nsP4 interacts with nsP1 during initiation of minus-strand RNA synthesis.     

Both of the Rab5 subfamily small GTPases of Trypanosoma brucei are essential and required for endocytosis

Endocytosis is an essential process in Trypanosoma brucei and all evidence suggests it is exclusively clathrin-mediated. The trypanosome genome encodes two Rab5 proteins, small GTPases that play a role in very early stages of endocytosis. In the mammalian bloodstream stage TbRAB5A localises to compartments containing internalised antibody, variant surface glycoprotein (VSG) and transferrin, whilst TbRAB5B localises to compartments containing the transmembrane protein ISG(100). Dominant-active forms of TbRAB5A stimulate endocytosis in procyclic forms and alter the kinetics of anti-VSG antibody and transferrin turnover in bloodstream stages. Similar mutants of TbRAB5B increase fluid phase uptake in procyclic cells but do not significantly affect endocytosis in bloodstream forms. Here, we use RNA interference to evaluate the relative importance of TbRAB5A and TbRAB5B and show that both GTPases are essential in the bloodstream form. Depletion of either TbRAB5A or TbRAB5B results in morphological abnormalities, including enlargement of the flagellar pocket, consistent with a potent block to endocytosis. Also, RNAi compromises transferrin accumulation in both cases but induces distinct patterns of mislocalisation of endosomal markers. Finally, RNAi of either TbRAB5A or TbRAB5B results in a decrease in levels of clathrin. Taken together, these data indicate that both TbRAB5A and TbRAB5B are required for endocytosis in trypanosomes and demonstrate that there are multiple essential endocytic routes in this organism.     

Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice

A/duck/Guangxi/53/2002 (DKGX/53) and A/duck/Fujian/01/2002 (DKFJ/01) are H5N1 avian influenza viruses that are lethal in chickens. In mice, however, DKFJ/01 is highly pathogenic, whereas DKGX/53 displays low pathogenicity. In this study, we used reverse genetics to demonstrate that two amino acid residues at positions 30 and 215 of the M1 protein of these two viruses are important determinants for pathogenicity in mice. We thus firstly prove the M1 protein contributes to the virulence of H5N1 viruses in mice, and the amino acid residues shown to attenuate the virulence could be targeted in influenza virus candidates for live vaccine development.     

JSAP1 and JLP are required for ARF6 localization to the midbody in cytokinesis

The ADP‐ribosylation factor 6 (ARF6) GTPase is important in cytokinesis and localizes to the midbody. However, the mechanism and regulation of ARF6's recruitment to the midbody are largely unknown. Here, we investigated the functions of two binding partners of active ARF6, c‐Jun NH₂‐terminal kinase (JNK)/stress‐activated protein kinase‐associated protein 1 (JSAP1) and JNK‐associated leucine zipper protein (JLP), by gene knockout and rescue experiments in mouse embryonic fibroblasts. Depleting both JSAP1 and JLP impaired ARF6's localization to the midbody and delayed cytokinesis. These defects were almost completely rescued by wild‐type JSAP1 or JLP, but not by JSAP1 or JLP mutants that were unable to interact with active ARF6 or with the kinesin heavy chain (KHC) of kinesin‐1. In transfected cells, a constitutively active form of ARF6 associated with KHC only when co‐expressed with wild‐type JSAP1 or JLP and not with a JSAP1 or JLP mutant. These findings suggest that JSAP1 and JLP, which might be paralogous to each other, are critical and functionally redundant in cytokinesis and control ARF6 localization to the midbody by forming a tripartite complex of JSAP1/JLP, active ARF6, and kinesin‐1.     

Both mutations in G6PD A- are necessary to produce the G6PD deficient phenotype.

The high prevalence of glucose 6-phosphate dehydrogenase (G6PD) deficiency in African populations is due almost entirely to the enzyme variant A-, which differs from the wild-type G6PD B by two amino acid replacements, 68 Val-->Met and 126 Asn-->Asp. The non-deficient polymorphic variant G6PD A contains only the mutation 126 Asn-->Asp. The frequencies of the G6PD A and of the G6PD A- genes in parts of Africa are both about 0.2. The 68 Val-->Met mutation has not been found in a B background. This could be because the 68 Val-->Met mutation happened to arise in an A gene in the first instance, or because the 68 Val-->Met mutation alone is not sufficient to cause G6PD deficiency. We have approached this question by producing G6PD B, A, A-, and G6PD 68 Val-->Met in a bacterial expression system and analysing their biochemical properties. With each single mutation we found a slight decrease in both the specific activity and the yield of enzyme when compared to G6PD B. When both mutations were introduced together, there was a roughly additive effect on specific activity, but a much more drastic effect on enzyme yield (4% of normal). This synergistic effect was also demonstrated on thermal stability, especially at low NADP concentrations. Comparable results were produced when the replacement 119 Gln-->Glu was studied instead of 126 Asn-->Asp. We infer that the coexistence of the two mutations is responsible for enzyme deficiency in G6PD A- because they act synergistically in causing instability of the enzyme.     

Both CD1d antigen presentation and interleukin-12 are required to activate natural killer T cells during Trypanosoma cruzi infection

Mechanisms of natural killer T (NKT)-cell activation remain unclear. Here, we report that during Trypanosoma cruzi infection, interleukin-12 (IL-12) deficiency or anti-CD1d antibody treatment prevents normal activation. The required IL-12 arises independently of MyD88. The data support a model of normal NKT-cell activation that requires IL-12 and TCR stimulation.     

alpha-Neurexins are required for efficient transmitter release and synaptic homeostasis at the mouse neuromuscular junction

Neurotransmission at chemical synapses of the brain involves alpha-neurexins, neuron-specific cell-surface molecules that are encoded by three genes in mammals. Deletion of alpha-neurexins in mice previously demonstrated an essential function, leading to early postnatal death of many double-knockout mice and all triple mutants. Neurotransmitter release at central synapses of newborn knockouts was severely reduced, a function of alpha-neurexins that requires their extracellular sequences. Here, we investigated the role of alpha-neurexins at neuromuscular junctions, presynaptic terminals that lack a neuronal postsynaptic partner, addressing an important question because the function of neurexins was hypothesized to involve cell-adhesion complexes between neurons. Using systems physiology, morphological analyses and electrophysiological recordings, we show that quantal content, i.e. the number of acetylcholine quanta released per nerve impulse from motor nerve terminals, and frequency of spontaneous miniature endplate potentials at the slow-twitch soleus muscle are reduced in adult alpha-neurexin double-knockouts, consistent with earlier data on central synapses. However, the same parameters at diaphragm muscle neuromuscular junctions showed no difference in basal neurotransmission. To reconcile these observations, we tested the capability of control and alpha-neurexin-deficient diaphragm neuromuscular junctions to compensate for an experimental reduction of postsynaptic acetylcholine receptors by a compensatory increase of presynaptic release: Knockout neuromuscular junctions produced significantly less upregulation of quantal content than synapses from control mice. Our data suggest that alpha-neurexins are required for efficient neurotransmitter release at neuromuscular junctions, and that they may perform a role in the molecular mechanism of synaptic homeostasis at these peripheral synapses.