Early Protein Synthesis Research Articles

Adaptive truncation of the S gene in IBV during chicken embryo passaging plays a crucial role in its attenuation.

Like all coronaviruses, infectious bronchitis virus, the causative agent of infectious bronchitis in chickens, exhibits a high mutation rate. Adaptive mutations that arise during the production of live attenuated vaccines against IBV often decrease virulence. The specific impact of these mutations on viral pathogenicity, however, has not been fully elucidated. In this study, we identified a mutation at the 3' end of the S gene in an IBV strain that was serially passaged in chicken embryos, and showed that this mutation resulted in a 9-aa truncation of the cytoplasmic tail (CT) of the S protein. This phenomenon of CT truncation has previously been observed in the production of attenuated vaccines against other coronaviruses such as the porcine epidemic diarrhea virus. We next discovered that the 9-aa truncation in the S protein CT resulted in the loss of the endoplasmic-reticulum-retention signal (KKSV). Rescue experiments with recombinant viruses confirmed that the deletion of the KKSV motif impaired the localization of the S protein to the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) and increased its expression on the cell surface. This significantly reduced the incorporation of the S protein into viral particles, impaired early subgenomic RNA and protein synthesis, and ultimately reduced viral invasion efficiency in CEK cells. In vivo experiments in chickens confirmed the reduced pathogenicity of the mutant IBV strains. Additionally, we showed that the adaptive mutation altered the TRS-B of ORF3 and impacted the transcriptional regulation of this gene. Our findings underscore the significance of this adaptive mutation in the attenuation of IBV infection and provide a novel strategy for the development of live attenuated IBV vaccines.

EIF2Ss, a Novel c-Myc-Correlated Gene Family, is Associated with Poor Prognosis and Immune Infiltration in Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor in urgent need of novel diagnostics, prognostic markers, and treatments. Eukaryotic translation initiation factor 2 subunits (EIF2Ss), comprising Eukaryotic translation initiation factor 2 subunit alpha (EIF2S1), Eukaryotic translation initiation factor 2 subunit beta (EIF2S2), and Eukaryotic translation initiation factor 2 subunit gamma (EIF2S3), is a family of eukaryotic initiation factors that participate in early protein synthesis and are crucial for tumor initiation and progression. However, the role of EIF2Ss in PAAD has yet to be reported. The aim of this study was therefore to analyze EIF2Ss in relation to the diagnosis, prognosis, and treatment of PAAD. The cancer genome atlas (TCGA) database was used to investigate gene expression and patient survival. Gene alterations, immune cell infiltration, and immune checkpoints in PAAD were also evaluated. Univariate and multivariate analysis, nomograms, calibration curves, and Decision Curve Analysis (DCA) diagrams were used to develop and evaluate a prediction model for patient outcome. Single-cell RNA-seq (scRNA) analysis, functional enrichment, co-IP assay, mass spectrometry, and western blot were used to study the relationship between EIF2Ss and c-myc in PAAD. EIF2Ss are over-expressed in PAAD tissue and are associated with poor prognosis. The frequency of EIF2S1, EIF2S2, and EIF2S3 gene alteration in PAAD was 0.2%, 0.4%, and 0.2%, respectively. High EIF2Ss expression was associated with Th2 cell infiltration, whereas low expression was associated with pDC infiltration. Moreover, EIF2Ss expression was positively correlated with the expression of the NT5E, ULBP1, PVR, CD44, IL10RB, and CD276 checkpoints. A prediction model developed using EIF2Ss and important clinicopathologic features showed good predictive value for the overall survival of PAAD patients. ScRNA-Seq data showed that EIF2Ss was associated with enrichment for endothelial cells, fibroblasts, malignant cells, and ductal cells. EIF2Ss expression was also correlated with adipogenesis, interferon-alpha response, epithelial-mesenchymal transition, myc targets, G2M checkpoint, oxidative phosphorylation, and hypoxia. Functional enrichment analysis of EIF2Ss showed a close correlation with the myc pathway, and interactions between EIF2Ss and c-myc were confirmed by co-IP assay and mass spectrometry. Importantly, knockdown of c-myc decreased the expression of EIF2S1, EIF2S2, and EIF2S3 in PAAD cells. EIF2Ss were found to have significant clinical implications for the prognosis and treatment of PAAD. Inhibition of c-myc caused the downregulation of EIF2S1, EIF2S2, and EIF2S3 expression.

Shwachman-Diamond syndromes: clinical, genetic, and biochemical insights from the rare variants.

Shwachman-Diamond syndrome is a rare inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. In 10-30% of cases, transformation to a myeloid neoplasm occurs. Approximately 90% of patients have biallelic pathogenic variants in the SBDS gene located on human chromosome 7q11. Over the past several years, pathogenic variants in three other genes have been identified to cause similar phenotypes; these are DNAJC21, EFL1, and SRP54. Clinical manifestations involve multiple organ systems and those classically associated with the Shwachman-Diamond syndrome (bone, blood, and pancreas). Neurocognitive, dermatologic, and retinal changes may also be found. There are specific gene-phenotype differences. To date, SBDS, DNAJC21, and SRP54 variants have been associated with myeloid neoplasia. Common to SBDS, EFL1, DNAJC21, and SRP54 is their involvement in ribosome biogenesis or early protein synthesis. These four genes constitute a common biochemical pathway conserved from yeast to humans that involve early stages of protein synthesis and demonstrate the importance of this synthetic pathway in myelopoiesis.

Ancestral Sequence Reconstruction of the Ribosomal Protein uS8 and Reduction of Amino Acid Usage to a Smaller Alphabet.

Understanding the origin and early evolution of proteins is important for unveiling how the RNA world developed into an RNA-protein world. Because the composition of organic molecules in the Earth's primitive environment was plausibly not as diverse as today, the number of different amino acids used in early protein synthesis is likely to be substantially less than the current 20 proteinogenic residues. In this study, we have explored the thermal stability and RNA binding of ancestral variants of the ribosomal protein uS8 constructed from a reduced-alphabet of amino acids. First, we built a phylogenetic tree based on the amino acid sequences of uS8 from multiple extant organisms and used the tree to infer two plausible amino acid sequences corresponding to the last bacterial common ancestor of uS8. Both ancestral proteins were thermally stable and bound to an RNA fragment. By eliminating individual amino acid letters and monitoring thermal stability and RNA binding in the resulting proteins, we reduced the size of the amino acid set constituting one of the ancestral proteins, eventually finding that convergent sequences consisting of 15- or 14-amino acid alphabets still folded into stable structures that bound to the RNA fragment. Furthermore, a simplified variant reconstructed from a 13-amino-acid alphabet retained affinity for the RNA fragment, although it lost conformational stability. Collectively, RNA-binding activity may be achieved with a subset of the current 20 amino acids, raising the possibility of a simpler composition of RNA-binding proteins in the earliest stage of protein evolution.

Bio-priming of seeds: Plant stress management and its underlying cellular, biochemical and molecular mechanisms

Exposure of crops to stress is the most significant barrier to agricultural output and food security worldwide. Stress induces changes in plants’ physiological functions, which leads to lower plant growth and agricultural yield. Plants undergo a variety of molecular, cellular, and physiological changes to combat various abiotic stresses. Plants suffer from increased osmotic pressure outside the root due to osmotic stresses like drought or salt stress that ultimately result in reduced water availability to plant roots. In addition to morphological modifications, physiological changes such as lower leaf osmotic potential, accumulation of osmoprotectants, increased antioxidant activities, and so on occur in response to such abiotic stresses. Seed priming is a novel yet simple technique that involves the use of beneficial and eco-friendly biological agents to improve the physiological functioning of seeds. This technique also plays a vital role in restoring agro-ecological balances through the improvement of soil fertility or by decreasing soil and water contamination. An understanding of the mechanisms involved in seed bio-priming with plant growth-promoting microbes is very crucial to efficiently utilise this technique towards agricultural sustainability. It is generally thought that the advancement of metabolic processes along with activation of repairing systems during the pre-germination stage are the central reasons behind growth promotion and acquired stress tolerance. The application of plant growth-promoting rhizobacteria in agriculture has been emerging to improve stress resilience. Bio-priming of seeds ensures early protein and DNA synthesis, and also helps in effective mitochondrial development. This review aims to summarise the current state of understanding of the mechanisms involved in this eco-friendly technique.

Branched-chain amino acid and branched-chain ketoacid ingestion increases muscle protein synthesis rates in vivo in older adults: a double-blind, randomized trial

ABSTRACTBackgroundProtein ingestion increases muscle protein synthesis rates. However, limited data are currently available on the effects of branched-chain amino acid (BCAA) and branched-chain ketoacid (BCKA) ingestion on postprandial muscle protein synthesis rates.ObjectiveThe aim of this study was to compare the impact of ingesting 6 g BCAA, 6 g BCKA, and 30 g milk protein (MILK) on the postprandial rise in circulating amino acid concentrations and subsequent myofibrillar protein synthesis rates in older males.MethodsIn a parallel design, 45 older males (age: 71 ± 1 y; BMI: 25.4 ± 0.8 kg/m2) were randomly assigned to ingest a drink containing 6 g BCAA, 6 g BCKA, or 30 g MILK. Basal and postprandial myofibrillar protein synthesis rates were assessed by primed continuous l-[ring-13C6]phenylalanine infusions with the collection of blood samples and muscle biopsies.ResultsPlasma BCAA concentrations increased following test drink ingestion in all groups, with greater increases in the BCAA and MILK groups compared with the BCKA group (P < 0.05). Plasma BCKA concentrations increased following test drink ingestion in all groups, with greater increases in the BCKA group compared with the BCAA and MILK groups (P < 0.05). Ingestion of MILK, BCAA, and BCKA significantly increased early myofibrillar protein synthesis rates (0–2 h) above basal rates (from 0.020 ± 0.002%/h to 0.042 ± 0.004%/h, 0.022 ± 0.002%/h to 0.044 ± 0.004%/h, and 0.023 ± 0.003%/h to 0.044 ± 0.004%/h, respectively; P < 0.001), with no differences between groups (P > 0.05). Myofibrillar protein synthesis rates during the late postprandial phase (2–5 h) remained elevated in the MILK group (0.039 ± 0.004%/h; P < 0.001), but returned to baseline values following BCAA and BCKA ingestion (0.024 ± 0.005%/h and 0.024 ± 0.005%/h, respectively; P > 0.05).ConclusionsIngestion of 6 g BCAA, 6 g BCKA, and 30 g MILK increases myofibrillar protein synthesis rates during the early postprandial phase (0–2 h) in vivo in healthy older males. The postprandial increase following the ingestion of 6 g BCAA and BCKA is short-lived, with higher myofibrillar protein synthesis rates only being maintained following the ingestion of an equivalent amount of intact milk protein. This trial was registered at Nederlands Trial Register (www.trialregister.nl) as NTR6047.

Isolation and Characterization of vΔI3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication.

Vaccinia virus is unusual among DNA viruses in replicating exclusively in the cytoplasm of infected cells. The single-stranded DNA (ssDNA) binding protein (SSB) I3 is among the replication machinery encoded by the 195-kb genome, although direct genetic analysis of I3 has been lacking. Herein, we describe a complementing cell line (CV1-I3) that fully supports the replication of a null virus (vΔI3) lacking the I3 open reading frame (ORF). In noncomplementing CV1-CAT cells, vΔI3 shows a severe defect in the production of infectious virus (≥200-fold reduction). Early protein synthesis and core disassembly occur normally. However, DNA replication is profoundly impaired (≤0.2% of wild-type [WT] levels), and late proteins do not accumulate. When several other noncomplementing cell lines are infected with vΔI3, the yield of infectious virus is also dramatically reduced (168- to 1,776-fold reduction). Surprisingly, the residual levels of DNA accumulation vary from 1 to 12% in the different cell lines (CV1-CAT < A549 < BSC40 < HeLa); however, any nascent DNA that can be detected is subgenomic in size. Although this subgenomic DNA supports late protein expression, it does not support the production of infectious virions. Electron microscopy (EM) analysis of vΔI3-infected BSC40 cells reveals that immature virions are abundant but no mature virions are observed. Aberrant virions characteristic of a block to genome encapsidation are seen instead. Finally, we demonstrate that a CV1 cell line encoding a previously described I3 variant with impaired ssDNA binding activity is unable to complement vΔI3. This report provides definitive evidence that the vaccinia virus I3 protein is the replicative SSB and is essential for productive viral replication.IMPORTANCE Poxviruses are of historical and contemporary importance as infectious agents, vaccines, and oncolytic therapeutics. The cytoplasmic replication of poxviruses is unique among DNA viruses of mammalian cells and necessitates that the double-stranded DNA (dsDNA) genome encode the viral replication machinery. This study focuses on the I3 protein. As a ssDNA binding protein (SSB), I3 has been presumed to play essential roles in genome replication, recombination, and repair, although genetic analysis has been lacking. Herein, we report the characterization of an I3 deletion virus. In the absence of I3 expression, DNA replication is severely compromised and viral yield profoundly decreased. The production of infectious virus can be restored in a cell line expressing WT I3 but not in a cell line expressing an I3 mutant that is defective in ssDNA binding activity. These data show conclusively that I3 is an essential viral protein and functions as the viral replicative SSB.

Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus.

Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

Interaction of Immunoglobulin with Cytomegalovirus-Infected Cells.

Intravenous immunoglobulin (IVIG) is used to treat or prevent severe viral infection, especially cytomegalovirus (CMV) infections. IVIG was characterized to understand its interaction with CMV-infected cells. IVIG retarded CMV spread and reduced virus yields depending on the neutralizing (NT) antibody titer. Immediate early protein synthesis was reduced by IVIG in 3 to 15 h, and IVIG specifically reduced the ratio of 66/68k protein synthesis among immediate early proteins in an NT antibody-dependent manner between 4 and 8 h after infection, indicating that antigenic modulation of CMV-infected cells by IVIG reduced viral protein synthesis and virus production. The half-life of antibody bound to CMV-infected cells was 3.8 h. NT antibody titers to varicella-zoster virus (VZV) and CMV in IVIG were dose dependently absorbed by cells infected with VZV and CMV, respectively, but the antibody titers to CMV and VZV, respectively, were not affected. NT antibody in 0.3 mL of IVIG (15 mg) was specifically absorbed by 108 CMV-infected cells and 107 VZV-infected cells, suggesting that the NT antibody in IVIG might be inactivated by one-tenth of a similar volume of CMV-infected or VZV-infected cells. Various antiviral activities of IVIG may contribute to control and alleviation of CMV infection.

Inactivation Kinetics and Replication Cycle Inhibition of Adenovirus by Monochloramine

Monochloramine is commonly used as a secondary disinfectant to maintain a residual in drinking water distribution systems in the United States. The mechanism by which waterborne viruses become inactivated by monochloramine remains widely unknown. A more fundamental understanding of how viruses become inactivated is necessary for better detection and control of viruses in drinking water. Human adenovirus (HAdV) is known to be the waterborne virus most resistant to monochloramine disinfection, and this study presents inactivation kinetics over a range of environmental conditions. Several steps in the HAdV replication cycle were investigated to determine which steps become inhibited by monochloramine disinfection. Interestingly, monochloramine-inactivated HAdV could bind to host cells, but genome replication and early and late mRNA transcription were inhibited. We conclude that monochloramine exposure inhibited a replication cycle event after binding but prior to early viral protein synthesis.

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90min post-infection, and the virus particles with size of about 50nm including more than 20nm of vacuum drying gold were observed on the infected cells surface. What׳s more, the replication lasted around 13h after the early protein synthesis for about 5h, and the release of DHAV-1 was in steady state after 32h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies.

Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

Early Dengue Virus Protein Synthesis Induces Extensive Rearrangement of the Endoplasmic Reticulum Independent of the UPR and SREBP-2 Pathway

The rearrangement of intracellular membranes has been long reported to be a common feature in diseased cells. In this study, we used dengue virus (DENV) to study the role of the unfolded protein response (UPR) and sterol-regulatory-element-binding-protein-2 (SREBP-2) pathway in the rearrangement and expansion of the endoplasmic reticulum (ER) early after infection. Using laser scanning confocal and differential interference contrast microscopy, we demonstrate that rearrangement and expansion of the ER occurs early after DENV-2 infection. Through the use of mouse embryonic fibroblast cells deficient in XBP1 and ATF6, we show that ER rearrangement early after DENV infection is independent of the UPR. We then demonstrate that enlargement of the ER is independent of the SREBP-2 activation and upregulation of 3-hydroxy-3-methylglutaryl-Coenzyme-A reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway. We further show that this ER rearrangement is not inhibited by the treatment of DENV-infected cells with the cholesterol-inhibiting drug lovastatin. Using the transcription inhibitor actinomycin D and the translation elongation inhibitor cycloheximide, we show that de novo viral protein synthesis but not host transcription is necessary for expansion and rearrangement of the ER. Lastly, we demonstrate that viral infection induces the reabsorption of lipid droplets into the ER. Together, these results demonstrate that modulation of intracellular membrane architecture of the cell early after DENV-2 infection is driven by viral protein expression and does not require the induction of the UPR and SREBP-2 pathways. This work paves the way for further study of virally-induced membrane rearrangements and formation of cubic membranes.

Coding Constraints Modulate Chemically Spontaneous Mutational Replication Gradients in Mitochondrial Genomes

Distances from heavy and light strand replication origins determine duration mitochondrial DNA remains singlestranded during replication. Hydrolytic deaminations from A->G and C->T occur more on single- than doublestranded DNA. Corresponding replicational nucleotide gradients exist across mitochondrial genomes, most at 3rd, least 2nd codon positions. DNA singlestrandedness during RNA transcription causes gradients mainly in long-lived species with relatively slow metabolism (high transcription/replication ratios). Third codon nucleotide contents, evolutionary results of mutation cumulation, follow replicational, not transcriptional gradients in Homo; observed human mutations follow transcriptional gradients. Synonymous third codon position transitions potentially alter adaptive off frame information. No mutational gradients occur at synonymous positions forming off frame stops (these adaptively stop early accidental frameshifted protein synthesis), nor in regions coding for putative overlapping genes according to an overlapping genetic code reassigning stop codons to amino acids. Deviation of 3rd codon nucleotide contents from deamination gradients increases with coding importance of main frame 3rd codon positions in overlapping genes (greatest if these are 2nd position in overlapping genes). Third codon position deamination gradients calculated separately for each codon family are strongest where synonymous transitions are rarely pathogenic; weakest where transitions are frequently pathogenic. Synonymous mutations affect translational accuracy, such as error compensation of misloaded tRNAs by codon-anticodon mismatches (prevents amino acid misinsertion despite tRNA misacylation), a potential cause of pathogenic mutations at synonymous codon positions. Indeed, codon-family-specific gradients are inversely proportional to error compensation associated with gradient-promoted transitions. Deamination gradients reflect spontaneous chemical reactions in singlestranded DNA, but functional coding constraints modulate gradients.

Role of the RNA recognition motif of the E1B 55 kDa protein in the adenovirus type 5 infectious cycle

Although the adenovirus type 5 (Ad5) E1B 55 kDa protein can bind to RNA in vitro, no UV-light-induced crosslinking of this E1B protein to RNA could be detected in infected cells, under conditions in which RNA binding by a known viral RNA-binding protein (the L4 100 kDa protein) was observed readily. Substitution mutations, including substitutions reported to inhibit RNA binding in vitro, did not impair synthesis of viral early or late proteins or alter significantly the efficiency of viral replication in transformed or normal human cells. However, substitutions of conserved residues in the C-terminal segment of an RNA recognition motif specifically inhibited degradation of Mre11. We conclude that, if the E1B 55 kDa protein binds to RNA in infected cells in the same manner as in in vitro assays, this activity is not required for such well established functions as induction of selective export of viral late mRNAs.

Comparative inhibitory activity of the stilbenes resveratrol and oxyresveratrol on African swine fever virus replication

Stilbenols are polyphenolic phytoalexins produced by plants in response to biotic or abiotic stress. These compounds have received much attention because of their significant biological effects. One of these is their antiviral action, which has previously been documented for two members of this class, namely resveratrol and oxyresveratrol. Here we tested the antiviral effect of these two compounds on African swine fever virus, the only member of the newly created family Asfarviridae and a serious limitation to porcine production worldwide. Our results show a potent, dose-dependent antiviral effect of resveratrol and oxyresveratrol in vitro. Interestingly, this antiviral activity was found for these synthetic compounds and also for oxyresveratrol extracted from new natural sources (mulberry twigs). The antiviral effect of these two drugs was demonstrated at concentrations that do not induce cytotoxicity in cultured cells. Moreover, these antivirals achieved a 98–100% reduction in viral titers. Both compounds allowed early protein synthesis but inhibited viral DNA replication, late viral protein synthesis and viral factory formation.

Alpha-COPI Coatomer Protein Is Required for Rough Endoplasmic Reticulum Whorl Formation in Mosquito Midgut Epithelial Cells

BackgroundOne of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.Methodology/Principal FindingsAdult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta'), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.Conclusionsalpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.

A reporter system for Epstein-Barr virus (EBV) lytic replication: Anti-EBV activity of the broad anti-herpesviral drug artesunate

Epstein-Barr virus (EBV) is associated with severe human diseases. Therapies with conventional anti-herpesviral drugs are mostly ineffective so that novel drugs are urgently needed. As cell culture-based evaluation systems are required, a GFP (green fluorescent protein) reporter system was generated, which was conceived for an easy quantitation of lytic EBV replication and the analysis of EBV drug sensitivity. A reporter construct was generated on the basis of an EBV plasmid mini-replicon which enabled an episomal maintenance and selection of stably transfected Raji and 293T cell clones. Controlled by the viral lytic origin of DNA replication (oriLyt), this reporter construct could be activated through experimental EBV infection or through chemically stimulated reactivation from EBV latency. Using this system, the sensitivity of EBV to the broad-spectrum anti-herpesviral drug artesunate could be demonstrated: (i) artesunate inhibits EBV in the low micromolar range, (ii) two different strains of EBV are equally artesunate-sensitive, (iii) inhibition is detectable similarly in EBV-infected epithelial cells or lymphocytes, and (iv) the mode of antiviral action is based on a block of viral immediate early protein synthesis. The data demonstrate the usefulness of this reporter system for the quantitation of EBV replication and for determining EBV drug sensitivity.

Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

BackgroundTo understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.ResultsTo determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells) and in vivo (BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.ConclusionsThe F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.

PKR-like endoplasmic reticulum kinase (PERK) activation following brain ischemia is independent of unfolded nascent proteins

Transient global brain ischemia results in an immediate inhibition of protein translation upon reperfusion. During early brain reperfusion protein synthesis is inhibited by alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation by the PKR-like endoplasmic reticulum kinase (PERK). Normally, PERK is held in an inactive, monomeric state by the binding of the endoplasmic reticulum (ER) chaperone GRP78 to the lumenal end of PERK. The prevailing view is that ER stress leads to the accumulation of unfolded proteins in the ER lumen. GRP78 dissociates from PERK to bind these accumulated unfolded proteins, leading to PERK activation, phosphorylation of eIF2α, and inhibition of translation. To determine if an increase in unfolded nascent proteins following transient brain ischemia contributes to PERK activation, protein synthesis was blocked by intracerebral injection of anisomycin prior to induction of ischemia. Anisomycin inhibited protein synthesis by over 99% and reduced newly synthesized proteins in the ER to ∼20% of controls. With an ER nearly devoid of newly synthesized proteins, PERK was still activated and was able to phosphorylate eIF2α in CA1 neurons during reperfusion. These data strongly argue that PERK activation is independent of the large increase in unfolded nascent proteins within the ER following transient global brain ischemia.