Paradoxical imbalance between activated lymphocyte protein synthesis capacity and rapid division rate.

Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.

A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay.

Despite the success of rotavirus vaccines, rotaviruses remain one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality, especially in low- and middle-income countries. The reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins, such as vaccine antigens and therapeutic payloads. Here, we demonstrate that three positions in rotavirus genome-the C terminus of NSP1, NSP3 and NSP5-can tolerate the insertion of reporter genes. By using rotavirus expressing GFP, we develop a high-throughput neutralization assay and reveal the pre-existing immunity against rotavirus in humans and other animal species. Our work shows the plasticity of the rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.

Genetic mapping and analysis of candidate leaf color genes in common winter wheat (Triticum aestivum L.).

Leaf color-related genes play key roles in chloroplast development and photosynthetic pigment biosynthesis and affect photosynthetic efficiency and grain yield in crops. In this study, a recessive homozygous individual displaying yellow leaf color (yl1) was identified in the progeny population derived from a cross between wheat cultivars Xingmai1 (XM1) and Yunong3114 (YN3114). Phenotypic identification showed that yl1 exhibited the yellow character state over the entire growth period. Compared with XM1, yl1 plants had significantly lower chlorophyll content and net photosynthetic rate, and similar results were found between the green-type lines and yellow-type lines in the BC2F3 XM1 × yl1 population. Gene mapping via the bulked segregant exome capture sequencing (BSE-seq) method showed that the target gene TaYL1 was located within the region of 582,556,971-600,837,326 bp on chromosome 7D. Further analysis by RNA-seq suggested TraesCS7D02G469200 as a candidate gene for yellow leaf color in common wheat, which encodes a protein containing the AP2 domain. Moreover, comparative transcriptome profiling revealed that most differentially expressed genes (DEGs) were enriched in chlorophyll metabolism and photosynthesis pathways. Together, these results indicate that TaYL1 potentially affects chlorophyll synthesis and photosynthesis. This study further elucidates the biological mechanism of chlorophyll synthesis, metabolism, and photosynthesis in wheat and provides a theoretical basis for high photosynthetic efficiency in wheat breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01395-z.

Association of Thyroid-Stimulating Hormone (TSH) Levels With the Prognosis of Patients Undergoing Heart Transplantation: A Retrospective Study.

Purpose: To investigate the impact of TSH levels using a more stringent cutoff of subclinical hypothyroidism (i.e., TSH > 2.5 mIU/L) on the short-term complications and long-term prognosis in patients who underwent heart transplantation (HTx). Methods: This is a retrospective study of consecutive patients with end-stage heart failure (HF) who underwent HTx. They were divided into three groups: thyroid-stimulating hormone (TSH) ≤ 2.50 mIU/L (L-TSH), 2.50 < TSH ≤ 4.91 mIU/L (M-TSH), and TSH > 4.91 mIU/L (H-TSH). The outcomes are all-cause death and cardiogenic death. Results: There are 63 (70%) males and 27 (30%) females. Nine (10%) patients died within 1 month after surgery, including five cardiogenic deaths. By 1 year, a total of 19 patients total were dead. The survival rate in the M-TSH group was significantly higher than that of the L-TSH group (P = 0.017). After adjusted by variables of sex, age, BMI, diabetes history, hypertension history, the multivariable Cox analysis showed that body mass index (HR = 0.804, 95%CI: 0.680-0.951, P = 0.011), and L-TSH (HR = 8.757, 95%CI: 1.786-42.948, P = 0.007 vs. M-TSH), and H-TSH (HR = 6.427, 95%CI: 1.137-36.327, P = 0.035 vs. M-TSH) were independently associated with all-cause death. The multivariable Cox analysis showed that body mass index (HR = 0.703, 95%CI: 0.564-0.878, P = 0.002), and L-TSH (HR = 17.717, 95%CI: 1.907-164.607, P = 0.011 vs. M-TSH) were independently associated with cardiogenic death. Conclusion: For patients with end-stage HF undergoing HTx, low and high baseline TSH levels are independently associated with 1-year all-cause death and low baseline TSH levels with cardiogenic death.

Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

An insight into the mechanism and molecular basis of dysfunctional immune response involved in cholestasis.

Cholestasis is one of the most common clinical symptom of liver diseases. If patients do not receive effective treatment, cholestasis can evolve into liver fibrosis, cirrhosis and ultimately liver failure requiring liver transplantation. Currently, only ursodeoxycholic acid, obeticholic acid and bezafibrate are FDA-approved drugs, thereby requiring a breakthrough in new mechanisms and therapeutic development. Inflammation is one of the common complications of cholestasis. Hepatic accumulation of toxic hydrophobic bile acids is a highly immunogenic process involving both resident and immigrating immune cells. And the resulting inflammation may further aggravate hepatocyte injury. Though, great investigations have been made in the immune responses during cholestasis, the relationship between immune responses and cholestasis remains unclear. Moreover, scarce reviews summarize the immune responses during cholestasis and the efficacy of therapies on immune response. The main purpose of this paper is to review the existing literature on dysfunctional immune response during cholestasis and the effect of treatment on immune response which may provide an insight for researchers and drug development.

Hybrid Gene Origination Creates Human-Virus Chimeric Proteins during Infection.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, because viral polymerases cleave 5'-m7G-capped host transcripts to prime viral mRNA synthesis ("cap-snatching"). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, which we named "start-snatching." Depending on the reading frame, start-snatching allows the translation of host and viral "untranslated regions" (UTRs) to create N-terminally extended viral proteins or entirely novel polypeptides by genetic overprinting. We show that both types of chimeric proteins are made in IAV-infected cells, generate T cell responses, and contribute to virulence. Our results indicate that during infection with IAV, and likely a multitude of other human, animal and plant viruses, a host-dependent mechanism allows the genesis of hybrid genes.

A SIINFEKL-Based System to Measure MHC Class I Antigen Presentation Efficiency and Kinetics.

Antigen presentation by classical MHC class I molecules to CD8+ T cells is a central aspect of the adaptive immune response. Here, we describe methods to monitor antigen presentation using the model ovalbumin Kb-binding peptide, SIINFEKL. SIINFEKL genetically incorporated into viral or cellular source proteins can be used to precisely probe various aspects of antigen presentation, including the kinetics of peptide generation, MHC class I surface stability, and presentation efficiency following pharmacological and genetic manipulations including genome wide and high throughput drug screening.

Ribosomal Proteins Regulate MHC Class I Peptide Generation for Immunosurveillance.

The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.

Immunoribosomes: Where's there's fire, there's fire.

The MHC class I antigen presentation pathway enables T cell immunosurveillance of cancer cells, viruses and other intracellular pathogens. Rapidly degraded newly synthesized proteins (DRiPs) are a major source of self-, and particularly, viral antigenic peptides. A number of findings support the idea that a substantial fraction of antigenic peptides are synthesized by "immunoribosomes", a subset of translating ribosomes that generate class I peptides with enhanced efficiency. Here, we review the evidence for the immunoribosome hypothesis.

Flu DRiPs in MHC Class I Immunosurveillance.

Since the publication of the DRiP (defective ribosomal product) hypothesis in 1996, numerous studies have addressed the contribution of DRiPs to generating viral antigenic peptides for CD8+ T cell immunosurveillance. Here, we review studies characterizing the generation of antigenic peptides from influenza A virus encoded DRiPs, discuss the many remaining mysteries regarding the nature of their co-translational generation, and speculate on where the future might lead.

Influenza A Virus Negative Strand RNA Is Translated for CD8+ T Cell Immunosurveillance.

Probing the limits of CD8+ T cell immunosurveillance, we inserted the SIINFEKL peptide into influenza A virus (IAV)-negative strand gene segments. Although IAV genomic RNA is considered noncoding, there is a conserved, relatively long open reading frame present in segment 8, encoding a potential protein termed NEG8. The biosynthesis of NEG8 from IAV has yet to be demonstrated. Although we failed to detect NEG8 protein expression in IAV-infected mouse cells, cell surface Kb-SIINFEKL complexes are generated when SIINFEKL is genetically appended to the predicted C terminus of NEG8, as shown by activation of OT-I T cells in vitro and in vivo. Moreover, recombinant IAV encoding of SIINFEKL embedded in the negative strand of the neuraminidase-stalk coding sequence also activates OT-I T cells in mice. Together, our findings demonstrate both the translation of sequences on the negative strand of a single-stranded RNA virus and its relevance in antiviral immunosurveillance.

Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature.

Mycolactone is a bacteria-derived macrolide that blocks the biogenesis of a large array of secretory and integral transmembrane proteins (TMP) through potent inhibition of the Sec61 translocon. Here, we used quantitative proteomics to delineate the direct and indirect effects of mycolactone-mediated Sec61 blockade in living cells. In T lymphocytes, dendritic cells and sensory neurons, Sec61 substrates downregulated by mycolactone were in order of incidence: secretory proteins (with a signal peptide but no transmembrane domain), TMPs with a signal peptide (Type I) and TMPs without signal peptide and a cytosolic N terminus (Type II). TMPs without a signal peptide and the opposite N terminus topology (Type III) were refractory to mycolactone inhibition. This rule applied comparably to single- and multi-pass TMPs, and extended to exogenous viral proteins. Parallel to its broad-spectrum inhibition of Sec61-mediated protein translocation, mycolactone rapidly induced cytosolic chaperones Hsp70/Hsp90. Moreover, it activated an atypical endoplasmic reticulum stress response, differing from conventional unfolded protein response by the down-regulation of Bip. In addition to refining our mechanistic understanding of Sec61 inhibition by mycolactone, our findings thus reveal that Sec61 blockade induces proteostatic stress in the cytosol and the endoplasmic reticulum.

Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity.

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitroIn vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression.IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

Varied Role of Ubiquitylation in Generating MHC Class I Peptide Ligands.

CD8+ T cell immunosurveillance is based on recognizing oligopeptides presented by MHC class I molecules. Despite decades of study, the importance of protein ubiquitylation to peptide generation remains uncertain. In this study, we examined the ability of MLN7243, a recently described ubiquitin-activating enzyme E1 inhibitor, to block overall cytosolic peptide generation and generation of specific peptides from vaccinia- and influenza A virus-encoded proteins. We show that MLN7243 rapidly inhibits ubiquitylation in a variety of cell lines and can profoundly reduce the generation of cytosolic peptides. Kinetic analysis of specific peptide generation reveals that ubiquitylation of defective ribosomal products is rate limiting in generating class I peptide complexes. More generally, our findings demonstrate that the requirement for ubiquitylation in MHC class I-restricted Ag processing varies with class I allomorph, cell type, source protein, and peptide context. Thus, ubiquitin-dependent and -independent pathways robustly contribute to MHC class I-based immunosurveillance.

Protein Translation Activity: A New Measure of Host Immune Cell Activation.

We describe the in vivo ribopuromycylation (RPM) method, which uses a puromycin-specific Ab to fluorescently label ribosome-bound puromycylated nascent chains, enabling measurement of translational activity via immunohistochemistry or flow cytometry. Tissue staining provides a unique view of virus-induced activation of adaptive, innate, and stromal immune cells. RPM flow precisely quantitates virus-induced activation of lymphocytes and innate immune cells, and it provides a unique measure of immune cell deactivation and quiescence. Using RPM we find that high endothelial cells in draining lymph nodes rapidly increase translation in the first day of vaccinia virus infection. We also find a population of constitutively activated splenic T cells in naive mice and further that most bone marrow T cells activate 3 d after vaccinia virus infection. Bone marrow T cell activation is nonspecific, IL-12-dependent, and induces innate memory T cell phenotypic markers. Thus, RPM measures translational activity to uniquely identify cell populations that participate in the immune response to pathogens, other foreign substances, and autoantigens.

Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance.

Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392-400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065-2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of K(b)-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished K(b)-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased K(b)-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685-4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More broadly, given the wide use of fluorescent proteins, their ubiquitous and abundant fragmentation must be considered when interpreting experiments using these extremely useful probes.