DEGENERATED LEMMA (DEL) Is a New Allele of OsRDR6 That Regulates Lemma Development and Affects Rice Grain Yield

The lemma and palea are floral organ structures unique to grasses, and their development affects grain size. However, information on the molecular mechanism of lemma development is limited. In this study, we investigated a rice spikelet mutant, degenerated lemma (del), which developed florets with a slightly degenerated or rod-like lemma. The results indicate that the mutation of the DEL gene interfered with lemma development. In addition, del also showed a significant reduction in grain length and width, seed setting rate, and 1000-grain weight, which led to a reduction in yield. The results indicate that the mutation of the DEL gene further affects rice grain yield. Map-based cloning shows a single-nucleotide substitution from T to A within Os01g0527600/DEL, causing an amino acid mutation of Leu-34 to His-34 in the del mutant. DEL is an allele of OsRDR6, encoding the RNA-dependent RNA polymerase 6, and is highly expressed in the spikelet. RT-qPCR results show that the expression of some floral organ identity genes was changed, which indicates that the DEL gene regulates lemma development by modulating the expression of these genes. The present results suggest that DEL plays an important role in lemma development and rice grain yield.

Use of the Codon Table to Quantify the Evolutionary Role of Random Mutations

The various biases affecting RNA mutations during evolution is the subject of intense research, leaving the extent of the role of random mutations undefined. To remedy this lacuna, using the codon table, the number of codons representing each amino acid was correlated with the amino acid frequencies in different branches of the evolutionary tree. The correlations were seen to increase as evolution progressed. Furthermore, the number of RNA mutations that resulted in a given amino acid mutation were found to be correlated with several widely used amino acid similarity tables (used in sequence alignments). These correlations were seen to increase when the observed codon usage was factored in.

Structural and Functional Analysis of Disease-Linked p97 ATPase Mutant Complexes

IBMPFD/ALS is a genetic disorder caused by a single amino acid mutation on the p97 ATPase, promoting ATPase activity and cofactor dysregulation. The disease mechanism underlying p97 ATPase malfunction remains unclear. To understand how the mutation alters the ATPase regulation, we assembled a full-length p97R155H with its p47 cofactor and first visualized their structures using single-particle cryo-EM. More than one-third of the population was the dodecameric form. Nucleotide presence dissociates the dodecamer into two hexamers for its highly elevated function. The N-domains of the p97R155H mutant all show up configurations in ADP- or ATPγS-bound states. Our functional and structural analyses showed that the p47 binding is likely to impact the p97R155H ATPase activities via changing the conformations of arginine fingers. These functional and structural analyses underline the ATPase dysregulation with the miscommunication between the functional modules of the p97R155H.

MLKL D144K mutation activates the necroptotic activity of the N-terminal MLKL domain

Mixed-lineage kinase domain-like protein (MLKL) is an essential effector protein of necroptotic cell death. The four-helix bundle domain (4HB) presented by the first 125 amino acids of the N terminal domain is sufficient for its necroptotic activity. However, it has been proposed that the subsequent helix H6 of the brace region has a regulatory effect on its necroptotic activity. How the brace region restrains the necroptotic activity of the N-terminal domain of MLKL is currently unknown. Here, we demonstrate the importance of helix H6 to constrain the necroptotic activity. A single amino acid mutation D144K was able to activate the necroptotic activity of the N terminal domain of MLKL by removing helix H6 away from 4HB domain. This enabled protein oligomerization and membrane translocation. Moreover, a biophysical comparison revealed that helix H6 becomes partially unstructured due to D144K mutation, leading to a lower overall thermodynamic stability of the mutant protein compared to the wild type.

Monitoring of SARS-CoV-2 B.1.1.7 variant early-phase spreading in South-Moravian Region in the Czech Republic and evaluation of its pathogenicity

SARS-CoV-2 emerged in Wuhan, China, in December 2019. Starting in January 2020, over a period of several months, the initial virus (Wuhan-Hu-1/2019; Wu et al. 2020) diverged in a descendant strain carrying D614G amino acid mutation in spike protein. By summer 2020 this novel coronavirus (nCoV) became the most dominant form of the virus circulating worldwide and raised serious international concern. Currently (April 2021), there are 3598 subsequent PANGO branched lineages recognized that carry numerous mutations. To date, the most emerging lineages of SARS-CoV-2 worldwide include B.1.1.7 lineage with a frequency of 48% followed by several dozens of others with frequencies 7.5% or less, such as B.1.351, B.1.1.28, B.1.2, B.1.1.519, P.1, R.1, etc. (www.nextrain.org, Centers for Disease Control and Prevention; CDC 2020 www.cdc.gov). In this study, we monitored the spreading of B.1.1.7 lineage from the early phase of its appearance until it became predominant in the South-Moravian region of the Czech Republic. We measured significantly associated clinical marker (Ct; cycle threshold) correlating with viral load in B.1.1.7 lineage. Interestingly, we found that the spreading of B.1.1.7 strain was associated with a shift in patients average age, as compared to the former predominant lineage. Finally, we calculated the impact of the B.1.1.7 lineage on hospitalization and case fatality of the patients on the intensive care unit in the central South-Moravian faculty hospital.

Development of a Genetically Stable Live Attenuated Influenza Vaccine Strain using an Engineered High-Fidelity Viral Polymerase

RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence post their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two times lower mutation rates and decreased misincorporation rates, as compared to the wild-type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live-attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus. Importance The LAIV currently in use is prescribed for actively immunizing individuals aged 2-49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1, which displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.

Differential Influence of Age on the Relationship between Genetic Mismatch and A(H1N1)pdm09 Vaccine Effectiveness

Assessment of influenza vaccine effectiveness (VE) and identification of relevant influencing factors are the current priorities for optimizing vaccines to reduce the impacts of influenza. To date, how the difference between epidemic strains and vaccine strains at genetic scale affects age-specific vaccine performance remains ambiguous. This study investigated the association between genetic mismatch on hemagglutinin and neuraminidase genes and A(H1N1)pdm09 VE in different age groups with a novel computational approach. We found significant linear relationships between VE and genetic mismatch in children, young adults, and middle-aged adults. In the children’s group, each 3-key amino acid mutation was associated with an average of 10% decrease in vaccine effectiveness in a given epidemic season, and genetic mismatch exerted no influence on VE for the elderly group. We demonstrated that present vaccines were most effective for children, while protection for the elderly was reduced and indifferent to vaccine component updates. Modeling such relationships is practical to inform timely evaluation of VE in different groups of populations during mass vaccination and may inform age-specific vaccination regimens.