Genomic Analysis of a Novel Active Prophage of Hafnia Paralvei

Relatively little is known about the prophages in Enterobacterales bacteria except Escherichia coli. A novel phage, Hafnia phage yong2, was induced from Hafnia paralvei by mitomycin C. The phage has an elliptical head of approximately 45 × 38 nm, a long noncontractile tail of approximately 157 × 4 nm. The complete genome of Hafnia phage yong2 is a 39,546 bp double-stranded DNA with a G+C content of 49.9%, containing 59 opening reading frames (ORFs), and having at least one fixed terminus (GGGGCAGCGACA) with R=107>100. In phylogenetic analysis, Hafnia phage yong2 clustered with 4 predicted Hafnia prophages and 1 predicted Enterobacteriaceae prophage. The prophages cluster and Drexlerviridae family together formed two distinct subclades nested within a clade, suggesting the existence of a novel class of prophages with conserved sequences and unique evolutionary status not yet studied before in Hafniaceae and Enterobacteriaceae bacteria.

Importance of Viral Late Domains in Budding and Release of Enveloped RNA Viruses

Late assembly (L) domains are conserved sequences that are necessary for the late steps of viral replication, acting like cellular adaptors to engage the ESCRT membrane fission machinery that promote virion release. These short sequences, whose mutation or deletion produce the accumulation of immature virions at the plasma membrane, were firstly identified within retroviral Gag precursors, and in a further step, also in structural proteins of many other enveloped RNA viruses including arenaviruses, filoviruses, rhabdoviruses, reoviruses, and paramyxoviruses. Three classes of L domains have been identified thus far (PT/SAP, YPXnL/LXXLF, and PPxY), even if it has recently been suggested that other motifs could act as L domains. Here, we summarize the current state of knowledge of the different types of L domains and their cellular partners in the budding events of RNA viruses, with a particular focus on retroviruses.

Conserved Sequence Analysis of Influenza A Virus HA Segment and Its Application in Rapid Typing

The high mutation rate of the influenza A virus hemagglutinin segment poses great challenges to its long-term effective testing and subtyping. Our conserved sequence searching method achieves high-specificity conserved sequences on H1–H9 subtypes. In addition, PCR experiments show that primers based on conserved sequences can be used in influenza A virus HA subtyping. Conserved sequence-based primers are expected to be long-term, effective subtyping tools for influenza A virus HA.

Elucidation of Novel cis-Regulatory Elements and Promoter Structures Involved in Iron Excess Response Mechanisms in Rice Using a Bioinformatics Approach

Iron (Fe) excess is a major constraint on crop production in flooded acidic soils, particularly in rice cultivation. Under Fe excess, plants activate a complex mechanism and network regulating Fe exclusion by roots and isolation in various tissues. In rice, the transcription factors and cis-regulatory elements (CREs) that regulate Fe excess response mechanisms remain largely elusive. We previously reported comprehensive microarray analyses of several rice tissues in response to various levels of Fe excess stress. In this study, we further explored novel CREs and promoter structures in rice using bioinformatics approaches with this microarray data. We first performed network analyses to predict Fe excess-related CREs through the categorization of the gene expression patterns of Fe excess-responsive transcriptional regulons, and found four major expression clusters: Fe storage type, Fe chelator type, Fe uptake type, and WRKY and other co-expression type. Next, we explored CREs within these four clusters of gene expression types using a machine-learning method called microarray-associated motif analyzer (MAMA), which we previously established. Through a comprehensive bioinformatics approach, we identified a total of 560 CRE candidates extracted by MAMA analyses and 42 important conserved sequences of CREs directly related to the Fe excess response in various rice tissues. We explored several novel cis-elements as candidate Fe excess CREs including GCWGCWGC, CGACACGC, and Myb binding-like motifs. Based on the presence or absence of candidate CREs using MAMA and known PLACE CREs, we found that the Boruta-XGBoost model explained expression patterns with high accuracy of about 83%. Enriched sequences of both novel MAMA CREs and known PLACE CREs led to high accuracy expression patterns. We also found new roles of known CREs in the Fe excess response, including the DCEp2 motif, IDEF1-, Zinc Finger-, WRKY-, Myb-, AP2/ERF-, MADS- box-, bZIP and bHLH- binding sequence-containing motifs among Fe excess-responsive genes. In addition, we built a molecular model and promoter structures regulating Fe excess-responsive genes based on new finding CREs. Together, our findings about Fe excess-related CREs and conserved sequences will provide a comprehensive resource for discovery of genes and transcription factors involved in Fe excess-responsive pathways, clarification of the Fe excess response mechanism in rice, and future application of the promoter sequences to produce genotypes tolerant of Fe excess.

Impact of Glycosylation on SARS-CoV-2 Infection and Broadly Protective Vaccine Design

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 167 million confirmed cases and over 3 million deaths so far. This global pandemic has led to great efforts directed toward the study of this virus and its infection mechanism as well as development of effective means to control this devastating infectious disease. Like many other viral surface proteins, the trimeric SARS-CoV-2 spike (S) protein is heavily glycosylated with 22 N- and 2 O-glycosites per monomer which are likely to influence S protein folding and evade host immune response. More than one million S protein sequences with over 1,000 sites of mutation in its 1,273 amino acids have been reported to the GISAID database, including the highly transmissible variant strains found in the UK and South Africa. This high frequency of transmission and mutation is a major challenge in the development of broadly protective vaccines to control the pandemic. We have studied the impact of glycosylation on receptor-ligand interaction through evaluation of ACE2 and S protein expressed in different cell lines. Of different S protein glycoforms, the one expressed from lung epithelial cells, the primary cells for infection, has more complex-type glycans and higher binding avidity to the receptor as compared with the S protein from HEK293T cells which have more high-mannose or hybrid-type glycoforms. We also found that most of the S protein glycosites are highly conserved and the glycosites at positions 801 and 1194 are essential for viral entry. In addition, the RBD of S1 and the HR regions of S2 contain most of highly conserved sequences, and removal of each glycosite on pseudotyped SARS-CoV-2 virus for evaluation of the impact on structure and function provides insights into the design of broadly protective vaccines. In an effort to develop such universal vaccines, we found that mice immunized with monoglycosylated S protein (Smg) elicited better antibody responses capable of neutralizing not only the wild type but also the variants from the UK and South Africa than those with the fully-glycosylated S protein (Sfg), and strikingly, Smg vaccination provides better survival for hACE2 transgenic mice when challenged with lethal dose of SARS-CoV-2. Moreover, using single B cell technology, we isolated a monoclonal antibody from Smg immunized mice which was also able to neutralize the wild type and variants, suggesting that removal of unnecessary glycans from S protein to better expose the highly conserved sequences is an effective approach to developing broadly protective vaccines against SARS-CoV-2 and variants.

Targeting conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus

Gene drives are genetic systems designed to efficiently spread a modification through a population. They have been designed almost exclusively in eukaryotic species, and especially in insects. We recently developed a CRISPR-based gene drive system in herpesviruses that relies on similar mechanisms and could efficiently spread into a population of wildtype viruses. A common consequence of gene drives in insects is the appearance and selection of drive-resistant sequences that are no longer recognized by CRISPR-Cas9. Here, we analyze in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that, after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications. Importance The use of defective viruses that interfere with the replication of their infectious parent after co-infecting the same cells – a therapeutic strategy known as viral interference – has recently generated a lot of interest. The CRISPR-based system that we recently reported in herpesviruses represents a novel interfering strategy that causes the conversion of wildtype viruses into new recombinant viruses and drives the native viral population to extinction. In this report, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we show that, after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show however that targeting evolutionary conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.

Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control

DNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated. Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control

ABSTRACTDNA sequences that are exactly conserved over long evolutionary time scales have been observed in a variety of taxa. Such sequences are likely under strong functional constraint and they have been useful in the field of comparative genomics for identifying genome regions with regulatory function. A potential new application for these ultra-conserved elements has emerged in the development of gene drives to control mosquito populations. Many gene drives work by recognising and inserting at a specific target sequence in the genome, often imposing a reproductive load as a consequence. They can therefore select for target sequence variants that provide resistance to the drive. Focusing on highly conserved, highly constrained sequences lowers the probability that variant, gene drive-resistant alleles can be tolerated.Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.

Large scale multiple sequence alignment of intraspecies samples: conserved sequence analysis of influenza A virus HA segment and its application in rapid typing

Background The high mutation rate of influenza A virus hemagglutinin segment brings great challenges to its long-term effective testing and subtyping. Method We analyzed the conserved sequences of hemagglutinin subtype H1-H9 by breadth first, and designed primers for HA subtyping based on conserved sequences. Results Our conserved sequence searching method get high specificity conserved sequences on H1-H9 subtypes respectively. And PCR experiments show that primers based on conserved sequences can be used in influenza A virus HA subtyping. Conclusions Conserved sequences based primers are expected to be the long-term effective influenza A virus HA subtyping tools.

Identification of enhancers that drive the spatially restricted expression of Vsx1 and Rx in the outer proliferation center of the developing Drosophila optic lobe

Combinatorial spatial and temporal patterning of stem cells is a powerful mechanism for the generation of neural diversity in insect and vertebrate nervous systems. In the developing Drosophila medulla, the neural stem cells of the outer proliferation center (OPC) are spatially patterned by the mutually exclusive expression of three homeobox transcription factors: Vsx1 in the center of the OPC crescent (cOPC), Optix in the main arms (mOPC), and Rx in the posterior tips (pOPC). These spatial factors act together with a temporal cascade of transcription factors in OPC neuroblasts to specify the greater than 80 medulla cell types. Here, we identify the enhancers that are sufficient to drive the spatially restricted expression of the Vsx1 and Rx genes in the OPC. We show that removal of the cOPC enhancer in the Muddled inversion mutant leads to the loss of Vsx1 expression in the cOPC. Analysis of the evolutionarily conserved sequences within these enhancers suggests that direct repression by Optix may restrict the expression of Vsx1 and Rx to the cOPC and pOPC, respectively.