Genes involved in the limited spread of SARS-CoV-2 in the lower respiratory airways of hamsters may be associated with adaptive evolution.

Novel respiratory viruses can cause a pandemic and then evolve to coexist with humans. The Omicron strain of severe acute respiratory syndrome coronavirus 2 has spread worldwide since its emergence in late 2021, and its sub-lineages are now established in human society. Compared to previous strains, Omicron is markedly less invasive in the lungs and causes less severe disease. One reason for this is that humans are acquiring immunity through previous infection and vaccination, but the nature of the virus itself is also changing. Using our newly established low-volume inoculation system, which reflects natural human infection, we show that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain. Furthermore, by characterizing chimeric viruses with the Omicron gene in the Wuhan strain genetic background and vice versa, we found that viral genes downstream of ORF3a, but not the S gene, were responsible for the limited spread of the Omicron strain in the lower airways of the virus-infected hamsters. Moreover, molecular evolutionary analysis of SARS-CoV-2 revealed a positive selection of genes downstream of ORF3a (M and E genes). Our findings provide insight into the adaptive evolution of the virus in humans during the pandemic convergence phase.IMPORTANCEThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread worldwide since its emergence in late 2021, and its sub-lineages are established in human society. Compared to previous strains, the Omicron strain is less invasive in the lower respiratory tract, including the lungs, and causes less severe disease; however, the mechanistic basis for its restricted replication in the lower airways is poorly understood. In this study, using a newly established low-volume inoculation system that reflects natural human infection, we demonstrated that the Omicron strain spreads less efficiently into the lungs of hamsters compared with an earlier Wuhan strain and found that viral genes downstream of ORF3a are responsible for replication restriction in the lower respiratory tract of Omicron-infected hamsters. Furthermore, we detected a positive selection of genes downstream of ORF3a (especially the M and E genes) in SARS-CoV-2, suggesting that these genes may undergo adaptive changes in humans.

Rare CRHR2 and GRM8 variants identified as candidate factors associated with eating disorders in Japanese patients by whole exome sequencing.

Eating disorders (EDs) are a type of psychiatric disorder characterized by pathological eating and related behavior and considered to be highly heritable. The purpose of this study was to explore rare variants expected to display biological functions associated with the etiology of EDs. We performed whole exome sequencing (WES) of affected sib-pairs corresponding to disease subtype through their lifetime and their parents. From those results, rare single nucleotide variants (SNVs) concordant with sib-pairs were extracted and estimated to be most deleterious in the examined families. Two non-synonymous SNVs located on corticotropin-releasing hormone receptor 2 (CRHR2) and glutamate metabotropic receptor 8 (GRM8) were identified as candidate disease susceptibility factors. The SNV of CRHR2 was included within the cholesterol binding motif of the transmembrane helix region, while the SNV of GRM8 was found to contribute to hydrogen bonds for an α-helix structure. CRHR2 plays important roles in the serotoninergic system of dorsal raphe nuclei, which is involved with feeding and stress-coping behavior, whereas GRM8 modulates glutamatergic neurotransmission. Moreover, GRM8 modulates glutamatergic neurotransmission, and is also considered to have effects on dopaminergic and adrenergic neurotransmission. Thus, identification of rare and deleterious variants in this study is expected to increase understanding and treatment of affected individuals. Further investigation regarding the biological function of these variants may provide an opportunity to elucidate the pathogenesis of EDs.

Two-step evolution of HIV-1 budding system leading to pandemic in the human population.

The pandemic HIV-1, HIV-1 group M, emerged from a single spillover event of its ancestral lentivirus from a chimpanzee. During human-to-human spread worldwide, HIV-1 diversified into multiple subtypes. Here, our interdisciplinary investigation mainly sheds light on the evolutionary scenario of the viral budding system of HIV-1 subtype C (HIV-1C), a most successfully spread subtype. Of the two amino acid motifs for HIV-1 budding, the P(T/S)AP and YPxL motifs, HIV-1C loses the YPxL motif. Our data imply that HIV-1C might lose this motif to evade immune pressure. Additionally, the P(T/S)AP motif is duplicated dependently of the level of HIV-1 spread in the human population, and >20% of HIV-1C harbored the duplicated P(T/S)AP motif. We further show that the duplication of the P(T/S)AP motif is caused by the expansion of the CTG triplet repeat. Altogether, our results suggest that HIV-1 has experienced a two-step evolution of the viral budding process during human-to-human spread worldwide.

Multiple mutations of SARS-CoV-2 Omicron BA.2 variant orchestrate its virological characteristics.

IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.

SARS-CoV-2 HaploGraph: visualization of SARS-CoV-2 haplotype spread in Japan.

Since the early phase of the coronavirus disease 2019 (COVID-19) pandemic, a number of research institutes have been sequencing and sharing high-quality severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes to trace the route of infection in Japan. To provide insight into the spread of COVID-19, we developed a web platform named SARS-CoV-2 HaploGraph to visualize the emergence timing and geographical transmission of SARS-CoV-2 haplotypes. Using data from the GISAID EpiCoV database as of June 4, 2022, we created a haplotype naming system by determining the ancestral haplotype for each epidemic wave and showed prefecture- or region-specific haplotypes in each of four waves in Japan. The SARS-CoV-2 HaploGraph allows for interactive tracking of virus evolution and of geographical prevalence of haplotypes, and aids in developing effective public health control strategies during the global pandemic. The code and the data used for this study are publicly available at: https://github.com/ktym/covid19/.

The hepatic niche leads to aggressive natural killer cell leukemia proliferation through the transferrin-transferrin receptor 1 axis.

Aggressive natural killer cell leukemia (ANKL) is a rare lymphoid neoplasm frequently associated with Epstein-Barr virus, with a disastrously poor prognosis. Owing to the lack of samples from patients with ANKL and relevant murine models, comprehensive investigation of its pathogenesis including the tumor microenvironment (TME) has been hindered. Here we established 3 xenograft mice derived from patients with ANKL (PDXs), which enabled extensive analysis of tumor cells and their TME. ANKL cells primarily engrafted and proliferated in the hepatic sinusoid. Hepatic ANKL cells were characterized by an enriched Myc-pathway and proliferated faster than those in other organs. Interactome analyses and in vivo CRISPR-Cas9 analyses revealed transferrin (Tf)-transferrin receptor 1 (TfR1) axis as a potential molecular interaction between the liver and ANKL. ANKL cells were rather vulnerable to iron deprivation. PPMX-T003, a humanized anti-TfR1 monoclonal antibody, showed remarkable therapeutic efficacy in a preclinical setting using ANKL-PDXs. These findings indicate that the liver, a noncanonical hematopoietic organ in adults, serves as a principal niche for ANKL and the inhibition of the Tf-TfR1 axis is a promising therapeutic strategy for ANKL.

Current trends in RNA virus detection through metatranscriptome sequencing data.

With advances in sequencing technology, metatranscriptome sequencing from a variety of environmental and biological sources has revealed the existence of various previously unknown RNA viruses. This review presents recent major RNA virome studies sampled from invertebrate and vertebrate species as well as aquatic environments. In particular, we focus on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and related RNA virus identification through metatranscriptome sequencing analyses. Recently developed bioinformatics software and databases for RNA virus identification are introduced. A relationship between newly identified RNA viruses and endogenous viral elements in host genomes is also discussed.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

Functional molecular evolution of a GTP sensing kinase: PI5P4Kß.

Over 4 billion years of evolution, multiple mutations, including nucleotide substitutions, gene and genome duplications and recombination, have established de novo genes that translate into proteins with novel properties essential for high-order cellular functions. However, molecular processes through which a protein evolutionarily acquires a novel function are mostly speculative. Recently, we have provided evidence for a potential evolutionary mechanism underlying how, in mammalian cells, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) evolved into a GTP sensor from ATP-utilizing kinase. Mechanistically, PI5P4Kß has acquired the guanine efficient association (GEA) motif by mutating its nucleotide base recognition sequence, enabling the evolutionary transition from an ATP-dependent kinase to a distinct GTP/ATP dual kinase with its KM for GTP falling into physiological GTP concentrations-the genesis of GTP sensing activity. Importantly, the GTP sensing activity of PI5P4Kß is critical for the manifestation of cellular metabolism and tumourigenic activity in the multicellular organism. The combination of structural, biochemical and biophysical analyses used in our study provides a novel framework for analysing how a protein can evolutionarily acquire a novel activity, which potentially introduces a critical function to the cell.

Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

Genomic diversity of SARS-CoV-2 can be accelerated by mutations in the nsp14 gene.

Coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a proofreading exonuclease, nonstructural protein 14 (nsp14), that helps ensure replication competence at a low evolutionary rate compared with other RNA viruses. In the current pandemic, SARS-CoV-2 has accumulated diverse genomic mutations including in nsp14. Here, to clarify whether amino acid substitutions in nsp14 affect the genomic diversity and evolution of SARS-CoV-2, we searched for amino acid substitutions in nature that may interfere with nsp14 function. We found that viruses carrying a proline-to-leucine change at position 203 (P203L) have a high evolutionary rate and that a recombinant SARS-CoV-2 virus with the P203L mutation acquired more diverse genomic mutations than wild-type virus during its replication in hamsters. Our findings suggest that substitutions, such as P203L, in nsp14 may accelerate the genomic diversity of SARS-CoV-2, contributing to virus evolution during the pandemic.

Delivering mRNAs to mouse tissues using the SEND system.

mRNAs produced in a cell are almost always translated within the same cell. Some mRNAs are transported to other cells of the organism through processes involving membrane nanotubes or extracellular vesicles. A recent report describes a surprising new phenomenon of encapsulating mRNAs inside virus-like particles (VLPs) to deliver them to other cells in a process that was named SEND (Selective Endogenous eNcapsidation for cellular Delivery). Although the seminal work demonstrates the SEND process in cultured cells, it is unknown whether this phenomenon occurs in vivo . Here, we demonstrate the SEND process in living organisms using specially designed genetically engineered mouse models. Our proof of principle study lays a foundation for the SEND-VLP system to potentially be used as a gene therapy tool to deliver therapeutically important mRNAs to tissues.

Nanopore Sequencing Data Analysis of 16S rRNA Genes Using the GenomeSync-GSTK System.

With the development of nanopore sequencing technology, long reads of DNA sequences can now be determined rapidly from various samples. This protocol introduces the GenomeSync-GSTK system for bacterial species identification in a given sample using nanopore sequencing data of 16S rRNA genes as an example. GenomeSync is a collection of genome sequences designed to provide easy access to genomic data of the species as demanded. GSTK (genome search toolkit) is a set of scripts for managing local homology searches using genomes obtained from the GenomeSync database. Based on this protocol, nanopore sequencing data analyses of metagenomes and amplicons could be efficiently performed. We also noted reanalysis in conjunction with future developments in nanopore sequencing technology and the accumulation of genome sequencing data.

Potentially reduced fusogenicity of syncytin-2 in New World monkeys.

Syncytin-2 is a membrane fusion protein involved in placenta development that is derived from the endogenous retrovirus envelope gene acquired in the common ancestral lineage of New World and Old World monkeys (OWMs). It is known that syncytin-2 is conserved between apes and OWMs, suggesting its functional importance; however, syncytin-2 of common marmosets (Callithrix jacchus) exhibits lower fusogenic activity than those of humans and OWMs in human cell lines. To obtain insight into the functional diversity of syncytin-2 genes in primates, we examined the syncytin-2 gene in New World monkeys (NWMs). We experimentally evaluated the cell fusion ability of syncytin-2 in humans, C. jacchus, and tufted capuchins (Sapajus apella). We found that the cell fusion ability of S. apella was lower than that of human syncytin-2. Chimeric syncytin-2 constructs revealed that the amino acid differences in the surface unit of S. apella syncytin-2 were responsible for the weak cell fusion activity. In addition, genomic sequence analyses of syncytin-2 revealed that the open reading frames (ORFs) of syncytin-2 were highly conserved in seven apes and 22 OWMs; however, the syncytin-2 ORFs of three of 12 NWM species were truncated. Our results suggest that syncytin-2 in several NWMs may be of less importance than in OWMs and apes, and other syncytin-like genes may be required for placental development in various NWM species.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Monotreme-specific conserved putative proteins derived from retroviral reverse transcriptase.

Endogenous retroviruses (ERVs) have played an essential role in the evolution of mammals. ERV-derived genes are reported in the therians, many of which are involved in placental development; however, the contribution of the ERV-derived genes in monotremes, which are oviparous mammals, remains to be uncovered. Here, we conducted a comprehensive search for possible ERV-derived genes in platypus and echidna genomes and identified three reverse transcriptase-like genes named RTOM1, RTOM2, and RTOM3 clustered in the GRIP2 intron. Comparative genomic analyses revealed that RTOM1, RTOM2, and RTOM3 are strongly conserved and are under purifying selection between these species. These could be generated by tandem duplications before the divergence of platypus and echidna. All RTOM transcripts were specifically expressed in the testis, possibly suggesting their physiological importance. This is the first study reporting monotreme-specific de novo gene candidates derived from ERVs, which provides new insights into the unique evolution of monotremes.

Endogenous Retroviruses and Placental Evolution, Development, and Diversity.

The main roles of placentas include physical protection, nutrient and oxygen import, export of gasses and fetal waste products, and endocrinological regulation. In addition to physical protection of the fetus, the placentas must provide immune protection throughout gestation. These basic functions are well-conserved; however, placentas are undoubtedly recent evolving organs with structural and cellular diversities. These differences have been explained for the last two decades through co-opting genes and gene control elements derived from transposable elements, including endogenous retroviruses (ERVs). However, the differences in placental structures have not been explained or characterized. This manuscript addresses the sorting of ERVs and their integration into the mammalian genomes and provides new ways to explain why placental structures have diverged.

NeoRdRp: A Comprehensive Dataset for Identifying RNA-dependent RNA Polymerases of Various RNA Viruses from Metatranscriptomic Data.

RNA viruses are distributed throughout various environments, and most have recently been identified by metatranscriptome sequencing. However, due to the high nucleotide diversity of RNA viruses, it is still challenging to identify novel RNA viruses from metatranscriptome data. To overcome this issue, we created a dataset of RNA-dependent RNA polymerase (RdRp) domains that are essential for all RNA viruses belonging to Orthornavirae. Genes with RdRp domains from various RNA viruses were clustered based on amino acid sequence similarities. A multiple sequence alignment was generated for each cluster, and a hidden Markov model (HMM) profile was created when the number of sequences was greater than three. We further refined 426 HMM profiles by detecting RefSeq RNA virus sequences and subsequently combined the hit sequences with the RdRp domains. As a result, 1,182 HMM profiles were generated from 12,502 RdRp domain sequences, and the dataset was named NeoRdRp. The majority of NeoRdRp HMM profiles successfully detected RdRp domains, specifically in the UniProt dataset. Furthermore, we compared the NeoRdRp dataset with two previously reported methods for RNA virus detection using metatranscriptome sequencing data. Our methods successfully identified the majority of RNA viruses in the datasets; however, some RNA viruses were not detected, similar to the other two methods. NeoRdRp may be repeatedly improved by the addition of new RdRp sequences and is applicable as a system for detecting various RNA viruses from diverse metatranscriptome data.

Efficient compression of SARS-CoV-2 genome data using Nucleotide Archival Format.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome data are essential for epidemiology, vaccine development, and tracking emerging variants. Millions of SARS-CoV-2 genomes have been sequenced during the pandemic. However, downloading SARS-CoV-2 genomes from databases is slow and unreliable, largely due to suboptimal choice of compression method. We evaluated the available compressors and found that Nucleotide Archival Format (NAF) would provide a drastic improvement compared with current methods. For Global Initiative on Sharing Avian Flu Data's (GISAID) pre-compressed datasets, NAF would increase efficiency 52.2 times for gzip-compressed data and 3.7 times for xz-compressed data. For DNA DataBank of Japan (DDBJ), NAF would improve throughput 40 times for gzip-compressed data. For GenBank and European Nucleotide Archive (ENA), NAF would accelerate data distribution by a factor of 29.3 times compared with uncompressed FASTA. This article provides a tutorial for installing and using NAF. Offering a NAF download option in sequence databases would provide a significant saving of time, bandwidth, and disk space and accelerate biological and medical research worldwide.

Convergent evolution of antiviral machinery derived from endogenous retrovirus truncated envelope genes in multiple species.

Host genetic resistance to viral infection controls the pathogenicity and epidemic dynamics of infectious diseases. Refrex-1 is a restriction factor against feline leukemia virus subgroup D (FeLV-D) and an endogenous retrovirus (ERV) in domestic cats (ERV-DC). Refrex-1 is encoded by a subset of ERV-DC loci with truncated envelope genes and secreted from cells as a soluble protein. Here, we identified the copper transporter CTR1 as the entry receptor for FeLV-D and genotype I ERV-DCs. We also identified CTR1 as a receptor for primate ERVs from crab-eating macaques and rhesus macaques, which were found in a search of intact envelope genes capable of forming infectious viruses. Refrex-1 counteracted infection by FeLV-D and ERV-DCs via competition for the entry receptor CTR1; the antiviral effects extended to primate ERVs with CTR1-dependent entry. Furthermore, truncated ERV envelope genes found in chimpanzee, bonobo, gorilla, crab-eating macaque, and rhesus macaque genomes could also block infection by feline and primate retroviruses. Genetic analyses showed that these ERV envelope genes were acquired in a species- or genus-specific manner during host evolution. These results indicated that soluble envelope proteins could suppress retroviral infection across species boundaries, suggesting that they function to control retroviral spread. Our findings revealed that several mammalian species acquired antiviral machinery from various ancient retroviruses, leading to convergent evolution for host defense.