ells expressing ACE2 are potential targets of SARS-CoV-2 infection 1,2. Studies based on single-cell RNA sequencing (scRNA-seq) of lung cells have identified type II pneumocytes, ciliated cells and transient secretory cells as the main types of ACE2-expressing cell 3,4. Furthermore, ACE2 was proposed to be an ISG, on the basis of its inducible expression in cells treated with interferons (IFNs) or infected by viruses that induce IFN responses, such as influenza 4,5. These findings implied that the induction of ACE2 expression in IFN-high conditions could result in an amplified risk of SARS-CoV-2 infection 4,5. Concerns could also be raised about possible ACE2-inducing side effects of IFN-based treatments proposed for COVID-19 (refs. 6-9). ACE2 plays multiple roles in normal physiological conditions and as part of the host tissue-protective machinery in damaging conditions, including viral infections. As a terminal carboxypeptidase, ACE2 cleaves a single carboxy-terminal residue from peptide hormones such as angiotensin II and des-Arg9-bradykinin. ACE and ACE2 belong to the renin-angiotensin-aldosterone system, which regulates blood pressure and fluid-electrolyte balance; dysfunction of this system contributes to comorbidities in COVID-19 (refs. 10,11). des-Arg9-bradykinin is generated from bradykinin and belongs to the kallikrein-kinin system, which is critical in regulating vascular leakage and pulmonary edema, early signs of severe COVID-19 (refs. 12,13). High plasma angiotensin II levels were found to be responsible for coronavirus-associated acute respiratory distress syndrome (ARDS), lung damage and high mortality in mouse models 14,15 and as a predictor of lethality in avian influenza in humans 16,17. In the same conditions, ACE2, which decreases the levels of angiotensin II, was identified as a protective factor. The hijacking of the normal host tissue-protective machinery guarded by ACE2 was suggested as a mechanism through which SARS-CoV-2 could infect more cells 4,5. Thus, it is critically important to identify factors affecting ACE2 expression in normal physiological processes and during viral infections and associated pathologies, such as in COVID-19. Herein, aiming to explore the IFN-inducible expression of ACE2 and its role in SARS-CoV-2 infection, we identified a novel, truncated isoform of ACE2, which we designate as dACE2. We then showed that dACE2, but not ACE2, is induced in various human cell types by IFNs and viruses; this information is important to consider for future therapeutic strategies and understanding COVID-19 susceptibility and outcomes. Results dACE2 is a novel inducible isoform of ACE2. To address the extent to which IFNs induce the expression of ACE2 in human cells, we used our existing RNA-seq dataset (NCBI Sequence Read Archive (SRA): PRJNA512015) of a breast cancer cell line T47D infected with Sendai virus (SeV), known to be a strong inducer of IFNs and ISGs 18-20. IFNs were not expressed in T47D cells at baseline, but SeV strongly induced expression of IFNB1, a type I IFN, an...

Mitochondrial (mt) function depends critically on optimal interactions between components encoded by mt and nuclear DNAs. mitochondrial DNA (mtDNA) inheritance (SMI) is thought to have evolved in animal species to maintain mito-nuclear complementarity by preventing the spread of selfish mt elements thus typically rendering mtDNA heteroplasmy evolutionarily ephemeral. Here, we show that mtDNA intraorganismal heteroplasmy can have deterministic underpinnings and persist for hundreds of millions of years. We demonstrate that the only exception to SMI in the animal kingdom, that is, the doubly uniparental mtDNA inheritance system in bivalves, with its three-way interactions among egg mt-, sperm mt- and nucleus-encoded gene products, is tightly associated with the maintenance of separate male and female sexes (dioecy) in freshwater mussels. Specifically, this mother-through-daughter and father-through-son mtDNA inheritance system, containing highly differentiated mt genomes, is found in all dioecious freshwater mussel species. Conversely, all hermaphroditic species lack the paternally transmitted mtDNA (=possess SMI) and have heterogeneous macromutations in the recently discovered, novel protein-coding gene (F-orf) in their maternally transmitted mt genomes. Using immunoelectron microscopy, we have localized the F-open reading frame (ORF) protein, likely involved in specifying separate sexes, in mitochondria and in the nucleus. Our results support the hypothesis that proteins coded by the highly divergent maternally and paternally transmitted mt genomes could be directly involved in sex determination in freshwater mussels. Concomitantly, our study demonstrates novel features for animal mt genomes: the existence of additional, lineage-specific, mtDNA-encoded proteins with functional significance and the involvement of mtDNA-encoded proteins in extra-mt functions. Our results open new avenues for the identification, characterization, and functional analyses of ORFs in the intergenic regions, previously defined as "noncoding," found in a large proportion of animal mt genomes.

Doubly uniparental inheritance (DUI) of mitochondrial DNA in marine mussels (Mytiloida), freshwater mussels (Unionoida), and marine clams (Veneroida) is the only known exception to the general rule of strict maternal transmission of mtDNA in animals. DUI is characterized by the presence of genderassociated mitochondrial DNA lineages that are inherited through males (male-transmitted or M types) or females (female-transmitted or F types), respectively. This unusual system constitutes an excellent model for studying basic aspects of mitochondrial DNA inheritance and the evolution of mtDNA genomes in general.Here we compare published mitochondrial genomes of unionoid bivalve species with DUI, with an emphasis on characterizing unassigned regions, to identify regions of the F and M mtDNA genomes that could (i) play a role in replication or transcription of the mtDNA molecule and/or (ii) determine whether a genome will be transmitted via the female or the male gamete. Our results reveal the presence of one Fspecific and one M-specific open reading frames (ORFs), and we hypothesize that they play a role in the transmission and/or gender-specific adaptive functions of the M and F mtDNA genomes in unionoid bivalves. Three major unassigned regions shared among all F and M unionoid genomes have also been identified, and our results indicate that (i) two of them are potential heavy-strand control regions (O H ) for regulating replication and/or transcription and that (ii) multiple and potentially bidirectional light-strand origins of replication (O L ) are present in unionoid F and M mitochondrial genomes. We propose that unassigned regions are the most promising candidate sequences in which to find regulatory and/or genderspecific sequences that could determine whether a mitochondrial genome will be maternally or paternally transmitted.

With the first reports on coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the scientific community working in the field of type III IFNs (IFN-λ) realized that this class of IFNs could play an important role in this and other emerging viral infections. In this Viewpoint, we present our opinion on the benefits and potential limitations of using IFN-λ to prevent, limit, and treat these dangerous viral infections.

Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (pS) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown that pS is generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level of pS values, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-anddeath evolution at the DNA level.

CD8؉ T lymphocytes (CD8-TL) select viral escape variants in both human immunodeficiency virus and simian immunodeficiency virus (SIV) infections. The frequency of CD8-TL viral escape as well as the contribution of escape to overall virus diversification has not been assessed. We quantified CD8-TL selection in SIV infections by sequencing viral genomes from 35 SIVmac239-infected animals at the time of euthanasia. Here we show that positive selection for sequences encoding 46 known CD8-TL epitopes is comparable to the positive selection observed for the variable loops of env. We also found that >60% of viral variation outside of the viral envelope occurs within recognized CD8-TL epitopes. Therefore, we conclude that CD8-TL selection is the dominant cause of SIV diversification outside of the envelope. CD8ϩ -T-lymphocyte (CD8-TL) responses against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) select viral escape variants that are poorly recognized by the immune system. The number of CD8-TL responses that select escape variants and the contribution of CD8-TL selection to viral diversification are unknown. CD8-TL responses against HIV and SIV are broadly directed, as they target epitopes in all viral proteins (11,26). If many of these responses select for viral variants, CD8-TL escape may be principally responsible for sequence changes that accumulate within infected individuals, an outcome recently hypothesized by Overbaugh and Bangham (40).Until recently, the molecular and immunologic tools that are necessary to analyze CD8-TL selection at the genome level have been unavailable. Most studies of CD8-TL escape have focused on single epitopes or small groups of epitopes clustered in single SIV and HIV genes (4,7,16,20,22,43,44). While they were instrumental in demonstrating that CD8-TL selection is a cardinal feature of SIV and HIV infections, these studies could not comprehensively evaluate the frequency of CD8-TL escape throughout the viral genome.High-throughput DNA sequencing technologies now enable the sequencing of entire SIV genomes from infected animals (38). Additionally, 46 CD8-TL epitopes bound by the highfrequency Indian rhesus macaque major histocompatibility complex class I (MHC class I) alleles Mamu-A‫10ء‬ (3), -A‫20ء‬ (28a), and -B‫71ء‬ (34) are now known. By sequencing complete viral genomes spanning these 46 CD8-TL epitopes, we can now directly measure the frequency of CD8-TL selection.With this study, we found that CD8-TL escape is common, with sequences encoding 23 of 46 epitopes accumulating variations that are consistent with escape. Within a single animal, Ͼ64% of the nonsynonymous viral variants outside of the envelope were located within recognized CD8-TL epitopes, suggesting that CD8-TL escape is a primary selector of withinhost diversity. MATERIALS AND METHODSViruses and animals. The 35 animals used for the bulk of the analyses were infected with SIVmac239 between 1998 and 2001. These animals were challenged with SIV in trials of vaccine efficacy and pathogenesis...

BackgroundIt is generally accepted that CD8+ T cell responses play an important role in control of immunodeficiency virus replication. The association of HLA-B27 and -B57 with control of viremia supports this conclusion. However, specific correlates of viral control in individuals expressing these alleles have been difficult to define. We recently reported that transient in vivo CD8+ cell depletion in simian immunodeficiency virus (SIV)-infected elite controller (EC) macaques resulted in a brief period of viral recrudescence. SIV replication was rapidly controlled with the reappearance of CD8+ cells, implicating that these cells actively suppress viral replication in ECs.Methods and FindingsHere we show that three ECs in that study made at least seven robust CD8+ T cell responses directed against novel epitopes in Vif, Rev, and Nef restricted by the MHC class I molecule Mamu-B*08. Two of these Mamu-B*08-positive animals subsequently lost control of SIV replication. Their breakthrough virus harbored substitutions in multiple Mamu-B*08-restricted epitopes. Indeed, we found evidence for selection pressure mediated by Mamu-B*08-restricted CD8+ T cells in all of the newly identified epitopes in a cohort of chronically infected macaques.ConclusionsTogether, our data suggest that Mamu-B*08-restricted CD8+ T cell responses effectively control replication of pathogenic SIVmac239. All seven regions encoding Mamu-B*08-restricted CD8+ T cell epitopes also exhibit amino acid replacements typically seen only in the presence of Mamu-B*08, suggesting that the variation we observe is indeed selected by CD8+ T cell responses. SIVmac239 infection of Indian rhesus macaques expressing Mamu-B*08 may therefore provide an animal model for understanding CD8+ T cell-mediated control of HIV replication in humans.

The association between particular major histocompatibility complex class I (MHC-I) alleles and control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication implies that certain CD8؉ T-lymphocyte (CD8-TL) responses are better able than others to control viral replication in vivo. However, possession of favorable alleles does not guarantee improved prognosis or viral control. In rhesus macaques, the MHC-I allele Mamu-B*17 is correlated with reduced viremia and is overrepresented in macaques that control SIVmac239, termed elite controllers (ECs). However, there is so far no mechanistic explanation for this phenomenon. Here we show that the chronic-phase Mamu-B*17-restricted repertoire is focused primarily against just five epitopes-VifHW8, EnvFW9, NefIW9, NefMW9, and env ARF cRW9-in both ECs and progressors. Interestingly, Mamu-B*17-restricted CD8-TL do not target epitopes in Gag. CD8-TL escape variation occurred in all targeted Mamu-B*17-restricted epitopes. However, recognition of escape variant peptides was commonly observed in both ECs and progressors. Wild-type sequences in the VifHW8 epitope tended to be conserved in ECs, but there was no evidence that this enhances viral control. In fact, no consistent differences were detected between ECs and progressors in any measured parameter. Our data suggest that the narrowly focused Mamu-B*17-restricted repertoire suppresses virus replication and drives viral evolution. It is, however, insufficient in the majority of individuals that express the "protective" Mamu-B*17 molecule. Most importantly, our data indicate that the important differences between Mamu-B*17-positive ECs and progressors are not readily discernible using standard assays to measure immune responses.