Entry inhibitors as arenavirus antivirals.

Arenaviruses belonging to the Arenaviridae family, genus mammarenavirus, are enveloped, single-stranded RNA viruses primarily found in rodent species, that cause severe hemorrhagic fever in humans. With high mortality rates and limited treatment options, the search for effective antivirals is imperative. Current treatments, notably ribavirin and other nucleoside inhibitors, are only partially effective and have significant side effects. The high lethality and lack of treatment, coupled with the absence of vaccines for all but Junín virus, has led to the classification of these viruses as Category A pathogens by the Centers for Disease Control (CDC). This review focuses on entry inhibitors as potential therapeutics against mammarenaviruses, which include both New World and Old World arenaviruses. Various entry inhibition strategies, including small molecule inhibitors and neutralizing antibodies, have been explored through high throughput screening, genome-wide studies, and drug repurposing. Notable progress has been made in identifying molecules that target receptor binding, internalization, or fusion steps. Despite promising preclinical results, the translation of entry inhibitors to approved human therapeutics has faced challenges. Many have only been tested in in vitro or animal models, and a number of candidates showed efficacy only against specific arenaviruses, limiting their broader applicability. The widespread existence of arenaviruses in various rodent species and their potential for their zoonotic transmission also underscores the need for rapid development and deployment of successful pan-arenavirus therapeutics. The diverse pool of candidate molecules in the pipeline provides hope for the eventual discovery of a broadly effective arenavirus antiviral.

Murine leukemia virus infection of non-dividing dendritic cells is dependent on nucleoporins.

Retroviral reverse transcription starts within the capsid and uncoating and reverse transcription are mutually dependent. There is still debate regarding the timing and cellular location of HIV's uncoating and reverse transcription and whether it occurs solely in the cytoplasm, nucleus or both. HIV can infect non-dividing cells because there is active transport of the preintegration complex (PIC) across the nuclear membrane, but Murine Leukemia Virus (MLV) is thought to depend on cell division for replication and whether MLV uncoating and reverse transcription is solely cytoplasmic has not been studied. Here, we used NIH3T3 and primary mouse dendritic cells to determine where the different stages of reverse transcription occur and whether cell division is needed for nuclear entry. Our data strongly suggest that in both NIH3T3 cells and dendritic cells (DCs), the initial step of reverse transcription occurs in the cytoplasm. However, we detected MLV RNA/DNA hybrid intermediates in the nucleus of dividing NIH3T3 cells and non-dividing DCs, suggesting that reverse transcription can continue after nuclear entry. We also confirmed that the MLV PIC requires cell division to enter the nucleus of NIH3T3 cells. In contrast, we show that MLV can infect non-dividing primary DCs, although integration of MLV DNA in DCs still required the viral p12 protein. Knockdown of several nuclear pore proteins dramatically reduced the appearance of integrated MLV DNA in DCs but not NIH3T3 cells. Additionally, MLV capsid associated with the nuclear pore proteins NUP358 and NUP62 during infection. These findings suggest that simple retroviruses, like the complex retrovirus HIV, gain nuclear entry by traversing the nuclear pore complex in non-mitotic cells.

DNA damage primes hematopoietic stem cells for direct megakaryopoiesis.

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all cells of the hematopoietic system. 1 Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs. 2 The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment specifically in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage associated with uracil misincorporation in vivo and in vitro . Consistent with this notion, thymidine attenuated DNA damage, rescued HSC maintenance and reduced the generation of CD41 + MK-committed HSCs in vitro . Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhanced in vitro maintenance of HSCs. We conclude that a DNA damage response drives direct megakaryopoiesis and that replication stress-induced direct megakaryopoiesis, at least in part caused by uracil misincorporation, is a barrier to HSC maintenance in vitro . DNA damage-induced direct megakaryopoiesis may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells.

IFI207, a young and fast-evolving antiviral factor, stabilizes STING.

Mammalian ALR proteins bind nucleic acids and initiate production of type I interferons or inflammasome assembly, thereby contributing to host innate immunity. ALR s are encoded at a single genetic locus. In mice, the Alr locus is highly polymorphic at the sequence and copy number level. We suggest that one rapidly evolving member of the Alr family, Ifi207 , was introduced to the Mus genome by a recent recombination event. Ifi207 has a large, distinctive repeat region that differs in sequence and length in different Mus strains. We show that IFI207 plays a key role in the STING-mediated response to cGAMP, DNA, and MLV, and that IFI207 controls MLV infection in vivo. Uniquely, IFI207 acts by stabilizing STING protein via its repeat region. Our studies suggest that under the pressure of host-pathogen coevolution, in a dynamic locus such as the Alr , recombination between gene family members creates new genes with novel and essential functions that play diverse roles in biological processes.

The Cytidine Deaminase APOBEC3G Contributes to Cancer Mutagenesis and Clonal Evolution in Bladder Cancer.

Mutagenic processes leave distinct signatures in cancer genomes. The mutational signatures attributed to APOBEC3 cytidine deaminases are pervasive in human cancers. However, data linking individual APOBEC3 proteins to cancer mutagenesis in vivo are limited. Here, we showed that transgenic expression of human APOBEC3G promotes mutagenesis, genomic instability, and kataegis, leading to shorter survival in a murine bladder cancer model. Acting as mutagenic fuel, APOBEC3G increased the clonal diversity of bladder cancer, driving divergent cancer evolution. Characterization of the single-base substitution signature induced by APOBEC3G in vivo established the induction of a mutational signature distinct from those caused by APOBEC3A and APOBEC3B. Analysis of thousands of human cancers revealed the contribution of APOBEC3G to the mutational profiles of multiple cancer types, including bladder cancer. Overall, this study dissects the mutagenic impact of APOBEC3G on the bladder cancer genome, identifying that it contributes to genomic instability, tumor mutational burden, copy-number loss events, and clonal diversity. SIGNIFICANCE: APOBEC3G plays a role in cancer mutagenesis and clonal heterogeneity, which can potentially inform future therapeutic efforts that restrict tumor evolution. See related commentary by Caswell and Swanton, p. 487.

DDX41 is required for cGAS-STING activation against DNA virus infection.

Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.

DDX41 is needed for pre- and postnatal hematopoietic stem cell differentiation in mice.

DDX41 is a tumor suppressor frequently mutated in human myeloid neoplasms, but whether it affects hematopoiesis is unknown. Using a knockout mouse, we demonstrate that DDX41 is required for mouse hematopoietic stem and progenitor cell (HSPC) survival and differentiation, particularly of myeloid lineage cells. Transplantation of Ddx41 knockout fetal liver and adult bone marrow (BM) cells was unable to rescue mice from lethal irradiation, and knockout stem cells were also defective in colony formation assays. RNA-seq analysis of Lin-/cKit+/Sca1+Ddx41 knockout cells from fetal liver demonstrated that the expression of many genes associated with hematopoietic differentiation were altered. Furthermore, differential splicing of genes involved in key biological processes was observed. Our data reveal a critical role for DDX41 in HSPC differentiation and myeloid progenitor development, likely through regulating gene expression programs and splicing.

Repair of APOBEC3G-Mutated Retroviral DNA In Vivo Is Facilitated by the Host Enzyme Uracil DNA Glycosylase 2.

Apolipoprotein B mRNA editing enzyme catalytic subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative-strand DNA during reverse transcription, leading to G-to-A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an Ung and mouse Apobec3 knockout background (UNG-/-APO-/-), and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared with A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA. IMPORTANCE While APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection-first, in unintegrated nuclear viral reverse-transcribed DNA, resulting in its degradation; and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.

Signal-regulatory protein alpha is an anti-viral entry factor targeting viruses using endocytic pathways.

Signal-regulatory protein alpha (SIRPA) is a well-known inhibitor of phagocytosis when it complexes with CD47 expressed on target cells. Here we show that SIRPA decreased in vitro infection by a number of pathogenic viruses, including New World and Old World arenaviruses, Zika virus, vesicular stomatitis virus and pseudoviruses bearing the Machupo virus, Ebola virus and SARS-CoV-2 glycoproteins, but not HSV-1, MLV or mNoV. Moreover, mice with targeted mutation of the Sirpa gene that renders it non-functional were more susceptible to infection with the New World arenaviruses Junín virus vaccine strain Candid 1 and Tacaribe virus, but not MLV or mNoV. All SIRPA-inhibited viruses have in common the requirement for trafficking to a low pH endosomal compartment. This was clearly demonstrated with SARS-CoV-2 pseudovirus, which was only inhibited by SIRPA in cells in which it required trafficking to the endosome. Similar to its role in phagocytosis inhibition, SIRPA decreased virus internalization but not binding to cell surface receptors. We also found that increasing SIRPA levels via treatment with IL-4 led to even greater anti-viral activity. These data suggest that enhancing SIRPA's activity could be a target for anti-viral therapies.

APOBEC3A drives deaminase domain-independent chromosomal instability to promote pancreatic cancer metastasis.

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.

Mouse APOBEC3 Restriction of Retroviruses.

Apolipoprotein B mRNA editing enzyme, catalytic peptide 3 (APOBEC3) proteins are critical host proteins that counteract and prevent the replication of retroviruses. Unlike the genome of humans and other species, the mouse genome encodes a single Apobec3 gene, which has undergone positive selection, as reflected by the allelic variants found in different inbred mouse strains. This positive selection was likely due to infection by various mouse retroviruses, which have persisted in their hosts for millions of years. While mouse retroviruses are inhibited by APOBEC3, they nonetheless still remain infectious, likely due to the actions of different viral proteins that counteract this host factor. The study of viruses in their natural hosts provides important insight into their co-evolution.

APOBEC3A catalyzes mutation and drives carcinogenesis in vivo.

The APOBEC3 family of antiviral DNA cytosine deaminases is implicated as the second largest source of mutation in cancer. This mutational process may be a causal driver or inconsequential passenger to the overall tumor phenotype. We show that human APOBEC3A expression in murine colon and liver tissues increases tumorigenesis. All other APOBEC3 family members, including APOBEC3B, fail to promote liver tumor formation. Tumor DNA sequences from APOBEC3A-expressing animals display hallmark APOBEC signature mutations in TCA/T motifs. Bioinformatic comparisons of the observed APOBEC3A mutation signature in murine tumors, previously reported APOBEC3A and APOBEC3B mutation signatures in yeast, and reanalyzed APOBEC mutation signatures in human tumor datasets support cause-and-effect relationships for APOBEC3A-catalyzed deamination and mutagenesis in driving multiple human cancers.

Insights into Sensing of Murine Retroviruses.

Retroviruses are major causes of disease in animals and human. Better understanding of the initial host immune response to these viruses could provide insight into how to limit infection. Mouse retroviruses that are endemic in their hosts provide an important genetic tool to dissect the different arms of the innate immune system that recognize retroviruses as foreign. Here, we review what is known about the major branches of the innate immune system that respond to mouse retrovirus infection, Toll-like receptors and nucleic acid sensors, and discuss the importance of these responses in activating adaptive immunity and controlling infection.

Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

CACNA1S haploinsufficiency confers resistance to New World arenavirus infection.

Understanding the genetics of susceptibility to infectious agents is of great importance to our ability to combat disease. Here, we show that voltage-gated calcium channels (VGCCs) are critical for cellular binding and entry of the New World arenaviruses Junín and Tacaribe virus, suggesting that zoonosis via these receptors could occur. Moreover, we demonstrate that α1s haploinsufficiency renders cells and mice more resistant to infection by these viruses. In addition to being more resistant to infection, haploinsufficient cells and mice required a lower dosage of VGCC antagonists to block infection. These studies underscore the importance of genetic variation in susceptibility to both viruses and pharmaceutics.

A recessive Trim2 mutation causes an axonal neuropathy in mice.

We analyzed Trim2A/A mice, generated by CRISPR-Cas9, which have a recessive, null mutation of Trim2. Trim2A/A mice develop ataxia that is associated with a severe loss of cerebellar Purkinje cells and a peripheral neuropathy. Myelinated axons in the CNS, including those in the deep cerebellar nuclei, have focal enlargements that contain mitochondria and neurofilaments. In the PNS, there is a loss of myelinated axons, particularly in the most distal nerves. The pathologically affected neuronal populations - primary sensory and motor neurons as well as cerebellar Purkinje cells - express TRIM2, suggesting that loss of TRIM2 in these neurons results in cell autonomous effects on their axons. In contrast, these pathological findings were not found in a second strain of Trim2 mutant mice (Trim2C/C), which has a partial deletion in the RING domain that is needed for ubiquitin ligase activity. Both the Trim2Aand the Trim2C alleles encode mutant TRIM2 proteins with reduced ubiquitination activity. In sum, Trim2A/A mice are a genetically authentic animal model of a recessive axonal neuropathy of humans, apparently for a function that does not depend on the ubiquitin ligase activity.

Human APOBEC3G Prevents Emergence of Infectious Endogenous Retrovirus in Mice.

Endogenous retroviruses (ERV) are found throughout vertebrate genomes, and failure to silence their activation can have deleterious consequences on the host. Mutation and subsequent disruption of ERV loci is therefore an indispensable component of the cell-intrinsic defenses that maintain the integrity of the host genome. Abundant in vitro and in silico evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G), can potently restrict retrotransposition; yet, in vivo data demonstrating such activity is lacking, since no replication-competent human ERV have been identified. In mice deficient for Toll-like receptor 7 (TLR7), transcribed ERV loci can recombine and generate infectious ERV. In this study, we show that ectopic expression of hA3G can prevent the emergence of replication-competent, infectious ERV in Tlr7-/- mice. Mice encode one copy of Apobec3 in their genome. ERV reactivation in Tlr7-/- mice was comparable in the presence or absence of Apobec3 In contrast, expression of a human APOBEC3G transgene abrogated emergence of infectious ERV in the Tlr7-/- background. No ERV RNA was detected in the plasma of hA3G+Apobec3-/-Tlr7-/- mice, and infectious ERV virions could not be amplified through coculture with permissive cells. These data reveal that hA3G can potently restrict active ERV in vivo and suggest that expansion of the APOBEC3 locus in primates may have helped to provide for the continued restraint of ERV in the human genome.IMPORTANCE Although APOBEC3 proteins are known to be important antiviral restriction factors in both mice and humans, their roles in the restriction of endogenous retroviruses (ERV) have been limited to in vitro studies. Here, we report that human APOBEC3G expressed as a transgene in mice prevents the emergence of infectious ERV from endogenous loci. This study reveals that APOBEC3G can powerfully restrict active retrotransposons in vivo and demonstrates how transgenic mice can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.

TRIM2, a novel member of the antiviral family, limits New World arenavirus entry.

Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot-Marie-Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.