Publications by authors named "Susan R Ross"

60 Publications

Mouse APOBEC3 Restriction of Retroviruses.

Viruses 2020 10 27;12(11). Epub 2020 Oct 27.

Department of Microbiology and Immunology, University of Illinois at Chicago College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.

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 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.
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http://dx.doi.org/10.3390/v12111217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692085PMC
October 2020

APOBEC3A catalyzes mutation and drives carcinogenesis in vivo.

J Exp Med 2020 12;217(12)

Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN.

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.
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http://dx.doi.org/10.1084/jem.20200261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953736PMC
December 2020

Insights into Sensing of Murine Retroviruses.

Viruses 2020 07 31;12(8). Epub 2020 Jul 31.

Department of Microbiology and Immunology, University of Illinois at Chicago College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612, USA.

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.
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http://dx.doi.org/10.3390/v12080836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472155PMC
July 2020

CACNA1S haploinsufficiency confers resistance to New World arenavirus infection.

Proc Natl Acad Sci U S A 2020 08 27;117(32):19497-19506. Epub 2020 Jul 27.

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612

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.
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http://dx.doi.org/10.1073/pnas.1920551117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431027PMC
August 2020

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

J Virol 2020 08 31;94(18). Epub 2020 Aug 31.

Department of Microbiology and Immunology, University of Illinois Chicago College of Medicine, Chicago, Illinois, USA

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 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 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. 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 , 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.
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http://dx.doi.org/10.1128/JVI.00032-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459557PMC
August 2020

A recessive Trim2 mutation causes an axonal neuropathy in mice.

Neurobiol Dis 2020 07 20;140:104845. Epub 2020 Mar 20.

Department of Neurology, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

We analyzed Trim2 mice, generated by CRISPR-Cas9, which have a recessive, null mutation of Trim2. Trim2 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 (Trim2), which has a partial deletion in the RING domain that is needed for ubiquitin ligase activity. Both the Trim2and the Trim2 alleles encode mutant TRIM2 proteins with reduced ubiquitination activity. In sum, Trim2 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.
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http://dx.doi.org/10.1016/j.nbd.2020.104845DOI Listing
July 2020

The KT Jeang Retrovirology Prize 2020: call for nominations.

Retrovirology 2020 02 10;17(1). Epub 2020 Feb 10.

Division of Infection & Immunity, The Rayne Institute, University College London, 5 University Street, London, WC1E 6BT, UK.

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http://dx.doi.org/10.1186/s12977-019-0509-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008541PMC
February 2020

Human APOBEC3G Prevents Emergence of Infectious Endogenous Retrovirus in Mice.

J Virol 2019 10 30;93(20). Epub 2019 Sep 30.

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

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 and evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G), can potently restrict retrotransposition; yet, 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 mice. Mice encode one copy of in their genome. ERV reactivation in mice was comparable in the presence or absence of In contrast, expression of a human transgene abrogated emergence of infectious ERV in the background. No ERV RNA was detected in the plasma of hA3G mice, and infectious ERV virions could not be amplified through coculture with permissive cells. These data reveal that hA3G can potently restrict active ERV and suggest that expansion of the locus in primates may have helped to provide for the continued restraint of ERV in the human genome. 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 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 and demonstrates how transgenic mice can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.
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http://dx.doi.org/10.1128/JVI.00728-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798113PMC
October 2019

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

PLoS Biol 2019 02 6;17(2):e3000137. Epub 2019 Feb 6.

Department of Microbiology and Immunology, UIC College of Medicine, Chicago, Illinois, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pbio.3000137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380604PMC
February 2019

DDX41 Recognizes RNA/DNA Retroviral Reverse Transcripts and Is Critical for Control of Murine Leukemia Virus Infection.

mBio 2018 06 5;9(3). Epub 2018 Jun 5.

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

Host recognition of viral nucleic acids generated during infection leads to the activation of innate immune responses essential for early control of virus. Retrovirus reverse transcription creates numerous potential ligands for cytosolic host sensors that recognize foreign nucleic acids, including single-stranded RNA (ssRNA), RNA/DNA hybrids, and double-stranded DNA (dsDNA). We and others recently showed that the sensors cyclic GMP-AMP synthase (cGAS), DEAD-box helicase 41 (DDX41), and members of the Aim2-like receptor (ALR) family participate in the recognition of retroviral reverse transcripts. However, why multiple sensors might be required and their relative importance in control of retroviral infection are not known. Here, we show that DDX41 primarily senses the DNA/RNA hybrid generated at the first step of reverse transcription, while cGAS recognizes dsDNA generated at the next step. We also show that both DDX41 and cGAS are needed for the antiretroviral innate immune response to murine leukemia virus (MLV) and HIV in primary mouse macrophages and dendritic cells (DCs). Using mice with cell type-specific knockout of the gene, we show that DDX41 sensing in DCs but not macrophages was critical for controlling MLV infection. This suggests that DCs are essential targets for infection, as well as for initiating the antiviral response. Our work demonstrates that the innate immune response to retrovirus infection depends on multiple host nucleic acid sensors that recognize different reverse transcription intermediates. Viruses are detected by many different host sensors of nucleic acid, which in turn trigger innate immune responses, such as type I interferon (IFN) production, required to control infection. We show here that at least two sensors are needed to initiate a highly effective innate immune response to retroviruses-DDX41, which preferentially senses the RNA/DNA hybrid generated at the first step of retrovirus replication, and cGAS, which recognizes double-stranded DNA generated at the second step. Importantly, we demonstrate using mice lacking DDX41 or cGAS that both sensors are needed for the full antiviral response needed to control MLV infection. These findings underscore the need for multiple host factors to counteract retroviral infection.
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http://dx.doi.org/10.1128/mBio.00923-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989071PMC
June 2018

The best laid plans of mice and women.

Authors:
Susan R Ross

PLoS Pathog 2018 04 12;14(4):e1006873. Epub 2018 Apr 12.

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.

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http://dx.doi.org/10.1371/journal.ppat.1006873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897020PMC
April 2018

Deaminase-Dead Mouse APOBEC3 Is an Retroviral Restriction Factor.

J Virol 2018 06 14;92(11). Epub 2018 May 14.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

The apolipoprotein B editing complex 3 (APOBEC3) proteins are potent retroviral restriction factors that are under strong positive selection, both in terms of gene copy number and sequence diversity. A common feature of all the members of the APOBEC3 family is the presence of one or two cytidine deamination domains, essential for cytidine deamination of retroviral reverse transcripts as well as packaging into virions. Several studies have indicated that human and mouse APOBEC3 proteins restrict retrovirus infection via cytidine deaminase (CD)-dependent and -independent means. To understand the relative contribution of CD-independent restriction , we created strains of transgenic mice on an APOBEC3 knockout background that express a deaminase-dead mouse APOBEC3 due to point mutations in both CD domains (E73Q/E253Q). Here, we show that the CD-dead APOBEC3 can restrict murine retroviruses Moreover, unlike the wild-type protein, the mutant APOBEC3 is not packaged into virions but acts only as a cell-intrinsic restriction factor that blocks reverse transcription by incoming viruses. Finally, we show that wild-type and CD-dead mouse APOBEC3 can bind to murine leukemia virus (MLV) reverse transcriptase. Our findings suggest that the mouse APOBEC3 cytidine deaminase activity is not required for retrovirus restriction. APOBEC3 proteins are important host cellular restriction factors essential for restricting retrovirus infection by causing mutations in the virus genome and by blocking reverse transcription. While both methods of restriction function , little is known about their role during infection. By developing transgenic mice with mutations in the cytidine deamination domains needed for enzymatic activity and interaction with viral RNA, we show that APOBEC3 proteins can still restrict infection by interacting with reverse transcriptase and blocking its activity. These studies demonstrate that APOBEC3 proteins have evolved multiple means for blocking retrovirus infection and that all of these means function .
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http://dx.doi.org/10.1128/JVI.00168-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952145PMC
June 2018

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA.

mBio 2017 07 5;8(4). Epub 2017 Jul 5.

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, USA

Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs) that stimulate the induction of interferons (IFNs) and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING). Absent in melanoma 2 (AIM2)-like receptors (ALRs) have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA. Autoimmune diseases like Aicardi-Goutières syndrome and lupus erythematosus arise when cells of the immune system become activated and attack host cells and tissues. We found that DNA generated by endogenous retroviruses and retroelements in inbred mice and mouse cells is recognized by several host proteins found in macrophages that are members of the ALR family and that these proteins both suppress and activate the pathways leading to the generation of cytokines and IFNs. We also show that there is great genetic diversity between different inbred mouse strains in the ALR genes, which might contribute to differential susceptibility to autoimmunity. Understanding how immune cells become activated is important to the control of disease.
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http://dx.doi.org/10.1128/mBio.00944-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573678PMC
July 2017

New World Arenavirus Biology.

Annu Rev Virol 2017 09 23;4(1):141-158. Epub 2017 Jun 23.

Department of Microbiology and Immunology, University of Illinois College of Medicine at Chicago, Chicago, Illinois 60612; email: ,

Hemorrhagic fevers caused by viruses were identified in the late 1950s in South America. These viruses have existed in their hosts, the New World rodents, for millions of years. Their emergence as infectious agents in humans coincided with changes in the environment and farming practices that caused explosions in their host rodent populations. Zoonosis into humans likely occurs because the pathogenic New World arenaviruses use human transferrin receptor 1 to enter cells. The mortality rate after infection with these viruses is high, but the mechanism by which disease is induced is still not clear. Possibilities include direct effects of cellular infection or the induction of high levels of cytokines by infected sentinel cells of the immune system, leading to endothelia and thrombocyte dysfunction and neurological disease. Here we provide a review of the ecology and molecular and cellular biology of New World arenaviruses, as well as a discussion of the current animal models of infection. The development of animal models, coupled with an improved understanding of the infection pathway and host response, should lead to the discovery of new drugs for treating infections.
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http://dx.doi.org/10.1146/annurev-virology-101416-042001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478856PMC
September 2017

The effect of HIV-1 Vif polymorphisms on A3G anti-viral activity in an in vivo mouse model.

Retrovirology 2016 06 30;13(1):45. Epub 2016 Jun 30.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.
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http://dx.doi.org/10.1186/s12977-016-0280-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929759PMC
June 2016

In Vivo Examination of Mouse APOBEC3- and Human APOBEC3A- and APOBEC3G-Mediated Restriction of Parvovirus and Herpesvirus Infection in Mouse Models.

J Virol 2016 09 12;90(17):8005-12. Epub 2016 Aug 12.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Unlabelled: APOBEC3 knockout and human APOBEC3A and -3G transgenic mice were tested for their ability to be infected by the herpesviruses herpes simplex virus 1 and murine herpesvirus 68 and the parvovirus minute virus of mice (MVM). Knockout, APOBEC3A and APOBEC3G transgenic, and wild-type mice were equally infected by the herpesviruses, while APOBEC3A but not mouse APOBEC3 conferred resistance to MVM. No viruses showed evidence of cytidine deamination by mouse or human APOBEC3s. These data suggest that in vitro studies implicating APOBEC3 proteins in virus resistance may not reflect their role in vivo

Importance: It is well established that APOBEC3 proteins in different species are a critical component of the host antiretroviral defense. Whether these proteins also function to inhibit other viruses is not clear. There have been a number of in vitro studies suggesting that different APOBEC3 proteins restrict herpesviruses and parvoviruses, among others, but whether they also work in vivo has not been demonstrated. Our studies looking at the role of mouse and human APOBEC3 proteins in transgenic and knockout mouse models of viral infection suggest that these restriction factors are not broadly antiviral and demonstrate the importance of testing their activity in vivo.
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http://dx.doi.org/10.1128/JVI.00973-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988146PMC
September 2016

Lessons Learned from Mouse Mammary Tumor Virus in Animal Models.

ILAR J 2016 ;57(1):12-23

Jaquelin P. Dudley, PhD, is a professor in the Department of Molecular Biosciences, Center for Infectious Disease and Institute for Cellular and Molecular Biology at the University of Texas at Austin. Tatyana V. Golovkina, PhD, is a professor in the Department of Microbiology at the University of Chicago in Chicago, Illinois. Susan R. Ross, PhD, is a professor in the Department of Microbiology in the Perelman School of Medicine of the University of Pennsylvania in Philadelphia, Pennsylvania.

Mouse mammary tumor virus (MMTV), which was discovered as a milk-transmitted, infectious, cancer-inducing agent in the 1930s, has been used as an animal model for the study of retroviral infection and transmission, antiviral immune responses, and breast cancer and lymphoma biology. The main target cells for MMTV infection in vivo are cells of the immune system and mammary epithelial cells. Although the host mounts an immune response to the virus, MMTV has evolved multiple means of evading this response. MMTV causes mammary tumors when the provirus integrates into the mammary epithelial and lymphoid cell genome during viral replication and thereby activates cellular oncogene expression. Thus, tumor induction is a by-product of the infection cycle. A number of important oncogenes have been discovered by carrying out MMTV integration site analysis, some of which may play a role in human breast cancer.
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http://dx.doi.org/10.1093/ilar/ilv044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007637PMC
January 2017

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor.

J Virol 2016 May 14;90(9):4494-4510. Epub 2016 Apr 14.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

Unlabelled: Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens.

Importance: The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.
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http://dx.doi.org/10.1128/JVI.00103-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836360PMC
May 2016

APOBEC3 Proteins in Viral Immunity.

J Immunol 2015 Nov;195(10):4565-70

Department of Microbiology, Abramson Cancer Center, Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6142

Apolipoprotein B editing complex 3 family members are cytidine deaminases that play important roles in intrinsic responses to infection by retroviruses and have been implicated in the control of other viruses, such as parvoviruses, herpesviruses, papillomaviruses, hepatitis B virus, and retrotransposons. Although their direct effect on modification of viral DNA has been clearly demonstrated, whether they play additional roles in innate and adaptive immunity to viruses is less clear. We review the data regarding the various steps in the innate and adaptive immune response to virus infection in which apolipoprotein B editing complex 3 proteins have been implicated.
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http://dx.doi.org/10.4049/jimmunol.1501504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4638160PMC
November 2015

Nucleic acid recognition orchestrates the anti-viral response to retroviruses.

Cell Host Microbe 2015 Apr 26;17(4):478-88. Epub 2015 Mar 26.

Department of Microbiology, Institute for Immunology and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Intrinsic restriction factors and viral nucleic acid sensors are important for the anti-viral response. Here, we show how upstream sensing of retroviral reverse transcripts integrates with the downstream effector APOBEC3, an IFN-induced cytidine deaminase that introduces lethal mutations during retroviral reverse transcription. Using a murine leukemia virus (MLV) variant with an unstable capsid that induces a strong IFNβ antiviral response, we identify three sensors, IFI203, DDX41, and cGAS, required for MLV nucleic acid recognition. These sensors then signal using the adaptor STING, leading to increased production of IFNβ and other targets downstream of the transcription factor IRF3. Using knockout and mutant mice, we show that APOBEC3 limits the levels of reverse transcripts that trigger cytosolic sensing, and that nucleic acid sensing in vivo increases expression of IFN-regulated restriction factors like APOBEC3 that in turn reduce viral load. These studies underscore the importance of the multiple layers of protection afforded by host factors.
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http://dx.doi.org/10.1016/j.chom.2015.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393365PMC
April 2015

Different modes of retrovirus restriction by human APOBEC3A and APOBEC3G in vivo.

PLoS Pathog 2014 May 22;10(5):e1004145. Epub 2014 May 22.

Department of Microbiology, Institute for Immunology and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.
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http://dx.doi.org/10.1371/journal.ppat.1004145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4031197PMC
May 2014

Toll-like receptor 2-mediated innate immune responses against Junín virus in mice lead to antiviral adaptive immune responses during systemic infection and do not affect viral replication in the brain.

J Virol 2014 Jul 23;88(14):7703-14. Epub 2014 Apr 23.

Department of Microbiology, Institute for Immunology, and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Successful adaptive immunity to virus infection often depends on the initial innate response. Previously, we demonstrated that Junín virus, the etiological agent responsible for Argentine hemorrhagic fever (AHF), activates an early innate immune response via an interaction between the viral glycoprotein and Toll-like receptor 2 (TLR2). Here we show that TLR2/6 but not TLR1/2 heterodimers sense Junín virus glycoprotein and induce a cytokine response, which in turn upregulates the expression of the RNA helicases RIG-I and MDA5. NF-κB and Erk1/2 were important in the cytokine response, since both proteins were phosphorylated as a result of the interaction of virus with TLR2, and treatment with an Erk1/2-specific inhibitor blocked cytokine production. We show that the Junín virus glycoprotein activates cytokine production in a human macrophage cell line as well. Moreover, we show that TLR2-mediated immune response plays a role in viral clearance because wild-type mice cleared Candid 1 (JUNV C1), the vaccine strain of Junín virus, more rapidly than did TLR2 knockout mice. This clearance correlated with the generation of Junín virus-specific CD8(+) T cells. However, infected wild-type and TLR2 knockout mice developed TLR2-independent blocking antibody responses with similar kinetics. We also show that microglia and astrocytes but not neurons are susceptible to infection with JUNV C1. Although JUNV C1 infection of the brain also triggered a TLR2-dependent cytokine response, virus levels were equivalent in wild-type and TLR2 knockout mice. Importance: Junín virus is transmitted by rodents native to Argentina and is associated with both systemic disease and, in some patients, neurological symptoms. Humans become infected when they inhale aerosolized Junín virus. AHF has a 15 to 30% mortality rate, and patients who clear the infection develop a strong antibody response to Junín virus. Here we investigated what factors determine the immune response to Junín virus. We show that a strong initial innate immune response to JUNV C1 determines how quickly mice can clear systemic infection and that this depended on the cellular immune response. In contrast, induction of an innate immune response in the brain had no effect on virus infection levels. These findings may explain how the initial immune response to Junín virus infection could determine different outcomes in humans.
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http://dx.doi.org/10.1128/JVI.00050-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097793PMC
July 2014

siRNA screen for genes that affect Junín virus entry uncovers voltage-gated calcium channels as a therapeutic target.

Sci Transl Med 2013 Sep;5(204):204ra131

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

New World hemorrhagic fever arenavirus infection results in 15 to 30% mortality in humans. We performed a high-throughput small interfering RNA screen with Junín virus glycoprotein-pseudotyped viruses to find potential host therapeutic targets. Voltage-gated calcium channel (VGCC) subunits, for which there are Food and Drug Administration (FDA)-approved drugs, were identified in the screen. Knockdown of VGCC subunits or treatment with channel blockers diminished Junín virus-cell fusion and entry into cells and thereby decreased infection. Gabapentin, an FDA-approved drug used to treat neuropathic pain that targets the α₂δ₂ subunit, inhibited infection of mice by the Candid 1 vaccine strain of the virus. These findings demonstrate that VGCCs play a role in virus infection and have the potential to lead to therapeutic intervention of New World arenavirus infection.
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http://dx.doi.org/10.1126/scitranslmed.3006827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104171PMC
September 2013

Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex.

Proc Natl Acad Sci U S A 2013 May 13;110(22):9078-83. Epub 2013 May 13.

Department of Microbiology, Abramson Cancer Center and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19103, USA.

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag-containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo--namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.
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http://dx.doi.org/10.1073/pnas.1217399110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670389PMC
May 2013

APOBEC3 inhibition of mouse mammary tumor virus infection: the role of cytidine deamination versus inhibition of reverse transcription.

J Virol 2013 May 28;87(9):4808-17. Epub 2013 Feb 28.

Department of Microbiology, Institute for Immunology and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3(BALB) and APOBEC3(BL/6)) and knockout mice to determine whether cytidine deamination was important for APOBEC3's anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo. Next, using an in vitro deamination assay, we determined that virion-packaged APOBEC3 retains its deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo. This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.
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http://dx.doi.org/10.1128/JVI.00112-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624289PMC
May 2013

Mouse mammary tumor virus suppresses apoptosis of mammary epithelial cells through ITAM-mediated signaling.

J Virol 2012 Dec 26;86(24):13232-40. Epub 2012 Sep 26.

Department of Microbiology, Perelman School of Medicine, University of Pennyslvania, Philadelphia, Pennsylvania, USA.

Many receptors in hematopoietic cells use a common signaling pathway that relies on a highly conserved immunoreceptor tyrosine-based activation motif (ITAM), which signals through Src family tyrosine kinases. ITAM-bearing proteins are also found in many oncogenic viruses, including the mouse mammary tumor virus (MMTV) envelope (Env). We previously showed that MMTV Env expression transformed normal mammary epithelial cells and that Src kinases were important mediators in this transformation. To study how ITAM signaling affects mammary cell transformation, we utilized mammary cell lines expressing two different ITAM-containing proteins, one encoding a MMTV provirus and the other a B cell receptor fusion protein. ITAM-expressing cells were resistant to both serum starvation- and chemotherapeutic drug-induced apoptosis, whereas cells transduced with these molecules bearing ITAM mutations were indistinguishable from untransduced cells in their sensitivity to these treatments. We also found that Src kinase was activated in the MMTV-expressing cells and that MMTV-induced apoptosis resistance was completely restored by the Src inhibitor PP2. In vivo, MMTV infection delayed involution-induced apoptosis in the mouse mammary gland. Our results show that MMTV suppresses apoptosis through ITAM-mediated Src tyrosine kinase signaling. These studies could lead to the development of effective treatment of nonhematopoietic cell cancers in which ITAM-mediated signaling plays a role.
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http://dx.doi.org/10.1128/JVI.02029-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503107PMC
December 2012

Novel common integration sites targeted by mouse mammary tumor virus insertion in mammary tumors have oncogenic activity.

PLoS One 2011 7;6(11):e27425. Epub 2011 Nov 7.

Department of Microbiology/Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Non-acute transforming retroviruses like mouse mammary tumor virus (MMTV) cause cancer, at least in part, through integration near cellular genes involved in growth control, thereby de-regulating their expression. It is well-established that MMTV commonly integrates near and activates expression of members of the Wnt and Fgf pathways in mammary tumors. However, there are a significant number of tumors for which the proviral integration sites have not been identified. Here, we used high through-put screening to identify common integration sites (CISs) in MMTV-induced tumors from C3H/HeN and BALB/c mice. As expected, members of both the Wnt and Fgf families were identified in this screen. In addition, a number of novel CISs were found, including Tcf7l2, Antxr1/Tem8, and Arhgap18. We show here that expression of these three putative oncogenes in normal murine mammary gland cells altered their growth kinetics and caused their morphological transformation when grown in three dimensional cultures. Additionally, expression of Tcf7l2 and Antxr1/Tem8 sensitized cells to exogenous WNT ligand. As Tcf7l2, Antxr1/Tem8, and Arhgap18 have been associated with human breast and other cancers, these data demonstrate that MMTV-induced insertional mutation remains an important means for identifying genes involved in breast cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210173PMC
May 2012

Junin virus infects mouse cells and induces innate immune responses.

J Virol 2011 Nov 31;85(21):11058-68. Epub 2011 Aug 31.

Department of Microbiology and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Junín virus is the causative agent for Argentine hemorrhagic fever, and its natural host is the New World rodent Calomys musculinus. The virus is transmitted to humans by aerosolization, and it is believed that many of the clinical symptoms are caused by cytokines produced by sentinel cells of the immune system. Here we used the Junín virus vaccine strain Candid 1 to determine whether mouse cells could be used to study virus entry and antiviral innate immune responses. We show that Candid 1 can infect and propagate in different mouse-derived cell lines through a low-pH-dependent, transferrin receptor 1-independent mechanism, suggesting that there is a second entry receptor. In addition, Candid 1 induced expression of the antiviral cytokines tumor necrosis factor alpha and beta interferon in macrophages, and this induction was independent of viral replication. Using Candid 1, as well as virus-like particles bearing the viral glycoprotein, to infect different primary cells and established macrophage cell lines with deletions in the Toll-like receptor (TLR) pathway, we show that TLR2 is a cellular sensor of both the Parodi and Candid 1 viral glycoproteins. Because Junín virus is highly lethal in humans, the use of an experimentally tractable model system, such as the mouse, could provide a better understanding of the antiviral innate cellular responses to Junín virus and the role of these responses in pathogenesis.
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http://dx.doi.org/10.1128/JVI.05304-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194972PMC
November 2011