Elucidating the Antiviral Mechanism of Different MARCH Factors.

The membrane-associated RING-CH (MARCH) proteins belong to a family of E3 ubiquitin ligases, whose main function is to remove transmembrane proteins from the plasma membrane. Recent work has shown that the human MARCH1, 2, and 8 are antiretroviral factors that target the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins by reducing their incorporation in the budding virions. Nevertheless, the dearth of information regarding the antiviral mechanism of this family of proteins necessitates further examination. In this study, using both the human MARCH proteins and their mouse homologues, we provide a comprehensive analysis of the antiretroviral mechanism of this family of proteins. Moreover, we show that human MARCH proteins restrict to various degrees the envelope glycoproteins of a diverse number of viruses. This report sheds light on the important antiviral function of MARCH proteins and their significance in cell intrinsic immunity.IMPORTANCE This study examines the mechanism utilized by different MARCH proteins to restrict retrovirus infection. MARCH proteins block the incorporation of envelope glycoproteins to the budding virions. In this report, by comparing the human and mouse MARCH genes and using murine leukemia virus (MLV) and HIV-1, we identify differences in the mechanism of restriction among MARCH proteins. Furthermore, we perform a comprehensive analysis on a number of envelope glycoproteins and show that MARCH proteins have broad antiviral functions.

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.

SERINC5 Potently Restricts Retrovirus Infection In Vivo.

The serine incorporator (SERINC) proteins are multipass transmembrane proteins that affect sphingolipid and phosphatidylserine synthesis. Human SERINC5 and SERINC3 were recently shown to possess antiretroviral activity for a number of retroviruses, including human immunodeficiency virus (HIV), murine leukemia virus (MLV), and equine infectious anemia virus (EIAV). In the case of MLV, the glycosylated Gag (glyco-Gag) protein was shown to counteract SERINC5-mediated restriction in in vitro experiments and the viral envelope was found to determine virion sensitivity or resistance to SERINC5. However, nothing is known about the in vivo function of SERINC5. Antiretroviral function of a host factor in vitro is not always associated with antiretroviral function in vivo Using SERINC5-/- mice that we had generated, we showed that mouse SERINC5 (mSERINC5) restriction of MLV infection in vivo is influenced not only by glyco-Gag but also by the retroviral envelope. Finally, we also examined the in vivo function of the other SERINC gene with known antiretroviral functions, SERINC3. By using SERINC3-/- mice, we found that the murine homologue, mSERINC3, had no antiretroviral role either in vivo or in vitro To our knowledge, this report provides the first data showing that SERINC5 restricts retrovirus infection in vivo and that restriction of retrovirus infectivity in vivo is dependent on the presence of both glyco-Gag and the viral envelope.IMPORTANCE This study examined for the first time the in vivo function of the serine incorporator (SERINC) proteins during retrovirus infection. SERINC3 and SERINC5 (SERINC3/5) restrict a number of retroviruses, including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV), by blocking their entry into cells. Nevertheless, HIV-1 and MLV encode factors, Nef and glycosylated Gag, respectively, that counteract SERINC3/5 in vitro We recently developed SERINC3 and SERINC5 knockout mice to examine the in vivo function of these genes. We found that SERINC5 restriction is dependent on the absence of glycosylated Gag and the expression of a specific viral envelope glycoprotein. On the other hand, SERINC3 had no antiviral function. Our findings have implications for the development of therapeutics that target SERINC5 during retrovirus infection.

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.

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

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 in vivo 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 Ddx41 gene, we show that DDX41 sensing in DCs but not macrophages was critical for controlling in vivo MLV infection. This suggests that DCs are essential in vivo 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.IMPORTANCE 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 in vivo MLV infection. These findings underscore the need for multiple host factors to counteract retroviral infection.

Deaminase-Dead Mouse APOBEC3 Is an In Vivo Retroviral Restriction Factor.

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 in vivo, 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 in vivo 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.IMPORTANCE 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 in vitro, little is known about their role during in vivo 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 in vivo 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 in vivo.

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

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.IMPORTANCE 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.

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

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.

APOBEC3 Proteins in Viral Immunity.

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.

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

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 IFNß 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 IFNß 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.

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

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.

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

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.

Theiler's murine encephalomyelitis virus L* amino acid position 93 is important for virus persistence and virus-induced demyelination.

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection associated with an inflammatory white matter demyelinating disease. TO subgroup strains synthesize an 18-kDa protein, L*, out of frame with the polyprotein from an initiation codon 13 nucleotides downstream from the polyprotein's AUG codon. We previously generated a mutant virus from our infectious DA full-length clone that has a change of the L* AUG codon to ACG (with no change in the polyprotein's amino acid sequence). Studies of this mutant virus showed that L* was key to the TO subgroup phenotype because the mutant had a decreased ability to persist and demyelinate. This work was initially called into question because a similar mutant derived from a different full-length DA infectious clone persisted and demyelinated similarly to wild-type DA virus (O. van Eyll and T. Michiels, J. Virol. 74:9071-9077, 2000). We now report that (i) the sequence of the L* coding region differs in the two infectious clones, resulting in a Ser or Leu as the predicted amino acid at position 93 of L* (with no change in the polyprotein's amino acid sequence), (ii) the difference in this amino acid is key to the phenotypic differences between the two mutants, and (iii) the change in amino acid 93 may affect L* phosphorylation. It is of interest that this amino acid only appears critical in determining the virus phenotype when L* is present in a significantly reduced amount (i.e., following translation from an ACG initiating codon).