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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., (Copyright © 2018 Stavrou et al.)

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 ., (Copyright © 2018 American Society for Microbiology.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)