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.United States. Public Health Service (Grant R01-AI-085015)United States. Public Health Service (Grant T32-CA115299 )United States. Public Health Service (Grant F32-AI100512

Deaminase-Dead Mouse APOBEC3 Is an <i>In Vivo</i> Retroviral Restriction Factor

ABSTRACT 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 . </jats:p

DDX41 recognizes RNA/DNA retroviral reverse transcripts and is critical for<i>in vivo</i>control of MLV infection

AbstractHost 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 inin vivocontrol of retroviral infection is 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 anti-retroviral innate immune response to MLV and HIV in primary mouse macrophages and dendritic cells (DC). Using mice with macrophage- or -specific knockout of the DDX41 gene, we show that DDX41 sensing in DCs but not macrophages was critical for controllingin vivoMLV infection. This suggests that DCs are essentialin vivotargets 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.ImportanceViruses are detected by many different host sensors of nucleic acid, which in turn trigger innate immune responses, such as type I 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 2ndstep. Importantly, we demonstrate using mice lacking DDX41 or cGAS, that both sensors are needed for the full antiviral response needed to controlin vivoMLV infection. These findings underscore the need for multiple host factors to counteract retroviral infection.</jats:sec

Nucleic Acid Recognition Orchestrates the Anti-Viral Response to Retroviruses

SummaryIntrinsic 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

454 Analysis of NY-ESO-1 expression in specimens from a Phase I/II NY-ESO-1 T-cell therapy clinical trial in non-small cell lung cancer and from exploratory studies in multiple tumor types

BackgroundThis analysis evaluates an NY-ESO-1 immunohistochemistry (IHC) clinical trial assay in multiple tumor types for the identification of patients who may be eligible for NY-ESO-1 TCR T-cell targeted therapy. We provide an analysis of NY-ESO-1 expression and prevalence in non-small cell lung carcinoma (NSCLC) tumor samples from a patient cohort of an early Phase I/II clinical trial assessing NY-ESO-1 TCR T-cell therapy. Furthermore, we describe exploratory analyses of NY-ESO-1 prevalence and expression in a preliminary set of multiple tumor types to identify new indications for NY-ESO-1 TCR T-cell therapy.MethodsAn IHC assay was developed to detect NY-ESO-1 expression in formalin-fixed paraffin-embedded (FFPE) specimens utilizing an anti-NY-ESO-1 monoclonal antibody, clone E978. NY-ESO-1 protein expression levels and diagnostic status were determined by pathological evaluation under light microscopy to capture the percentage of tumor cell staining across all tumor cells in specimens at staining intensities 0, 1+, 2+ and 3+. NY-ESO-1 expression data were assessed for: prevalence using a ≥10% cutoff at ≥ 1+ intensity to assign positivity, and prevalence across classification (primary and metastatic) and subtype (adenocarcinoma and squamous cell carcinoma) for the NSCLC specimens.ResultsThe overall prevalence for NSCLC specimens from the Phase I/II trial was 15% (49/325) for NY-ESO-1. A prevalence of 15% (29/191) for primary and 14% (19/132) for metastatic samples, 13% (20/159) for adenocarcinoma, and 14% (5/35) for squamous cell carcinoma was observed. No significant difference was observed between subtype or%Tumor at each intensity. The preliminary set of indications used in exploratory studies had an observed prevalence as follows: gastric adenocarcinoma, 14 (4/28)%; esophageal adenocarcinoma &amp; gastric esophageal junction, 9% (3/35); urothelial, 19% (6/31); head and neck squamous cell carcinoma, 10% (3/30); triple negative breast, 10% (3/30); hepatocellular carcinoma, 3%(1/30); and melanoma, 11% (3/27). NY-ESO-1 protein expression was localized in the cells’ nuclei and surrounding cytoplasm.ConclusionsMultiple indications assessed by the IHC clinical trial assay demonstrated similar NY-ESO-1 expression across the range of staining intensities and percentage of positive tumor observed as that in NSCLC, therefore warranting further development and validation of an IHC assay for NY-ESO-1 detection in these additional tumor types for use in clinical trials. These data support the use of IHC as a tool for the identification of patients whose tumors upregulate NY-ESO-1 in NSCLC and further encourage the investigation of multiple tumor types that may upregulate NY-ESO-1 as potential targets for NY-ESO-1 TCR T-cell therapies.AcknowledgementsThis study (NCT03709706) was funded by GlaxoSmithKline.Trial RegistrationNCT03709706ReferencesThomas R, et al. Front Immunol 2018;9:947Ethics ApprovalThis study was approved by the appropriate institutional review boards and independent ethics committees.</jats:sec

A3G but not A3A is packaged into M-MLV and inhibits reverse transcription.

<p>A) and B) Virions were isolated from the spleens of A3G (A), A3A (B), wild type and KO mice and EnRT assays were performed. Shown is the average of 3 independent experiments using different virus preparations; error bars show standard deviation. C) Western blot analysis of M-MLV virions from the transgenic mice, using anti-myc antisera (top panel). The blots were stripped and reprobed with anti-MLV antisera (bottom panel). Abbreviations: CA, capsid; +, extracts from 293T cells transfected with the A3A or A3G transgenes.</p

Cellular lysates from splenocytes derived from uninfected transgenic, wild type (BL/6) or mA3 knockout (KO) mice or from 293T cell lines over-expressing A3A or A3G were incubated with a 3′-fluorophore labeled 50-mer single-stranded oligonucleotide (S50) containing cytosine in the sequence context preferred by A3G (S50-CCC) or A3A (S50-TTC).

<p>Deamination was detected by uracil excision by UDG followed by fragmentation of the resulting abasic site by NaOH and heat, resulting in a 35-mer product (P35). High levels of activity in the 293/A3A samples result in deamination at multiple potential cytosines in the S50-CCC substrate (P37). This experiment was performed several times with the same lysates, with similar results.</p

Expression of A3A and A3G transgenes.

<p>A) RT-qPCR analysis of RNA isolated from different tissues of the A3G^high and A3G^low strains. B) RT-qPCR analysis of RNA isolated from different tissues of the A3A^high and A3A^low strains. Shown for comparison for both graphs are the endogenous A3 levels in nontransgenic C57BL/6 mice (mA3), as well as A3A and A3G expression in human H9 cells and human PBMCs (average of 2 individuals). The mice used for this analysis were uninfected. Both panels are representative of 2 independent experiments with a different mouse of each genotype. Error bars denote standard deviation of technical replicates.</p

Mutation frequencies in MLV and MMTV proviruses in splenic DNA of infected A3A and A3G mice.

<p>Analysis was performed on the cloned DNA represented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004145#ppat-1004145-g004" target="_blank">Figure 4</a> (3–4 mice/group; 10–15 sequences/mouse). Unique clones were distinguished by their mutation pattern. M-MLV has a total of 47 GG and 33 GA motifs in the 549bp target sequence, MMTV has 34 GGs and 56 GAs in 673bp and F-MLV has 58 GG and 29 GA motifs in 586bp.</p