Blood-Based Immune Protein Markers of Disease Progression in Murine Models of Acute and Chronic Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a chronic ailment afflicting millions of people worldwide, with the majority of recognized cases within industrialized countries. The impacts of IBD at the individual level are long-lasting with few effective treatments available, resulting in a large burden on the health care system. A number of existing animal models are utilized to evaluate novel treatment strategies. Two commonly used models are (1) acute colitis mediated by dextran sulphate sodium (DSS) treatment of wild-type mice and (2) chronic colitis mediated by the transfer of proinflammatory T cells into immunodeficient mice. Despite the wide use of these particular systems to evaluate IBD therapeutics, the typical readouts of clinical disease progression vary depending on the model used, which may be reflective of mechanistic differences of disease induction. The most reliable indicator of disease in both models remains intestinal damage which is typically evaluated upon experimental endpoint. Herein, we evaluated the expression profile of a panel of cytokines and chemokines in both DSS and T cell transfer models in an effort to identify a number of inflammatory markers in the blood that could serve as reliable indicators of the relative disease state. Out of the panel of 25 markers tested, 6 showed statistically significant shifts with the DSS model, compared to 11 in the T cell transfer model with IL-6, IL-13, IL-22, TNF-α and IFN-γ being common markers of disease in both models. Our data highlights biological differences between animal models of IBD and helps to guide future studies when selecting efficacy readouts during the evaluation of experimental IBD therapeutics.

Intact Viral Particle Counts Measured by Flow Virometry Provide Insight into the Infectivity and Genome Packaging Efficiency of Moloney Murine Leukemia Virus.

Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus-infected cells are well known to release soluble viral proteins, defective viruses, and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small-particle flow cytometry, is an emerging analytical method enabling high-throughput single-virus phenotypic characterizations. By utilizing the viral envelope glycoprotein (Env) and monodisperse light scattering characteristics as discerning parameters of intact virus particles, here, we analyzed the basic properties of Moloney MLV (M-MLV). We show that <24% of the total p30 capsid protein measured in infected cell supernatants is associated with intact viruses. We calculate that about one in five M-MLV particles contains a viral RNA genome pair and that individual intact particle infectivity is about 0.4%. These findings provide new insights into the characteristics of an extensively studied prototypical retrovirus while highlighting the benefits of flow virometry for the field of virology.IMPORTANCE Gammaretroviruses, or, more specifically, murine leukemia viruses (MLVs), have been a longstanding model for studying retroviruses. Although being extensively analyzed and dissected for decades, several facets of MLV biology are still poorly understood. One of the primary challenges has been enumerating total intact virus particles in a sample. While several analytical methods can precisely measure virus protein amounts, MLVs are known to induce the secretion of soluble and vesicle-associated viral proteins that can skew these measurements. With recent technological advances in flow cytometry, it is now possible to analyze viruses down to 90 nm in diameter with an approach called flow virometry. The technique has the added benefit of being able to discriminate viruses from extracellular vesicles and free viral proteins in order to confidently provide an intact viral particle count. Here, we used flow virometry to provide new insights into the basic characteristics of Moloney MLV.

Characterization of molecular attributes that influence LINE-1 restriction by all seven human APOBEC3 proteins.

LINE-1 (L1) is a non-long terminal repeat (LTR) retrotransposon inserted throughout the human genome. APOBEC3 (A3) proteins are part of a network of host intrinsic defenses capable of restricting retroviruses and the replication of L1 retroelements. These enzymes inactivate retroviruses primarily through deamination of single-stranded viral DNA. In contrast, only A3A deaminates L1 DNA, while the other six A3 proteins restrict L1 to varying degrees through yet poorly defined mechanisms. Here we provide further insight into the molecular attributes of L1 restriction by A3 proteins. We specifically investigated the roles of A3 protein oligomerization, interactions with RNA and their binding to the various L1 proteins. Our results show that compromising the ability of A3 proteins to oligomerize or interact with a nucleic acid substrate diminished L1 restriction to varying degrees. However the efficiency of their binding to L1 proteins did not predict restriction or the potency of the restriction.

Full-Length Glycosylated Gag of Murine Leukemia Virus Can Associate with the Viral Envelope as a Type I Integral Membrane Protein.

The glycosylated Gag protein (gPr80) of murine leukemia viruses (MLVs) has been shown to exhibit multiple roles in facilitating retrovirus release, infection, and resistance to host-encoded retroviral restriction factors, such as APOBEC3, SERINC3, and SERINC5. One way in which gPr80 helps MLVs to escape host innate immune restriction is by increasing capsid stability, a feature that protects viral replication intermediates from being detected by cytosolic DNA sensors. gPr80 also increases the resistance of MLVs to deamination and restriction by mouse APOBEC3 (mA3). How the gPr80 accessory protein, with its three N-linked glycosylation sites, contributes to these resistance mechanisms is still not fully understood. Here we further characterized the function of gPr80 and, more specifically, revealed that the asparagines targeted for glycosylation in gPr80 also contribute to capsid stability through their parallel involvement in the Pr65 Gag structural polyprotein. In fact, we demonstrate that sensitivity to deamination by the mA3 and human A3 proteins is directly linked to capsid stability. We also show that full-length gPr80 is detected in purified viruses. However, our results suggest that gPr80 is inserted in the NexoCcyto orientation of a type I integral membrane protein. Additionally, our experiments have revealed the existence of a large population of Env-deficient virus-like particles (VLPs) harboring gPr80 inserted in the opposite (NcytoCexo) polarity, which is typical of type II integral membrane proteins. Overall this study provides new insight into the complex nature of the MLV gPr80 accessory protein.IMPORTANCE Viruses have evolved numerous strategies to infect, spread in, and persist in their hosts. Here we analyze the details of how the MLV-encoded glycosylated Gag (gPr80) protein protects the virus from being restricted by host innate immune defenses. gPr80 is a variant of the structural Pr65 Gag protein with an 88-amino-acid extended leader sequence that directs the protein for translation and glycosylation in the endoplasmic reticulum. This study dissects the specific contributions of gPr80 glycans and capsid stability in helping the virus to infect cells, spread, and counteract the effects of the host intrinsic restriction factor APOBEC3. Overall this study provides further insight into the elusive role of the gPr80 protein.

Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation.

There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly all purification methods due to nearly indistinguishable biophysical characteristics such as size, buoyant density and nucleic acid content. Recently, our group has illustrated a means of isolating intact and highly enriched retroviral virions from EV-containing cell supernatants using an immunoprecipitation approach targeting the viral envelope glycoprotein of the Moloney Murine Leukemia Virus ( Renner et al., 2018 ). This technique, that we call intact virion immunoprecipitation (IVIP), enabled us to characterize the accessibility of epitopes on the surface of these retroviruses and assess the orientation of the virus-encoded integral membrane protein Glycogag (gPr80) in the viral envelope. Proper implementation of this protocol enables fast, simple and reproducible preparations of intact and highly purified retroviral particles devoid of detectable EV contaminants.

Retroviral Capsid Core Stability Assay.

Structural stability of the capsid core is a critical parameter for the productive infection of a cell by a retrovirus. Compromised stability can lead to premature core disassembly, exposure of replication intermediates to cytosolic nucleic acid sensors that can trigger innate antiviral responses, and failure to integrate the proviral genome into the host DNA. Thus, core stability is a critical feature of viral replicative fitness. While there are several well-described techniques to assess viral capsid core stability, most are generally time and labor intensive. Recently, our group compared the relative stability of murine leukemia virus capsid cores using an in vitro detergent-based approach combined with ultracentrifugation against the popular fate of capsid assay. We found that both methods reached similar conclusions, albeit the first method was a significantly simpler and faster way to assess relative capsid core stability when comparing viral mutants exhibiting differences in core stability.

N-linked glycosylation protects gammaretroviruses against deamination by APOBEC3 proteins.

UNLABELLED: Retroviruses are pathogens with rapid infection cycles that can be a source of disease, genome instability, and tumor development in their hosts. Host intrinsic restriction factors, such as APOBEC3 (A3) proteins, are constitutively expressed and dedicated to interfering with the replication cycle of retroviruses. To survive, propagate, and persist, retroviruses must counteract these restriction factors, often by way of virus genome-encoded accessory proteins. Glycosylated Gag, also called glycosylated Pr80 Gag (gPr80), is a gammaretrovirus genome-encoded protein that inhibits the antiretroviral activity of mouse A3 (mA3). Here we show that gPr80 exerts two distinct inhibitory effects on mA3: one that antagonizes deamination-independent restriction and another one that inhibits its deaminase activity. More specifically, we find that the number of N-glycosylated residues in gPr80 inversely correlates with the sensitivity of a gammaretrovirus to deamination by mouse A3 and also, surprisingly, by human A3G. Finally, our work highlights that retroviruses which have successfully integrated into the mouse germ line generally express a gPr80 with fewer glycosylated sites than exogenous retroviruses. This observation supports the suggestion that modulation of A3 deamination intensity could be a desirable attribute for retroviruses to increase genetic diversification and avoid immune detection. Overall, we present here the first description of how gammaretroviruses employ posttranslational modification to antagonize and modulate the activity of a host genome-encoded retroviral restriction factor. IMPORTANCE: APOBEC3 proteins are host factors that have a major role in protecting humans and other mammals against retroviruses. These enzymes hinder their replication and intensely mutate their DNA, thereby inactivating viral progeny and the spread of infection. Here we describe a newly recognized way in which some retroviruses protect themselves against the mutator activity of APOBEC3 proteins. We show that gammaretroviruses expressing an accessory protein called glycosylated Gag, or gPr80, use the host's posttranslational machinery and, more specifically, N-linked glycosylation as a way to modulate their sensitivity to mutations by APOBEC3 proteins. By carefully controlling the amount of mutations caused by APOBEC3 proteins, gammaretroviruses can find a balance that helps them evolve and persist.

Deamination intensity profiling of human APOBEC3 protein activity along the near full-length genomes of HIV-1 and MoMLV by HyperHRM analysis.

Enzymatic deamination of cytidines in DNA is an intrinsic component of antibody maturation and retroviral resistance, but can also be a source of HIV drug resistance and cancer-causing mutations. Here, we developed a high-throughput method based on high resolution melt (HRM) analysis called HyperHRM that can screen genomic DNA for rare hypermutated proviral sequences and accurately quantify the number of C-to-T or G-to-A mutations in each sequence. We demonstrate the effectiveness of the approach by profiling in parallel the intensity of the DNA mutator activity of all seven human APOBEC3 proteins on the near full-length sequence of HIV-1 and the Moloney murine leukemia virus. Additionally, HRM was successfully used to identify hypermutated proviral sequences in peripheral blood mononuclear cells from an HIV-1 patient. These results exemplify the effectiveness of HRM-based approaches for hypermutation quantification and for the detection of hypermutated DNA sequences potentially associated with disease or retroviral drug resistance.