Characterization of molecular attributes that influence LINE-1 restriction by all seven human APOBEC3 proteins
Tyler M. Renner, Kasandra Bélanger, Laura Rose Goodwin, Mark Campbell, Maria Rosales Gerpe, , Marc-André Langlois
- All seven human A3 proteins restrict L1 to varying degrees.
- Disruption of A3 oligomerization correlates with reduced L1 restriction.
- A3A exhibits both deamination-dependent and -independent L1 restriction.
- Binding of A3 proteins to L1 proteins ORF0p, ORF1p and ORF2p was assessed.
- Binding of A3 proteins to L1 proteins does not predict restriction intensity.
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.
Tyler M. Renner, Kasandra Bélanger, Cindy Lam, Maria Rosales Gerpe, Joanne E. McBane, Marc-André Langlois
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 gPr80 helps MLVs 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 against 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 have 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 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) harbouring 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 and persist in their host. 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 infect, 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.
Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry
Vera A. Tang, Tyler M. Renner, Anna K. Fritzche, Dylan Burger, Marc-André Langlois
Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115–130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.
Single-particle characterization of oncolytic vaccinia virus by flow virometry
Vera A. Tang, Tyler M. Renner, Oliver Varette, Fabrice Lebeouf, Jiahu Wang, Jean-Simon Diallo, Marc-André Langlois
Vaccine. 2016 Sep 30;34(42):5082-5089.
Vaccinia virus (VV) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase I, II and III clinical trials. Although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (SSPs) present in the sample. SSPs are comprised of infectious virus and non-infectious viral particles, but also cell contaminants derived from the virus isolation procedures, such as cellular vesicles and debris. Here we have employed flow virometry (FV) analysis and sorting to isolate and characterize distinct SSP populations in therapeutic oncolytic VV preparations. We show that VV preparations contain SSPs heterogeneous in size and include large numbers of non-infectious VV particles. Furthermore, we used FV to illustrate how VV has a propensity to aggregate over time and under various handling and storage procedures. Accordingly, we find that together the infectious titer, the total number of SSPs, the number of viral genomes and the level of particle aggregation in a sample constitute useful parameters that greatly facilitate inter-sample assessment of physical quality, and also provides a means to monitor sample deterioration over time. Additionally, we have successfully employed FV sorting to further isolate virus from other particles by identifying a lipophilic dye that preferentially stains VV over other SSPs in the sample. Overall, we demonstrate that FV is a fast and effective tool that can be used to perform quality, and consistency control assessments of oncolytic VV vaccine preparations.
- Renner, T.M., Bélanger, K., Goodwin, L.R., Campbell, M. and Langlois, M.A. (2018). Characterization of molecular attributes that influence LINE-1 restriction by all seven human APOBEC3 proteins. Virology. Volume 520, July 2018, Pages 127–136.
- Renner, T.M., Bélanger, K., Rosales Gerpe, M.C. and Langlois, M.A. (2018). Full-Length Glycosylated Gag of Murine Leukemia Virus Can Associate With The Viral Envelope as a Type I Integral Membrane Protein. Journal of Virology. 2018 Feb 26;92(6).
- Tang, V.A., Renner, T.M., Fritzsche, A. Burger, D., and Langlois, M.A. (2017). Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by NanoScale Flow Cytometry. Scientific Reports. 7: 17769.
- Tang, V.A Renner, T.M. Varette, O. Wang, J. Le Boeuf, F. Diallo, J.S. Bell, J.C. and Langlois, M.A. (2016). Single-Particle Characterization of Oncolytic Vaccinia Virus by Flow Virometry. Vaccine. 34: 5082-5089.
- Bélanger, K. and Langlois, M.A. (2015) Comparative analysis of the gene-inactivating potential of retroviral restriction factors APOBEC3F and APOBEC3G. Journal of General Virology. Volume 96, Sept. 2015, Pages 2878-2887.
- Bélanger, K. and Langlois, M.A. (2015) RNA-Binding Residues in the N-Terminus of APOBEC3G Influence its DNA Sequence Specificity and Retrovirus Restriction Efficiency. Virology. Volume 483, Sept. 2015, Pages 141-148.
- Rosales Gerpe, M.C., Renner, T.M., Bélanger, K., Lam, C., Aydin, H., and Langlois, M.A. (2015) N-Linked Glycosylation Protects Gammaretroviruses Against Deamination by APOBEC3 Proteins. Journal of Virology. Feb; 89 (4): 2342-57.
- Bélanger, K., Savoie, M., Aydin, H., Renner, T., Montazeri, Z., and Langlois, M.A. (2014) Deamination intensity profiling of human APOBEC3 protein activity along the near full-length genomes of HIV-1 and MoMLV by HyperHRM analysis. Virology, 448, 168-175.
- Bélanger, K., Savoie, M., Rosales Gerpe, M.C., Couture, J.F., and Langlois, M.A. (2013) Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses. Nucleic Acid Research, 41, 7438-7452.
- Langlois, M.A. (2010) Mother’s Milk and Intrinsic Immunity. Cell Host and Microbe. Dec; 8 (6): 467-469.
- Langlois, M.A., Kemmerich, K., Rada, C. and Neuberger, M.S. (2009) The AKV murine leukemia virus is restricted and hypermutated by mouse APOBEC3. Journal of Virology. November, 83 (22): 430-439.