Virus-producing Cells Research Articles

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Role of Heparanase and Syndecan-1 in HSV-1 Release from Infected Cells.

Herpes Simplex Virus 1 (HSV-1) is a neurotropic human virus that belongs to the Alphaherpesvirinae subfamily of Herpesviridae. Establishment of its productive infection and progression of disease pathologies depend largely on successful release of virions from the virus-producing cells. HSV-1 is known to exploit many host factors for its release. Recent studies have shown that heparanase (HPSE) is one such host enzyme that is recruited for this purpose. It is an endoglycosidase that cleaves heparan sulfate (HS) from the surface of infected cells. HS is a virus attachment coreceptor that is commonly found on cell surfaces as HS proteoglycans e.g., syndecan-1 (SDC-1). The current model suggests that HSV-1 during the late stage of infection upregulates HPSE, which in turn enhances viral release by removing the virus-trapping HS moieties. In addition to its role in directly enabling viral release, HPSE accelerates the shedding of HS-containing ectodomains of SDC-1, which enhances HSV-1 release via a similar mechanism by upregulating CREB3 and COPII proteins. This review outlines the role of HPSE and SDC-1 as newly assigned host factors that facilitate HSV-1 release during a lytic infection cycle.

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

SARS-CoV-2 spike N-terminal domain modulates TMPRSS2-dependent viral entry and fusogenicity.

SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) remains poorly characterized despite enrichment of mutations in this region across variants of concern (VOCs). Here, we examine the contribution of the NTD to infection and cell-cell fusion by constructing chimeric spikes bearing B.1.617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus. We find that the Delta NTD on a Kappa or wild-type (WT) background increases S1/S2 cleavage efficiency and virus entry, specifically in lung cells and airway organoids, through use of TMPRSS2. Delta exhibits increased cell-cell fusogenicity that could be conferred to WT and Kappa spikes by Delta NTD transfer. However, chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD do not show more efficient TMPRSS2 use or fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions in a spike context-dependent manner, and allosteric interactions may be lost when combining regions from more distantly related VOCs.

Epstein–Barr virus tegument protein BGLF2 in exosomes released from virus-producing cells facilitates de novo infection

BackgroundViruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear.ResultsWe screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein–Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection.ConclusionsThe EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes.Graphical abstract7EPPJ43CM5W95Q8y98YowkVideo abstract

Genetic tools for the stable overexpression of circular RNAs

ABSTRACT Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

SARS-coronavirus-2 infections: biological instabilities characterized by order parameters

A four-variable virus dynamics TIIV model was considered that involves infected cells in an eclipse phase. The state space description of the model was transferred into an amplitude space description which is the appropriate general, nonlinear physics framework to describe instabilities. In this context, the unstable eigenvector or order parameter of the model was determined. Subsequently, a model-based analysis of viral load data from eight symptomatic COVID-19 patients was conducted. For all patients, it was found that the initial SARS-CoV-2 infection evolved along the respective patient-specific order parameter, as expected by theoretical considerations. The order parameter amplitude that described the initial virus multiplication showed doubling times between 30 min and 3 h. Peak viral loads of patients were linearly related to the amplitudes of the patient order parameters. Finally, it was found that the patient order parameters determined qualitatively and quantitatively the relationships between the increases in virus-producing infected cells and infected cells in the eclipse phase. Overall, the study echoes the 40 years old suggestion by Mackey and Glass to consider diseases as instabilities.

A Feasible Alternative Strategy Targeting Furin Disrupts SARS-CoV-2 Infection Cycle.

ABSTRACTThe COVID-19 causing coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human lung cells via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). Notably, the cleavage of the spike by the host cell protease furin in virus-producing cells is critical for subsequent spike-driven entry into lung cells. Thus, effective targeted therapies blocking the spike cleavage and activation in viral producing cells may provide an alternate strategy to break the viral transmission cycle and to overcome disease pathology. Here we engineered and described an antibody-based targeted strategy, which directly competes with the furin mediated proteolytic activation of the spike in virus-producing cells. The described approach involves engineering competitive furin substrate residues in the IgG1 Fc-extended flexible linker domain of SARS-CoV-2 spike targeting antibodies. Considering the site of spike furin cleavage and SARS-CoV-2 egress remains uncertain, the experimental strategy pursued here revealed novel mechanistic insights into proteolytic processing of the spike protein, which suggest that processing does not occur in the constitutive secretory pathway. Furthermore, our results show blockade of furin-mediated cleavage of the spike protein for membrane fusion activation and virus host-cell entry function. These findings provide an alternate insight of targeting applicability to SARS-CoV-2 and the future coronaviridae family members, exploiting the host protease system to gain cellular entry and subsequent chain of infections.IMPORTANCE Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants. As host cell-enriched furin-mediated cleavage of SARS-CoV-2 spike protein is critical for viral entry and chain of the infection cycle, the solution described here of an antibody Fc-conjugated furin competing peptide is significant. In a scenario where spike mutational drifts do not interfere with the Fc-conjugated antibody's epitope, the proposed furin competing strategy confers a broad-spectrum targeting design to impede the production of efficiently transmissible SARS-CoV-2 viral particles. In addition, the proposed approach is plug-and-play against other potentially deadly viruses that exploit secretory pathway independent host protease machinery to gain cellular entry and subsequent transmissions to host cells.

The "LLQY" Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2′ sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike’s conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not vice versa. Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2′ cleavage sites in S protein synthesis and processing as well as virus assembly and infection.IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2′ sites are very important, like the S2′ cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants.

Selection and identification of an RNA aptamer that specifically binds the HIV-1 capsid lattice and inhibits viral replication.

The HIV-1 capsid core participates in several replication processes. The mature capsid core is a lattice composed of capsid (CA) monomers thought to assemble first into CA dimers, then into ∼250 CA hexamers and 12 CA pentamers. CA assembly requires conformational flexibility of each unit, resulting in the presence of unique, solvent-accessible surfaces. Significant advances have improved our understanding of the roles of the capsid core in replication; however, the contributions of individual CA assembly forms remain unclear and there are limited tools available to evaluate these forms in vivo. Here, we have selected aptamers that bind CA lattice tubes. We describe aptamer CA15-2, which selectively binds CA lattice, but not CA monomer or CA hexamer, suggesting that it targets an interface present and accessible only on CA lattice. CA15-2 does not compete with PF74 for binding, indicating that it likely binds a non-overlapping site. Furthermore, CA15-2 inhibits HIV-1 replication when expressed in virus producer cells, but not target cells, suggesting that it binds a biologically-relevant site during virus production that is either not accessible during post-entry replication steps or is accessible but unaltered by aptamer binding. Importantly, CA15-2 represents the first aptamer that specifically recognizes the HIV-1 CA lattice.

EXTH-63. A NOVEL MOUSE MODEL OF DIFFUSE MIDLINE GLIOMA FOR TARGETED IMMUNOTHERAPY

Abstract In children, diffuse midline gliomas retain poor outcomes, failing to have a durable response to conventional therapies. Immunotherapies hold promise, with the integration of the host's immune system fundamental to their design. Here, we describe a novel genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen, to evaluate the antitumor activity of IL13Rα2-CAR T cell and bispecific T cell engager (BiTE) therapies in preclinical studies. The RCAS-Tva delivery system was utilized to induce gliomagenesis through p53 loss and the constitutive expression of PDGFB and human IL13Rα2 in Nestin-Tva;p53fl/fl mice. Pups were injected with virus-producing DF1 cells, encoding either for RCAS-Cre and PDGFB+IL13Rα2 or RCAS-Cre and PDGFB. Kaplan-Meier survival curves established and compared tumor growth dynamics in both models. Tumor tissue was characterized through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing tissue were used for orthotopic injection and in vitro studies. Expression of PDGFB and IL13Rα2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between RCAS:PDGFB (n=25, 40 days) and RCAS:PDGB+IL13Rα2 (n=32, 39 days). Tumors demonstrated characteristics of high-grade glioma such as infiltration, pseudopalisading necrosis, microvascular proliferation, high Ki-67 index, heterogenous IL13Rα2 expression, with notable presence of CD11b+ macrophages and low count of CD3+ T cells. Orthotopic tumors from developed cell lines were histologically similar to de novo tumors. Treatment of generated cell lines with IL13Rα2-targeting BiTE protein resulted in a loss of glioma cell viability and target-specific activation of T cells. Engineered de novo tumors possess histopathologic features common to diffused midline gliomas. IL13Rα2-positive cell lines derived from de novo tumors were responsive to targeted treatment, opening the opportunity for preclinical assessment of IL13Rα2-directed immunotherapies, with the potential for clinical translation.

Rapid and inexpensive purification of adenovirus vectors using an optimised aqueous two-phase technology

Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at −20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines.

The Current Status of Latency Reversing Agents for HIV-1 Remission.

Combinatory antiretroviral therapy (cART) reduces human immunodeficiency virus type 1 (HIV-1) replication but is not curative because cART interruption almost invariably leads to a rapid rebound of viremia due to the persistence of stable HIV-1-infected cellular reservoirs. These reservoirs are mainly composed of CD4+ T cells harboring replication-competent latent proviruses. A broadly explored approach to reduce the HIV-1 reservoir size, the shock and kill strategy, consists of reactivating HIV-1 gene expression from the latently infected cellular reservoirs (the shock), followed by killing of the virus-producing infected cells (the kill). Based on improved understanding of the multiple molecular mechanisms controlling HIV-1 latency, distinct classes of latency reversing agents (LRAs) have been studied for their efficiency to reactivate viral gene expression in in vitro and ex vivo cell models. Here, we provide an up-to-date review of these different mechanistic classes of LRAs and discuss optimizations of the shock strategy by combining several LRAs simultaneously or sequentially.

T cell derived HIV-1 is present in the CSF in the face of suppressive antiretroviral therapy.

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from in vitro infected T cells or macrophages. The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.

Genome-wide activation screens to increase adeno-associated virus production

We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging.

Different Neutralization Sensitivity of SARS-CoV-2 Cell-to-Cell and Cell-Free Modes of Infection to Convalescent Sera.

The COVID-19 pandemic caused by SARS-CoV-2 has posed a global threat to human lives and economics. One of the best ways to determine protection against the infection is to quantify the neutralizing activity of serum antibodies. Multiple assays have been developed to validate SARS-CoV-2 neutralization; most of them utilized lentiviral or vesicular stomatitis virus-based particles pseudotyped with the spike (S) protein, making them safe and acceptable to work with in many labs. However, these systems are only capable of measuring infection with purified particles. This study has developed a pseudoviral assay with replication-dependent reporter vectors that can accurately quantify the level of infection directly from the virus producing cell to the permissive target cell. Comparative analysis of cell-free and cell-to-cell infection revealed that the neutralizing activity of convalescent sera was more than tenfold lower in cell cocultures than in the cell-free mode of infection. As the pseudoviral system could not properly model the mechanisms of SARS-CoV-2 transmission, similar experiments were performed with replication-competent coronavirus, which detected nearly complete SARS-CoV-2 cell-to-cell infection resistance to neutralization by convalescent sera. These findings suggest that the cell-to-cell mode of SARS-CoV-2 transmission, for which the mechanisms are largely unknown, could be of great importance for treatment and prevention of COVID-19.

The Engineered MARCH8-Resistant Vesicular Stomatitis Virus Glycoprotein Enhances Lentiviral Vector Transduction.

Lentiviral vectors are one of the most commonly used viral delivery systems for gene therapy. Vesicular stomatitis virus-G envelope glycoprotein (VSV G)-pseudotyped lentiviral vectors have been widely used in clinical studies for treatment of virus infections and genetic deficient diseases. However, the efficiency of lentiviral vector transduction has been long recognized as a limiting factor in clinical gene therapy application, especially in transducing hematopoietic stem cells. MARCH8 (membrane-associated RING-CH 8), an E3 ubiquitin ligase, has been reported to target and downregulate VSV G. Results in this study show that MARCH8 induces ubiquitination and lysosome degradation of VSV G, and knockout of MARCH8 in virus-producing cells increases lentiviral vector transduction by elevating the level of VSV G protein. We then engineered VSV G mutant that has the lysine residues in the cytoplasmic domain substituted for arginine, and showed that this G mutant resists degradation by MARCH8, and allows the enhancement of transduction efficiency of lentiviral vector particles than the parental VSV G protein. This engineered VSV G mutant thus further advances the lentiviral vector system as a powerful tool in gene therapy.

Abstract PO-039: Radiation therapy enhances anti-tumor activity of a MET CAR T-based immunotherapy approach for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most prevalent primary malignant brain tumor, which has an aggressive phenotype and mostly fatal recurrence after standard therapy. MET, the hepatocyte growth factor (HGF) receptor is a relevant target for GBM treatment as it is expressed in up to 50% of cases and its expression, which may be induced by radiation therapy (RT), can potentially contribute to RT-resistance of GBM stem cells, and tumor recurrence. MET-based chimeric antigen (CAR) T cell therapy is currently being evaluated in several solid tumor working settings. A combination of T cell-based therapies with RT may improve the efficacy of the CAR T cell therapy through by the RT-induced immune activation via release of cytokines and induction of antigen expression, similarly as has been observed when combining RT with immune checkpoint inhibitors. Here we investigated the combination of MET-targeting CAR T cells with RT for GBM treatment, hypothesizing enhanced anti-tumor effects. The current study used a panel of MET-proficient and MET-deficient human GBM and GBM stem-like cell lines. Cells have been irradiated with a single dose of 0, 2, 5, or 10 Gy and RT impact on MET expression has been assessed at various time points after RT administration. Results show MET increase after RT in some cell lines. To test MET-targeting CAR T effect on the GBM cell lines, CAR constructs that are based on HGF-MET binding elements have been introduced into virus-producing cells and used for human T cells transduction to generate MET-targeting CAR T cells. CAR T cells co-cultured with GBM cell lines in vitro have specifically and significantly decreased viability of MET-positive cancer cells. Data resulting from combination of RT and CAR T cells treatment suggest that radiation exhibits an enhancement of CAR T cells anti-tumor killing activity, indicating a synergism between the two modalities. In conclusion, our data are the first to indicate the efficacy of a MET-based CAR T immunotherapy approach in GBM cell lines. The results also demonstrate a basis for the combination of a MET CAR T modality together with RT. The mechanisms for understanding the interaction between RT and the MET CAR T cells are under investigation. Citation Format: Lusine Hovhannisyan, Daniel M. Aebersold, John Maher, Adrian F. Ochsenbein, Carsten Riether, Michaela Medova, Yitzhak Zimmer. Radiation therapy enhances anti-tumor activity of a MET CAR T-based immunotherapy approach for glioblastoma multiforme [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-039.

Structural Domains of the Herpes Simplex Type 1 gD Protein that Restrict HIV-1 Particle Infectivity.

Previously, we showed that the presence of the herpes simplex virus type 1 (HSV-1) gD glycoprotein but not gB potently restricted HIV-1 particle infectivity. This restriction was characterized by incorporation of HSV-1 gD and the exclusion of the HIV-1 gp120/gp41 from budding virus particles. To determine the structural domains involved in gD restriction of HIV-1, a series of deletion mutants and chimeric proteins between gD and the non-restrictive gB were generated. Our results show that deletion of the cytoplasmic tail domain (CTD) of gD or that replacement of the transmembrane domain (TMD) with the TMD from gB slightly reduced restriction activity. However, replacement of the gD CTD with that of gB resulted in lower cell surface expression, significantly less incorporation into HIV-1 particles, and inefficient restriction of the release of infectious HIV-1. Analysis of gB/gD chimeric proteins revealed that removal of the gB CTD or replacement with gD CTD resulted in enhanced surface expression and an increase in restriction activity. Finally, we show that expression of gD without other HSV-1 proteins resulted in gD fractionation into detergent resistant membranes (DRM) and that gD co-localized with the raft marker GM1, which may partially explain its incorporation into budding virus particles. Taken together, our results suggest that expression of gD at the cell surface is likely a major factor but that other intrinsic properties are also involved in the gD-mediated restriction of HIV-1 particle infectivity.IMPORTANCE Previously, we showed that unlike the HSV-1, the presence of the gD glycoprotein in virus producer cells but not gB potently restricted HIV-1 particle infectivity. To better understand the relationship between cell surface expression, virus incorporation and restriction of HIV-1, we analyzed a series of deletion mutants and chimeric proteins in which domains of gD and gB were swapped. Our results indicate that: a) gD/gB chimeras having the cytoplasmic domain (CTD) of gB significantly reduced cell surface expression, release from cells, incorporation into virus, and reduced HIV-1 restriction; b) removal of the gB CTD or replacement with the gD CTD resulted in better surface expression, incorporation into HIV-1, and enhanced restriction; and c) the transmembrane domain of gB can influence transport and ultimately effect incorporation of gB into HIV-1. Overall, these data support a role for gD surface expression as crucial to restriction of infectious HIV-1 release.

N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.