Experimental Evolution of an Oncolytic Vesicular Stomatitis Virus with Increased Selectivity for p53-Deficient Cells

Experimental evolution has been used for various biotechnological applications including protein and microbial cell engineering, but less commonly in the field of oncolytic virotherapy. Here, we sought to adapt a rapidly evolving RNA virus to cells deficient for the tumor suppressor gene p53, a hallmark of cancer cells. To achieve this goal, we established four independent evolution lines of the vesicular stomatitis virus (VSV) in p53-knockout mouse embryonic fibroblasts (p53−/− MEFs) under conditions favoring the action of natural selection. We found that some evolved viruses showed increased fitness and cytotoxicity in p53−/− cells but not in isogenic p53+/+ cells, indicating gene-specific adaptation. However, full-length sequencing revealed no obvious or previously described genetic changes associated with oncolytic activity. Half-maximal effective dose (EC50) assays in mouse p53-positive colon cancer (CT26) and p53-deficient breast cancer (4T1) cells indicated that the evolved viruses were more effective against 4T1 cells than the parental virus or a reference oncolytic VSV (MΔ51), but showed no increased efficacy against CT26 cells. In vivo assays using 4T1 syngeneic tumor models showed that one of the evolved lines significantly delayed tumor growth compared to mice treated with the parental virus or untreated controls, and was able to induce transient tumor suppression. Our results show that RNA viruses can be specifically adapted typical cancer features such as p53 inactivation, and illustrate the usefulness of experimental evolution for oncolytic virotherapy

The RNA binding protein La promotes RIG-I-mediated type I and type III IFN responses following Sendai viral infection

Abstract La/SS-B (or La) is a 48 kDa RNA-binding protein and an autoantigen in autoimmune disorders such as systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SS). La involvement in regulating the type I interferon (IFN) response is controversial - acting through both positive and negative regulatory mechanisms; inhibiting the IFN response and enhancing viral growth, or directly inhibiting viral replication. We therefore sought to clarify how La regulates IFN production in response to viral infection. ShRNA knockdown of La in HEK 293 T cells increased Sendai virus infection efficiency, decreased IFN-β, IFN-λ1, and interferon-stimulated chemokine gene expression. In addition, knockdown attenuated CCL-5 and IFN-λ1 secretion. Thus, La has a positive role in enhancing type I and type III IFN production. Mechanistically, we show that La directly binds RIG-I and have mapped this interaction to the CARD domains of RIG-I and the N terminal domain of La. In addition, we showed that this interaction is induced following RIG-I activation and that overexpression of La enhances RIG-I-ligand binding. Together, our results demonstrate a novel role for La in mediating RIG-I-driven responses downstream of viral RNA detection, ultimately leading to enhanced type I and III IFN production and positive regulation of the anti-viral response

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts

High frequency of cytomegalovirus-specific cytotoxic T-effector cells in HLA-A*0201-positive subjects during multiple viral coinfections.

How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-specific CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-specific CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strengths

Cytokine Homologs of Human Gammaherpesviruses

Gammaherpesviruses such as Epstein-Barr virus (EBV, human herpesvirus 4) and Kaposi sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) establish lifelong infection in the host. To further this lifestyle, they encode homologs of cellular cytokines and cytokine receptors with the overarching goal to escape from or to blunt host antiviral defenses. EBV encodes mimics of human interleukin (hIL)-10 and a G protein-coupled receptor protein with sequence similarity to CXCR, whereas KSHV encodes homologs of hIL-6, 3 CC chemokine ligands, and a G protein-coupled receptor with sequence similarity to IL8 receptor alpha. This review focuses on the EBV IL-10 homolog and the KSHV IL-6 homolog with respect to virus biology and pathogenesis of the virus-associated diseases

The human autoantigen La/SS-B accelerates herpes simplex virus type 1 replication in transfected mouse 3T3 cells

Permanently transfected mouse cell lines which expressed different levels of the human autoantigen La/SS-B were infected with different strains of herpes simplex virus type 1, including the strains ANG, HSZP, 17syn+ and HFEM. During infection the localization of the human La protein was followed using an anti-La MoAb, which recognized only the human La protein but did not cross-react with either the endogenous mouse La protein or any viral encoded protein. After infection La protein was transported from the nucleus to the cytoplasm. The time course of translocation was dependent on the amount of human La protein expressed in the respective cell line. Moreover, acceleration of viral replication was dependent on the level of expression of human La protein, suggesting that La protein is a cellular factor that facilitates virus replication