The role of non-coding single-stranded oligonucleotides on Respiratory syncytial virus infection

Abstract

Respiratory syncytial virus (RSV) is an enveloped RNA virus of the Pneumoviridae family. RSV is reported to infect host cells by receptor-mediated endocytosis, also called clathrin-dependent endocytosis. Many reports indicate that the virus utilizes the host receptor nucleolin for entry. RSV is one of the leading causes of acute lower respiratory tract infection in children, the elderly and immunocompromised individuals. It is therefore important to find viable treatments against RSV infections. To this date, the development of an RSV vaccine has unfortunately been unsuccessful. Therefore, a lot of research is instead focusing on developing entry or replication inhibitors against RSV.

We have previously discovered that a 35 bases long single-stranded oligonucleotide (ssON) can inhibit certain endocytic pathways, such as clathrin- and caveolin-dependent endocytosis. Based on this, the overall aim of the projects in this thesis is to investigate if ssON can work as an entry inhibitor against RSV and to study the overall effect that ssON has on RSV infection in vivo in a murine RSV challenge model. Furthermore, we aim to study the effect that proteins present in the surrounding extracellular environment of the virus have on the viral infectivity and pathogenesis.

In Paper I we show that single-stranded oligonucleotides can inhibit RSV infection in vitro and in vivo. We demonstrate that ssON shields nucleolin in vitro. Nucleolin is a receptor involved in RSV entry and by shielding nucleolin present in the cell membrane, ssON prevents RSV from binding to the cells. Furthermore, we show that ssON inhibits RSV infection in vivo. ssON treatment of RSV infection was associated with enhanced expression of RSV-induced Interferon-stimulated genes (ISGs), suggesting that interferon responses likely contributed to reduced RSV infection. As a continuation to Paper I, in Paper II we investigate if small non-coding RNAs (sncRNAs) exhibit the same antiviral capacity as ssON. We demonstrate that sncRNAs, of similar size as ssON, can inhibit RSV infection in vitro and similarly to ssON, they bind to the entry co-receptor nucleolin. In Paper III we study the pathogenesis of RSV. We show that proteins from the extracellular environment of the virus can bind to the virus and affect the viral infectivity as well as the interaction of the virus with host cells. We determine that RSV accumulates a rich and distinctive protein corona in different biological fluids including human plasma (HP), human bronchoalveolar lavage fluid (BALF), non-human primate plasma (MP) and fetal bovine serum (FBS). We show that RSV incubated with BALF has an increased viral infectivity in epithelial HEp-2 and monocytederived dendritic cells (moDCs). Moreover, RSV surrounded by a BALF-derived corona induce the activation of moDCs as assessed by increased expression of co-stimulatory molecules.

In summary, the projects in this thesis aim to assess the hypothesis that single-stranded oligonucleotides of DNA or RNA origin can be used to inhibit RSV infection. Furthermore, we aim to determine the effects, in terms of viral infectivity and pathogenesis, of the differential protein compositions present in biological fluids surrounding viruses.

Keywords: Respiratory syncytial virus (RSV), single-stranded oligonucleotide (ssON), small non-coding RNA (sncRNA), nucleolin, virus infection, antiviral.