Investigating host intracellular proteins with antiviral activity against influenza and herpesviruses

Abstract

Human pathogenic viruses present an ongoing global public health problem, as reflected by continuing endemic infections, seasonal epidemics, and pandemics caused by newly emerging viruses. While many intracellular host proteins fulfil defined roles in normal cellular physiology and function, a subset exert intrinsic antiviral activity and are termed host restriction factors. Host restriction factors represent an important component of innate antiviral immunity and can inhibit infection and/or replication of different DNA and/or RNA viruses at distinct stages in their viral replication cycle. This thesis has focussed on studying how two host restriction factors, namely SAM domain and HD domain-containing protein 1 (SAMHD1) and Membrane-associated RING-CH-Type Finger 8 protein (MARCHF8), restrict different RNA and DNA viruses. <br/> Studies described in Chapter 3 investigated the antiviral activity of SAMHD1 against respiratory RNA viruses in human macrophage-like and epithelial cells. Using human herpes simplex type 1 virus (HSV-1) as a control to validate the functionality of our approaches, we demonstrated that neither endogenous nor overexpressed SAMHD restricted seasonal influenza A virus (IAV), human metapneumovirus (HMPV) and human parainfluenza virus type 3 (hPIV3) replication in either cell type. However, overexpression of SAMHD1 inhibited highly pathogenic avian influenza virus (HPAI) A(H5N1) in human macrophage-like cells. <br/> HSV-1 infects a wide range of cell types, including epithelial cells, keratinocytes, astrocytes, neurons and immune cells, yet most studies published to date have investigated the role of SAMHD1 in restricting HSV-1 replication in monocytes and macrophages. In Chapter 4, we focussed on assessment of SAMHD1 expression and its role in restricting HSV-1 replication in different cell types relevant to HSV-1 pathogenesis, including keratinocytes, neurons and astrocytes. We report that endogenous SAMHD1 did not restrict HSV-1 replication in any of the neuron, astrocyte or keratinocyte cell models tested, whereas restriction was observed in differentiated THP-1 macrophages. In contrast, inducible overexpression of SAMHD1 in neuronal, but not in keratinocyte cell lines, resulted in HSV-1 restriction, with variable results observed following overexpression in an astrocytic cell line. We also used human small molecule neural precursor cells (smNPCs), derived from human pluripotent stem cells (iPSCs) and differentiated into neurons or astrocytes, to confirm that endogenous SAMHD1 did not restrict HSV-1 replication in either cell type. Together, these results indicate that the capacity of both endogenous and overexpressed SAMHD1 to restrict HSV-1 replication is cell-type dependent. <br/> We extended our studies of human SAMHD1 and herpesviruses to investigate the ability of bovine SAMHD1 to restrict replication of human and bovine herpesviruses (Chapter 5). We used an inducible overexpression system to express bovine SAMHD1 and, for comparison, human SAMHD1 in bovine epithelial- and macrophage-like cells, enabling assessment of their ability to restrict bovine herpesvirus type 1 (BHV-1) replication. We demonstrate that inducible expression of either bovine or human SAMHD1 restricted the replication of BHV-1, as well as HSV-1. Restriction of BHV-1 by bovine SAMHD1 correlated with the inhibition of viral genomic replication and late viral gene expression. Furthermore, by introducing mutations known to impact the nuclear localisation, dNTPase activity or antiviral activity against lentiviruses, we show that only dNTPase activity was required for restriction of BHV-1 by bovine SAMHD1. <br/> In Chapter 6, we report the ability of MARCHF8 to restrict HSV-1 replication in macrophage- or epithelial-like cells. While it is widely established that MARCHF8 can restrict replication of several RNA viruses, this represents the first report of its ability to act against a DNA virus. For many RNA viruses, MARCHF8-mediated restriction has been associated with downregulation of viral envelope glycoproteins from the cell surface, thereby limiting their subsequent incorporation into nascent virions. However, while MARCHF8 expression did not affect virus entry, translocation of viral genome to the nucleus and immediate early (IE) gene expression, it did inhibit HSV-1 genomic replication, and therefore subsequent late viral gene expression and release of infectious virions. MARCHF8-mediated restriction of HSV-1 occurred independent of other cellular factors known to impact genomic replication of HSV-1, namely SAMHD1 and CD81, and could also proceed efficiently in cells expressing a functional cGAS-STING pathway. To our knowledge, these studies are the first to demonstrate MARCHF8-mediated restriction of a human DNA virus. Moreover, inhibition of HSV-1 genomic replication represents a novel mechanism of MARCHF8-mediated virus restriction that is distinct to its reported antiviral activity against RNA viruses. <br/> Overall, our studies indicate that SAMHD1 and MARCHF8 represent broad-spectrum host restriction factors with the ability to act across different classes of viruses with distinct viral genomes, likely via mechanisms distinct to those previously described. Furthermore, we demonstrate the potential of bovine SAMHD1 to restrict replication of BHV-1, a bovine herpesvirus associated with significant economic impact in the cattle industry. Targeting host restriction factors such as SAMHD1 and MARCHF8 represents a promising approach for the development of novel host-directed antiviral therapies.