Cassandra T. David’s research while affiliated with Monash University (Australia) and other places

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease. IMPORTANCE Barmah Forest virus (BFV) is Australia’s second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.

Multifunctionality of viral genes is critical for processes in replication and modulation of infected cells. P gene of rabies virus generates the full-length protein, P1, and the truncated isoform, P3, which gains unique phenotypes lacking in P1, including interactions with multiple cellular membrane-less organelles (MLOs, liquid-liquid phase-separated (LLPS) structures), important to immune evasion. The gain-of-function by P3 proposes that multifunctionality of P isoforms is not merely due to their complement of independent modules, but is regulated by complex interactions of globular and intrinsically disordered regions (IDRs). However, the molecular basis of gain-of-function is unknown. Here we report biophysical and cellular analyses of P1 and P3, identifying a network of intra-protomer interactions involving the globular C-terminal domain and N-terminal IDRs, which differ between the isoforms. Mutagenesis of P3 identified substitutions causing gain- and loss-of-function for MLO interactions, associated with altered interactions of N- and C-terminal regions. Despite reduced MLO association of P1 and P3-loss-of-function mutants compared with wild-type P3, they retain capacity for LLPS in vitro, suggesting that specific inter-molecular interactions enable MLO targeting. P3 and P1 interact similarly with multiple MLO-associated proteins, but RNA binding is only observed for P3, and is enhanced or diminished by gain- and loss-of-function mutations, respectively. These data indicate that differences in interfaces formed between distant regions in P protein isoforms regulate protein-RNA interactions as a principal mechanism in the acquisition of unique functions/MLO interactions by P3, identifying a novel strategy in viral protein multifunctionality.

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.

Hendra virus (HeV) is a highly pathogenic member of the Henipavirus genus (order Mononegavirales ), the replication cycle of which occurs primarily in the cytoplasm. The HeV matrix protein (HeV M) plays critical roles in viral assembly and budding at the plasma membrane, but also undergoes nuclear/nucleolar trafficking, to accumulate in nucleoli early in infection and, later, localise predominantly at the plasma membrane. Previously we found that HeV M protein targets specific sub-nucleolar compartments (corresponding to the FC-DFC (fibrillar centre (FC)/dense fibrillar component (DFC)) where it interacts with the nucleolar protein Treacle and modulates rRNA biogenesis by subverting the host nucleolar DNA damage response, indicating the importance of specific sub-nucleolar trafficking to infection. However, the mechanisms underlying targeting and movement between sub-nucleolar compartments by viral or cellular proteins remain poorly defined. Here, we assessed the molecular regulation of HeV M protein nucleolar/sub-nucleolar trafficking, finding that in infected cells and in cells expressing HeV M protein alone, M protein localizes into Treacle-enriched FC-DFC at early time points, and that FC-DFC localization is subsequently lost due to relocalization into the surrounding granular component (GC) of the nucleolus. Analysis using mutated M proteins and pharmacological modulation of ubiquitination indicate that this dynamic localization is regulated by ubiquitination and oligomerisation, with ubiquitination required for retention of HeV M in Treacle-enriched sub-nucleolar compartments, and oligomerisation required for egress. To our knowledge, this study provides the first direct insights into the dynamics and mechanisms of viral protein trafficking between sub-nucleolar compartments, important to the interplay between HeV M protein and host cell factors during infection. AUTHOR SUMMARY Henipaviruses, including Hendra (HeV) and Nipah viruses, cause deadly diseases in humans and livestock and are considered priority diseases by the World Health Organization due to their epidemic potential and lack of effective treatments. Understanding how these viruses interact with host cells is essential for developing new therapeutics. Our study examines the matrix (M) protein of henipaviruses and its interaction with the nucleolus, a cell structure that mediates ribosome production, and is a common target for various viruses, although their functions are largely unresolved. Previously, we showed that the HeV M protein targets a sub-nucleolar structure, called the FC-DFC, to modulate ribosome biogenesis. Here, we report that the M protein’s movement between sub-nucleolar compartments is controlled by two processes: ubiquitination, which causes accumulation of the protein in the FC-DFC, and oligomerization, which is associated with exit. Similar mechanisms are also observed in other henipaviruses. Our findings reveal mechanisms regulating the hijacking of host cell functions by henipaviruses and suggest new potential targets for antiviral therapies. This study is the first to investigate how viral proteins move within the nucleolus, offering new insights into interactions that may be significant to multiple viruses.

There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following eight hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage internalization led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a food source to promote cellular growth and metabolism.

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically‐replicating negative‐sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA‐damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub‐nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.

Viral hijacking of microtubule (MT)-dependent transport is well understood, but several viruses also express discrete MT-associated proteins (vMAPs), potentially to modulate MT-dependent processes in the host cell. Specific roles for vMAP-MT interactions include subversion of antiviral responses by P3, an isoform of the P protein of rabies virus (RABV; genus Lyssavirus), which mediates MT-dependent antagonism of interferon (IFN)-dependent signal transducers and activators of transcription 1 (STAT1) signaling. P3 also undergoes nucleocytoplasmic trafficking and inhibits STAT1-DNA binding, indicative of intranuclear roles in a multipronged antagonistic strategy. MT association/STAT1 antagonist functions of P3 correlate with pathogenesis, indicating potential as therapeutic targets. However, key questions remain, including whether other P protein isoforms interact with MTs, the relationship of these interactions with pathogenesis, and the extent of conservation of P3-MT interactions between diverse pathogenic lyssaviruses. Using super-resolution microscopy, live-cell imaging, and immune signaling analyses, we find that multiple P protein isoforms associate with MTs and that association correlates with pathogenesis. Furthermore, P3 proteins from different lyssaviruses exhibit variation in intracellular localization phenotypes that are associated with STAT1 antagonist function, whereby P3-MT association is conserved among lyssaviruses of phylogroup I but not phylogroup II, while nucleocytoplasmic localization varies between P3 proteins of the same phylogroup within both phylogroup I and II. Nevertheless, the divergent P3 proteins retain significant IFN antagonist function, indicative of adaptation to favor different inhibitory mechanisms, with MT interaction important to phylogroup I viruses. IMPORTANCE Lyssaviruses, including rabies virus, cause rabies, a progressive encephalomyelitis that is almost invariably fatal. There are no effective antivirals for symptomatic infection, and effective application of current vaccines is limited in areas of endemicity, such that rabies causes ~59,000 deaths per year. Viral subversion of host cell functions, including antiviral immunity, is critical to disease, and isoforms of the lyssavirus P protein are central to the virus-host interface underpinning immune evasion. Here, we show that specific cellular interactions of P protein isoforms involved in immune evasion vary significantly between different lyssaviruses, indicative of distinct strategies to evade immune responses. These findings highlight the diversity of the virus-host interface, an important consideration in the development of pan-lyssavirus therapeutic approaches.