In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5′ phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently. Full article

Japanese encephalitis (JE) is inflammation and swelling of the brain caused by the JE virus (JEV), a mosquito-borne member of the Flavivirus family. There are around 68,000 JE cases worldwide each year, many of which result in permanent brain damage and death. There is no specific treatment for JE. Here we present the crystal structure of the JEV capsid protein, a potential drug target, at 1.98 Å, and compare it to other flavivirus capsid proteins. The JEV capsid has a helical secondary structure (α helixes 1–4) and a similar protein fold to the dengue virus (DENV), the West Nile virus (WNV), and the Zika virus (ZIKV) capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘) through α-helix 1-1’, 2-2’, and 4-4’ interactions. This dimeric form is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix-1 allows the formation of closed and open conformations with possible functional importance. The basic C-terminal pairing of α4-4’ forms a coiled-coil-like structure, indicating possible nucleic acid binding functionality. However, a comparison with other nucleic acid interacting domains indicates that homodimerization would preclude binding. This is the first JEV capsid protein to be described and is an addition to the structural biology of the Flavivirus. Full article

Respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. However, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). Respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. Furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza A and SARS-CoV, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. For a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (CNS). Viruses infecting human CNS cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. Like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the CNS as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to CNS cells (virus-induced neuropathology). The etiological agent of several neurological disorders remains unidentified. Opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. Herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in CNS infection, with a special emphasis on human coronaviruses. Full article

In 2014, the United States (US) experienced an unprecedented epidemic of enterovirus D68 (EV-D68)-induced respiratory disease that was temporally associated with the emergence of acute flaccid myelitis (AFM), a paralytic disease occurring predominantly in children, that has a striking resemblance to poliomyelitis. Although a definitive causal link between EV-D68 infection and AFM has not been unequivocally established, rapidly accumulating clinical, immunological, and epidemiological evidence points to EV-D68 as the major causative agent of recent seasonal childhood AFM outbreaks in the US. This review summarizes evidence, gained from in vivo and in vitro models of EV-D68-induced disease, which demonstrates that contemporary EV-D68 strains isolated during and since the 2014 outbreak differ from historical EV-D68 in several factors influencing neurovirulence, including their genomic sequence, their receptor utilization, their ability to infect neurons, and their neuropathogenicity in mice. These findings provide biological plausibility that EV-D68 is a causal agent of AFM and provide important experimental models for studies of pathogenesis and treatment that are likely to be difficult or impossible in humans. Full article

The California serogroup (CSG) comprises 18 serologically and genetically related mosquito-borne orthobunyaviruses. Of these viruses, at least seven have been shown to cause neurological disease in humans, including the leading cause of pediatric arboviral encephalitis in the USA, La Crosse virus. Despite the disease burden from these viruses, much is still unknown about the CSG viruses. This review summarizes our current knowledge of the CSG viruses, including human disease and the mechanisms of neuropathogenesis. Full article