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The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) poses a persistent threat to global public health. Although primarily a respiratory illness, extrapulmonary manifestations of COVID-19 include gastrointestinal, cardiovascular, renal and neurological diseases. Recent studies suggest that dysfunction of the endothelium during COVID-19 may exacerbate these deleterious events by inciting inflammatory and microvascular thrombotic processes. Although controversial, there is evidence that SARS-CoV-2 may infect endothelial cells by binding to the angiotensin-converting enzyme 2 (ACE2) cellular receptor using the viral Spike protein. In this review, we explore current insights into the relationship between SARS-CoV-2 infection, endothelial dysfunction due to ACE2 downregulation, and deleterious pulmonary and extra-pulmonary immunothrombotic complications in severe COVID-19. We also discuss preclinical and clinical development of therapeutic agents targeting SARS-CoV-2-mediated endothelial dysfunction. Finally, we present evidence of SARS-CoV-2 replication in primary human lung and cardiac microvascular endothelial cells. Accordingly, in striving to understand the parameters that lead to severe disease in COVID-19 patients, it is important to consider how direct infection of endothelial cells by SARS-CoV-2 may contribute to this process.

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

Pneumonia resulting from infection is one of the leading causes of death worldwide. Pulmonary infection by the respiratory syncytial virus (RSV) is a large burden on human health, for which there are few therapeutic options 1 . RSV targets ciliated epithelial cells in the airways, but how viruses such as RSV interact with receptors on these cells is not understood. Nucleolin is an entry coreceptor for RSV 2 and also mediates the cellular entry of influenza, the parainfluenza virus, some enteroviruses and the bacterium that causes tularaemia 3 , 4 . Here we show a mechanism of RSV entry into cells in which outside-in signalling, involving binding of the prefusion RSV-F glycoprotein with the insulin-like growth factor-1 receptor, triggers the activation of protein kinase C zeta (PKCζ). This cellular signalling cascade recruits nucleolin from the nuclei of cells to the plasma membrane, where it also binds to RSV-F on virions. We find that inhibiting PKCζ activation prevents the trafficking of nucleolin to RSV particles on airway organoid cultures, and reduces viral replication and pathology in RSV-infected mice. These findings reveal a mechanism of virus entry in which receptor engagement and signal transduction bring the coreceptor to viral particles at the cell surface, and could form the basis of new therapeutics to treat RSV infection. Respiratory syncytial virus enters cells by binding to cell-surface IGFR1, which activates PKCζ and induces trafficking of the NCL coreceptor to the RSV particles at the cell surface.

HIV-associated neurocognitive disorders (HAND) represent a spectrum neurological syndrome that affects up to 25% of patients with HIV/AIDS. Multiple pathogenic mechanisms contribute to the development of HAND symptoms including chronic neuroinflammation and neurodegeneration. Among the factors linked to development of HAND is altered expression of host cell microRNAs (miRNAs) in brain. Here, we examined brain miRNA profiles among HIV/AIDS patients with and without HAND. Our analyses revealed differential expression of 17 miRNAs in brain tissue from HAND patients. A subset of the upregulated miRNAs (miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p), are predicted to target peroxisome biogenesis factors (PEX2, PEX7, PEX11B and PEX13). Expression of these miRNAs in transfected cells significantly decreased levels of peroxisomal proteins and concomitantly decreased peroxisome numbers or affected their morphology. The levels of miR-500a-5p, miR-34c-3p, miR-93-3p and miR-381-3p were not only elevated in the brains of HAND patients, but were also upregulated during HIV infection of primary macrophages. Moreover, concomitant loss of peroxisomal proteins was observed in HIV-infected macrophages as well as in brain tissue from HIV-infected patients. HIV-induced loss of peroxisomes was abrogated by blocking the functions of the upregulated miRNAs. Overall, these findings point to previously unrecognized miRNA expression patterns in the brains of HIV patients. Targeting peroxisomes by up-regulating miRNAs that repress peroxisome biogenesis factors may represent a novel mechanism by which HIV-1 subverts innate immune responses and/or causes neurocognitive dysfunction.

Zika virus is a teratogenic mosquito-transmitted flavivirus that is associated with birth defects in newborns and Guillain–Barré syndrome in adults. The virus can also be sexually transmitted, but currently, very little is known about the cell types supporting virus replication and persistence in human testes. Using primary cell cultures, we observed that Sertoli but not Leydig cells are highly susceptible to Zika virus infection, a process that is dependent on the TAM family receptor Axl. In cell culture, Sertoli cells could be productively infected with Zika virus for at least 6-weeks. Infection of Sertoli cells resulted in dramatic changes to the transcriptional profile of these cells. The most upregulated mRNA in infected cells was basic fibroblast growth factor (FGF2), a cytokine that was found to enhance Zika virus replication and support viral persistence. Together these findings provide key insights into understanding how Zika virus persists in the male reproductive tract and in turn may aid in developing antiviral therapies or strategies to minimize sexual transmission of this pathogen.

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3 to 5 million cases of severe illness and ∼290,000 to 645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannosedependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity.

Peroxisomes are membrane-bounded organelles that contribute to a range of physiological functions in eukaryotic cells. In the central nervous system (CNS), peroxisomes are implicated in several vital homeostatic functions including, but not limited to, reactive oxygen species signaling and homeostasis; generation of critical myelin sheath components (including ether phospholipids); biosynthesis of neuroprotective docosahexaenoic acid; breakdown of neurotoxic metabolites (such as very-long chain fatty acids); and, intriguingly, glial activation and response to inflammatory stimuli. Indeed, peroxisomes play a critical role in modulating inflammatory responses and are key regulators of the mitochondrial antiviral signaling (MAVS) protein-mediated response to infections. The importance of peroxisomes in CNS physiology is exemplified by the peroxisome biogenesis disorders (PBDs), a spectrum of inherited disorders of peroxisome assembly and/or abundance, that are characterized in part by neurological manifestations ranging from severe cerebral malformations to vision and hearing loss, depending on the individual disorder. Recently, peroxisome dysfunction has been implicated in neurological diseases associated with neuroinflammation including Alzheimer’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and Parkinson’s disease while also contributing to the pathogenesis of neurotropic viruses including SARS-CoV-2, Human Pegivirus, HIV-1 and Zika virus. In the present review, we examine the diverse roles that peroxisomes serve in CNS health before reviewing more recent studies investigating peroxisome dysfunction in inflammatory brain disorders and also highlight potential peroxisomal targets for diagnostic biomarkers and therapeutic interventions.

People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis. Human immunodeficiency virus type 1 (HIV-1) establishes lifelong infections in humans, a process that relies on its ability to thwart innate and adaptive immune defenses of the host. Recently, we reported that HIV-1 infection results in a dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic organelles that also function as signaling platforms in the innate immune response. Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient for the depletion of cellular peroxisomes during infection. Vpu induces the expression of four microRNAs that target mRNAs encoding proteins required for peroxisome formation and metabolic function. The ability of Vpu to downregulate peroxisomes was found to be dependent upon the Wnt/β-catenin signaling pathway. Given the importance of peroxisomes in innate immune signaling and central nervous system function, the roles of Vpu in dampening antiviral signaling appear to be more diverse than previously realized. Finally, our findings highlight a potential role for Wnt/β-catenin signaling in peroxisome homeostasis through modulating the production of biogenesis factors. IMPORTANCE People living with HIV can experience accelerated aging and the development of neurological disorders. Recently, we reported that HIV-1 infection results in a dramatic loss of peroxisomes in macrophages and brain tissue. This is significant because (i) peroxisomes are important for the innate immune response and (ii) loss of peroxisome function is associated with cellular aging and neurodegeneration. Accordingly, understanding how HIV-1 infection causes peroxisome depletion may provide clues regarding how the virus establishes persistent infections and, potentially, the development of neurological disorders. Here, we show that the accessory protein Vpu is necessary and sufficient for the induction of microRNAs that target peroxisome biogenesis factors. The ability of Vpu to downregulate peroxisome formation depends on the Wnt/β-catenin pathway. Thus, in addition to revealing a novel mechanism by which HIV-1 uses intracellular signaling pathways to target antiviral signaling platforms (peroxisomes), we have uncovered a previously unknown link between the Wnt/β-catenin pathway and peroxisome homeostasis.

Zika virus (ZIKV) infection and persistence during pregnancy can lead to microcephaly and other fetal neurological disorders collectively known as Congenital Zika Syndrome. The immunological and virological events that contribute to the establishment of persistent ZIKV infection in humans are unclear though. Here we show that human fetal astrocytes (HFAs), the most abundant cell type in the central nervous system, become persistently infected with ZIKV resulting in continuous viral shedding for at least one month; a process that is facilitated by TIM/TAM receptors. HFAs are relatively resistant to ZIKV-induced apoptosis, a factor that may be important for chronic infection of these cells. Once infection was established, interferon treatment did not reduce virus replication. Moreover, the fact that the innate immune system was highly activated in persistently infected HFAs indicates that the virus can thrive in the presence of a sustained antiviral response. RNAseq analyses of persistently infected cells revealed that ZIKV alters host gene expression in a manner that could affect developmental processes. Conversely, data from sequencing of ZIKV genomes in persistently infected HFAs suggest that adaptive mutations were not required for establishing chronic infection. Based on these results, we postulate that HFAs are reservoirs for ZIKV in the fetal brain and that moderate apoptosis combined with inefficient antiviral response from these cells may contribute to the establishment of chronic brain infection associated with the ZIKV neurodevelopmental abnormalities.

The type 1 interferon-regulated E3 ubiquitin ligase MARCH1 reduces surface expression of HIV-1 envelope glycoproteins (Env) and their packaging into nascent virions, a condition that restricts viral infectivity. However, how HIV-1 counters this restriction, notably during infection of macrophages, remains unclear. Here, we show that the HIV-1 accessory protein Vpu increases the levels of microRNAs-25 and -93 to target MARCH1 mRNA. By recruiting β-TRCP, a component of the SCF β-TRCP E3 ligase complex that targets phosphorylated β-catenin for degradation, Vpu increases β-catenin levels, which, in concert with TCF4/LEF, drives transcription of the MARCH1 -targeting microRNAs. This potentiates HIV-1 infectivity as a result of increased Env incorporation into nascent virions. Pharmacological targeting of the β-catenin pathway inhibits Vpu-mediated upregulation of microRNAs-25 and -93 and restores MARCH1 restriction on HIV-1 infectivity. Overall, our findings highlight a novel mechanism by which HIV-1 counteracts MARCH1 by downregulating its expression via Vpu-mediated induction of microRNAs-25 and -93. In order to efficiently produce infectious viral particles, HIV must counter several restrictions exerted by host cell antiviral proteins. MARCH1 is a member of the MARCH protein family that restricts HIV infection by limiting the incorporation of viral envelope glycoproteins into nascent virions. Here, we identified two regulatory RNAs, microRNAs-25 and -93, induced by the HIV-1 accessory protein Vpu, that downregulate MARCH1 mRNA. We also show that Vpu induces these cellular microRNAs in macrophages by hijacking the cellular β-catenin pathway. The notion that HIV-1 has evolved a mechanism to counteract MARCH1 restriction on viral infectivity underlines the importance of MARCH1 in the host antiviral response.