Irreversible Inactivation of SARS-CoV-2 by Lectin Engagement with Two Glycan Clusters on the Spike Protein.

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins cyanovirin-N (CV-N) and griffithsin (GRFT). We discovered in this study that CV-N not only inhibits SARS-CoV-2 infection but also leads to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with CV-N and then washed to remove all soluble lectin did not recover infectivity. The infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition: one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable spike before host cell encounter.

Surfactant-Impregnated MOF-Coated Fabric for Antimicrobial Applications.

Since the onset of the SARS-CoV-2 pandemic, the world has witnessed over 617 million confirmed cases and more than 6.54 million confirmed deaths, but the actual totals are likely much higher. The virus has mutated at a significantly faster rate than initially projected, and positive cases continue to surge with the emergence of ever more transmissible variants. According to the CDC, and at the time of this manuscript submission, more than 77% of all current US cases are a result of the B.5 (omicron). The continued emergence of highly transmissible variants makes clear the need for more effective methods of mitigating disease spread. Herein, we have developed an antimicrobial fabric capable of destroying a myriad of microbes including betacoronaviruses. We have demonstrated the capability of this highly porous and nontoxic metal organic framework (MOF), γ-CD-MOF-1, to serve as a host for varied-length benzalkonium chlorides (BACs; active ingredient in Lysol). Molecular docking simulations predicted a binding affinity of up to -4.12 kcal·mol-1, which is comparable to that of other reported guest molecules for this MOF. Similar Raman spectra and powder X-ray diffraction patterns between the unloaded and loaded MOFs, accompanied by a decrease in the Brunauer-Emmett-Teller surface area from 616.20 and 155.55 m2 g-1 respectively, corroborate the suggested potential for pore occupation with BAC. The MOF was grown on polypropylene fabric, exposed to a BAC-loading bath, washed to remove excess BAC from the external surface, and evaluated for its microbicidal activity against various bacterial and viral classes. Significant antimicrobial character was observed against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, bacteriophage, and betacoronavirus. This study shows that a common mask material (polypropylene) can be coated with BAC-loaded γ-CD-MOF-1 while maintaining the guest molecule's antimicrobial effects.

Induction of a Senescence-Like Phenotype in Cultured Human Fetal Microglia During HIV-1 Infection.

HIV-1 causes premature aging in chronically infected patients. Despite effective anti-retroviral therapy, around 50% of patients suffer HIV-associated neurocognitive disorders (HAND), which likely potentiate aging-associated neurocognitive decline. Microglia support productive HIV-1 infection in the brain. Elevated markers of cellular senescence, including p53 and p21, have been detected in brain tissues from patients with HAND, but the potential for microglia senescence during HIV-1 infection has not been investigated. We hypothesized that HIV-1 can induce senescence in microglia. Primary human fetal microglia were exposed to single-round infectious HIV-1 pseudotypes or controls, and examined for markers of senescence. Post-infection, microglia had significantly elevated: senescence-associated ß-galactosidase activity, p21 levels, and production of cytokines such as IL-6 and IL-8, potentially indicative of a senescence-associated secretory phenotype. We also found increased detection of p53-binding protein foci in microglia nuclei post-infection. Additionally, we examined mitochondrial reactive oxygen species (ROS) and respiration, and found significantly increased mitochondrial ROS levels and decreased ATP-linked respiration during HIV-1 infection. Supernatant transfer from infected cultures to naïve microglia resulted in elevated p21 and caveolin-1 levels, and IL-8 production. Finally, nucleoside treatment reduced senescence markers induction in microglia. Overall, HIV-1 induces a senescence-like phenotype in human microglia, which could play a role in HAND.

Interplay between microRNAs, Toll-like receptors, and HIV-1: potential implications in HIV-1 replication and chronic immune activation.

MicroRNAs (miRNAs) are important cellular, small non-coding RNAs that regulate host gene expression and have well-characterized roles in inflammation and infectious diseases. It has become apparent as well that cellular miRNAs can play crucial roles in controlling HIV-1 infection and replication. Whether HIV-1 encodes and is able to express viral miRNAs in infected cells remains controversial. HIV-1 can manipulate the biogenesis of miRNAs as well as the expression profiles of cellular miRNAs. Toll-Like receptors (TLRs) are important pathogen recognition receptors that sense invading pathogens orchestrating innate and adaptive immune responses. Innate immune recognition of HIV-1 infection leads to activation of TLR7/8. Recent evidence has shown that certain miRNAs can also be recognized by TLR7/8 leading to immune activation. However, the potential TLR7/8-mediated recognition of HIV-1 encoded miRNAs and/or cellular miRNAs modulated in HIV-1 infected cells has not been experimentally explored. In this review, we summarize the current literature on HIV-1 infection and miRNAs. Furthermore, we underscore the need for future research on potential miRNA-induced activation of TLR7/8, which might contribute to the chronic immune activation observed in HIV-1 infected patients.

Hepatitis C virus core protein enhances HIV-1 replication in human macrophages through TLR2, JNK, and MEK1/2-dependent upregulation of TNF-α and IL-6.

Despite their differential cell tropisms, HIV-1 and HCV dramatically influence disease progression in coinfected patients. Macrophages are important target cells of HIV-1. We hypothesized that secreted HCV core protein might modulate HIV-1 replication. We demonstrate that HCV core significantly enhances HIV-1 replication in human macrophages by upregulating TNF-α and IL-6 via TLR2-, JNK-, and MEK1/2-dependent pathways. Furthermore, we show that TNF-α and IL-6 secreted from HCV core-treated macrophages reactivates monocytic U1 cells latently infected with HIV-1. Our studies reveal a previously unrecognized role of HCV core by enhancing HIV-1 infection in macrophages.

MicroRNAs and HIV-1 infection: antiviral activities and beyond.

Cellular microRNAs (miRNAs) are an important class of small, non-coding RNAs that bind to host mRNAs based on sequence complementarity and regulate protein expression. They play important roles in controlling key cellular processes including cellular inception, differentiation and death. While several viruses have been shown to encode for viral miRNAs, controversy persists over the expression of a functional miRNA encoded in the human immunodeficiency virus type 1 (HIV-1) genome. However, it has been reported that HIV-1 infectivity is influenced by cellular miRNAs. Either through directly targeting the viral genome or by targeting host cellular proteins required for successful virus replication, multiple cellular miRNAs seem to modulate HIV-1 infection and replication. Perhaps as a survival strategy, HIV-1 may modulate proteins in the miRNA biogenesis pathway to subvert miRNA-induced antiviral effects. Global expression profiles of cellular miRNAs have also identified alterations of specific miRNAs post-HIV-1 infection both in vitro and in vivo (in various infected patient cohorts), suggesting potential roles for miRNAs in pathogenesis and disease progression. However, little attention has been devoted in understanding the roles played by these miRNAs at a cellular level. In this manuscript, we review past and current findings pertaining to the field of miRNA and HIV-1 interplay. In addition, we suggest strategies to exploit miRNAs therapeutically for curbing HIV-1 infectivity, replication and latency since they hold an untapped potential that deserves further investigation.

RNA viruses and microRNAs: challenging discoveries for the 21st century.

RNA viruses represent the predominant cause of many clinically relevant viral diseases in humans. Among several evolutionary advantages acquired by RNA viruses, the ability to usurp host cellular machinery and evade antiviral immune responses is imperative. During the past decade, RNA interference mechanisms, especially microRNA (miRNA)-mediated regulation of cellular protein expression, have revolutionized our understanding of host-viral interactions. Although it is well established that several DNA viruses express miRNAs that play crucial roles in their pathogenesis, expression of miRNAs by RNA viruses remains controversial. However, modulation of the miRNA machinery by RNA viruses may confer multiple benefits for enhanced viral replication and survival in host cells. In this review, we discuss the current literature on RNA viruses that may encode miRNAs and the varied advantages of engineering RNA viruses to express miRNAs as potential vectors for gene therapy. In addition, we review how different families of RNA viruses can alter miRNA machinery for productive replication, evasion of antiviral immune responses, and prolonged survival. We underscore the need to further explore the complex interactions of RNA viruses with host miRNAs to augment our understanding of host-virus interplay.

Toll-like receptor 3 in viral pathogenesis: friend or foe?

Viral infections frequently induce acute and chronic inflammatory diseases, yet the contribution of the innate immune response to a detrimental host response remains poorly understood. In virus-infected cells, double-stranded RNA (dsRNA) is generated as an intermediate during viral replication. Cell necrosis (and the release of endogenous dsRNA) is a common event during both sterile and infectious inflammatory processes. The discovery of Toll-like receptor 3 (TLR3) as an interferon-inducing dsRNA sensor led to the assumption that TLR3 was the master sentinel against viral infections. This simplistic view has been challenged by the discovery of at least three members of the DExd/H-box helicase cytosolic sensors of dsRNA that share with TLR3 the Toll-interleukin-1 receptor (TIR) -adapter molecule TIR domain-containing adaptor protein interferon-ß (TRIF) for downstream type I interferon signalling. Data are conflicting on the role of TLR3 in protective immunity against viruses in the mouse model. Varying susceptibility to infection and disease outcomes have been reported in TLR3-immunodeficient mice. Surprisingly, the susceptibility to develop herpes simplex virus-1 encephalitis in humans with inborn defects of the TLR3 pathway varies, and TLR3-deficient humans do not show increased susceptibility to other viral infections. Therefore, a current challenge is to understand the protective versus pathogenic contribution of TLR3 in viral infections. We review recent advances in the identification of TLR3-signalling pathways, endogenous and virus-induced negative regulators of the TLR3 cascade, and discuss the protective versus pathogenic role of TLR3 in viral pathogenesis.

MicroRNAs, hepatitis C virus, and HCV/HIV-1 co-infection: new insights in pathogenesis and therapy.

MicroRNAs (miRNAs) can exert a profound effect on Hepatitis C virus (HCV) replication. The interaction of HCV with the highly liver-enriched miRNA, miR-122 represents one such unique example of viruses having evolved mechanism(s) to usurp the host miRNA machinery to support viral life cycle. Furthermore, HCV infection can also trigger changes in the cellular miRNA profile, which may ultimately contribute to the outcome of viral infection. Accumulating knowledge on HCV-host miRNA interactions has ultimately influenced the design of therapeutic interventions against chronic HCV infection. The importance of microRNA modulation in Human Immunodeficiency Virus (HIV-1) replication has been reported, albeit only in the context of HIV-1 mono-infection. The development of HCV infection is dramatically influenced during co-infection with HIV-1. Here, we review the current knowledge on miRNAs in HCV mono-infection. In addition, we discuss the potential role of some miRNAs, identified from the analyses of public data, in HCV/HIV-1 co-infection.

A role for microRNA-155 modulation in the anti-HIV-1 effects of Toll-like receptor 3 stimulation in macrophages.

HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I:C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood. We found that poly(I:C)- and LPS-stimulation of MDMs abrogated infection by CCR5-using, macrophage-tropic HIV-1, and by vesicular stomatitis virus glycoprotein-pseudotyped HIV-1 virions, while TLR2, TLR7 or TLR9 agonists only partially reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines or ß-chemokines. Integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). Using microarray analysis and quantitative reverse transcription (RT)-PCR, we found increased microRNA (miR)-155 levels in MDMs upon TLR3/4- but not TLR7-stimulation, and a miR-155 specific inhibitor (but not a scrambled control) partially restored infectivity in poly(I:C)-stimulated MDMs. Ectopic miR-155 expression remarkably diminished HIV-1 infection in primary MDMs and cell lines. Furthermore, poly(I:C)-stimulation and ectopic miR-155 expression did not alter detection of early viral RT products, but both resulted in an accumulation of late RT products and in undetectable or extremely low levels of integrated pro-viruses and 2-LTR circles. Reduced mRNA and protein levels of several HIV-1 dependency factors involved in trafficking and/or nuclear import of pre-integration complexes (ADAM10, TNPO3, Nup153, LEDGF/p75) were found in poly(I:C)-stimulated and miR-155-transfected MDMs, and a reporter assay suggested they are authentic miR-155 targets. Our findings provide evidence that miR-155 exerts an anti-HIV-1 effect by targeting several HIV-1 dependency factors involved in post-entry, pre-integration events, leading to severely diminished HIV-1 infection.

Protective role of Toll-like Receptor 3-induced type I interferon in murine coronavirus infection of macrophages.

Toll-like Receptors (TLRs) sense viral infections and induce production of type I interferons (IFNs), other cytokines, and chemokines. Viral recognition by TLRs and other pattern recognition receptors (PRRs) has been proven to be cell-type specific. Triggering of TLRs with selected ligands can be beneficial against some viral infections. Macrophages are antigen-presenting cells that express TLRs and have a key role in the innate and adaptive immunity against viruses. Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses that cause acute and chronic infections and can productively infect macrophages. Investigation of the interplay between CoVs and PRRs is in its infancy. We assessed the effect of triggering TLR2, TLR3, TLR4, and TLR7 with selected ligands on the susceptibility of the J774A.1 macrophage cell line to infection with murine coronavirus (mouse hepatitis virus, [MHV]). Stimulation of TLR2, TLR4, or TLR7 did not affect MHV production. In contrast, pre-stimulation of TLR3 with polyinosinic-polycytidylic acid (poly I:C) hindered MHV infection through induction of IFN-ß in macrophages. We demonstrate that activation of TLR3 with the synthetic ligand poly I:C mediates antiviral immunity that diminishes (MHV-A59) or suppresses (MHV-JHM, MHV-3) virus production in macrophages.

Hepatitis B and C virus hepatocarcinogenesis: lessons learned and future challenges.

Worldwide, hepatocellular carcinoma (HCC) is one of the most common cancers. It is thought that 80% of hepatocellular carcinomas are linked to chronic infections with the hepatitis B (HBV) or hepatitis C (HCV) viruses. Chronic HBV and HCV infections can alter hepatocyte physiology in similar ways and may utilize similar mechanisms to influence the development of HCC. There has been significant progress towards understanding the molecular biology of HBV and HCV and identifying the cellular signal transduction pathways that are altered by HBV and HCV infections. Although the precise molecular mechanisms that link HBV and HCV infections to the development of HCC are not entirely understood, there is considerable evidence that both inflammatory responses to infections with these viruses, and associated destruction and regeneration of hepatocytes, as well as activities of HBV- or HCV-encoded proteins, contribute to hepatocyte transformation. In this review, we summarize progress in defining mechanisms that may link HBV and HCV infections to the development of HCC, discuss the challenges of directly defining the processes that underlie HBV- and HCV-associated HCC, and describe areas that remain to be explored.

The V1-V3 region of a brain-derived HIV-1 envelope glycoprotein determines macrophage tropism, low CD4 dependence, increased fusogenicity and altered sensitivity to entry inhibitors.

BACKGROUND: HIV-1 infects macrophages and microglia in the brain and can cause neurological disorders in infected patients. We and others have shown that brain-derived envelope glycoproteins (Env) have lower CD4 dependence and higher avidity for CD4 than those from peripheral isolates, and we have also observed increased fusogenicity and reduced sensitivity to the fusion inhibitor T-1249. Due to the genetic differences between brain and spleen env from one individual throughout gp120 and in gp41's heptad repeat 2 (HR2), we investigated the viral determinants for the phenotypic differences by performing functional studies with chimeric and mutant Env. RESULTS: Chimeric Env showed that the V1/V2-C2-V3 region in brain's gp120 determines the low CD4 dependence and high avidity for CD4, as well as macrophage tropism and reduced sensitivity to the small molecule BMS-378806. Changes in brain gp41's HR2 region did not contribute to the increased fusogenicity or to the reduced sensitivity to T-1249, since a T-1249-based peptide containing residues found in brain's but not in spleen's HR2 had similar potency than T-1249 and interacted similarly with an immobilized heptad repeat 1-derived peptide in surface plasmon resonance analysis. However, the increased fusogenicity and reduced T-1249 sensitivity of brain and certain chimeric Env mostly correlated with the low CD4 dependence and high avidity for CD4 determined by brain's V1-V3 region. Remarkably, most but not all of these low CD4-dependent, macrophage tropic envelopes glycoproteins also had increased sensitivity to the novel allosteric entry inhibitor HNG-105. The gp120's C2 region asparagine 283 (N283) has been previously associated with macrophage tropism, brain infection, lower CD4 dependence and higher CD4 affinity. Therefore, we introduced the N283T mutation into an env clone from a brain-derived isolate and into a brain tissue-derived env clone, and the T283N change into a spleen-derived env from the same individual; however, we found that their phenotypes were not affected. CONCLUSION: We have identified that the V1-V3 region of a brain-derived envelope glycoprotein seems to play a crucial role in determining not only the low CD4 dependence and increased macrophage tropism, but also the augmented fusogenicity and reduced sensitivity to T-1249 and BMS-378806. By contrast, increased sensitivity to HNG-105 mostly correlated with low CD4 dependence and macrophage tropism but was not determined by the presence of the brain's V1-V3 region, confirming that viral determinants of phenotypic changes in brain-derived envelope glycoproteins are likely complex and context-dependent.

Replicase genes of murine coronavirus strains A59 and JHM are interchangeable: differences in pathogenesis map to the 3' one-third of the genome.

The important roles of the spike protein and other structural proteins in murine coronavirus (MHV) pathogenesis have been demonstrated; however, the role of the replicase gene remains unexplored. We assessed the influence of the replicase genes of the highly neurovirulent MHV-JHM strain and the hepatotropic and mildly neurovirulent A59 strain in acute infection of the mouse. Analysis of chimeric A59/JHM recombinant viruses indicates that the replicase genes are interchangeable and that it is the 3' end of the genome, encoding the structural proteins, rather than the replicase gene, that determines the pathogenic properties of these chimeras.

Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus.

Coronaviruses are a family of enveloped, single-stranded, positive-strand RNA viruses classified within the Nidovirales order. This coronavirus family consists of pathogens of many animal species and of humans, including the recently isolated severe acute respiratory syndrome coronavirus (SARS-CoV). This review is divided into two main parts; the first concerns the animal coronaviruses and their pathogenesis, with an emphasis on the functions of individual viral genes, and the second discusses the newly described human emerging pathogen, SARS-CoV. The coronavirus part covers (i) a description of a group of coronaviruses and the diseases they cause, including the prototype coronavirus, murine hepatitis virus, which is one of the recognized animal models for multiple sclerosis, as well as viruses of veterinary importance that infect the pig, chicken, and cat and a summary of the human viruses; (ii) a short summary of the replication cycle of coronaviruses in cell culture; (iii) the development and application of reverse genetics systems; and (iv) the roles of individual coronavirus proteins in replication and pathogenesis. The SARS-CoV part covers the pathogenesis of SARS, the developing animal models for infection, and the progress in vaccine development and antiviral therapies. The data gathered on the animal coronaviruses continue to be helpful in understanding SARS-CoV.

Expression and purification of SARS coronavirus proteins using SUMO-fusions.

Severe acute respiratory syndrome coronavirus (SARS-CoV) proteins belong to a large group of proteins that is difficult to express in traditional expression systems. The ability to express and purify SARS-CoV proteins in large quantities is critical for basic research and for development of pharmaceutical agents. The work reported here demonstrates: (1) fusion of SUMO (small ubiquitin-related modifier), a 100 amino acid polypeptide, to the N-termini of SARS-CoV proteins dramatically enhances expression in Escherichia coli cells and (2) 6x His-tagged SUMO-fusions facilitate rapid purification of the viral proteins on a large scale. We have exploited the natural chaperoning properties of SUMO to develop an expression system suitable for proteins that cannot be expressed by traditional methodologies. A unique feature of the system is the SUMO tag, which enhances expression, facilitates purification, and can be efficiently cleaved by a SUMO-specific protease to generate native protein with a desired N-terminus. We have purified various SARS-CoV proteins under either native or denaturing conditions. These purified proteins have been used to generate highly specific polyclonal antibodies. Our study suggests that the SUMO-fusion technology will be useful for enhancing expression and purification of the viral proteins for structural and functional studies as well as for therapeutic uses.

Murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

Murine coronavirus A59 strain causes mild to moderate hepatitis in mice. We have previously shown that mutants of A59, unable to induce hepatitis, may be selected by persistent infection of primary glial cells in vitro. These in vitro isolated mutants encoded two amino acids substitutions in the spike (S) gene: Q159L lies in the putative receptor binding domain of S, and H716D, within the cleavage signal of S. Here, we show that hepatotropic revertant variants may be selected from these in vitro isolated mutants (Q159L-H716D) by multiple passages in the mouse liver. One of these mutants, hr2, was chosen for more in-depth study based on a more hepatovirulent phenotype. The S gene of hr2 (Q159L-R654H-H716D-E1035D) differed from the in vitro isolates (Q159L-H716D) in only 2 amino acids (R654H and E1035D). Using targeted RNA recombination, we have constructed isogenic recombinant MHV-A59 viruses differing only in these specific amino acids in S (Q159L-R654H-H716D-E1035D). We demonstrate that specific amino acid substitutions within the spike gene of the hr2 isolate determine the ability of the virus to cause lethal hepatitis and replicate to significantly higher titers in the liver compared to wild-type A59. Our results provide compelling evidence of the ability of coronaviruses to rapidly evolve in vivo to highly virulent phenotypes by functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein.

Coronavirus replication and pathogenesis: Implications for the recent outbreak of severe acute respiratory syndrome (SARS), and the challenge for vaccine development.

A novel coronavirus has been recently identified as the causative agent of the severe acute respiratory syndrome (SARS) outbreak that has accounted for more than 8000 infected people worldwide. This review will discuss current knowledge on coronavirus replication, pathogenesis, evolution, and vaccine strategies, as well as the most recent findings on SARS coronavirus.

Sabadinine: a potential non-peptide anti-severe acute-respiratory-syndrome agent identified using structure-aided design.

A novel human coronavirus has been reported to be the causative agent of severe acute respiratory syndrome (SARS). Since replication of HcoVs depends on extensive proteolytic processing, the main proteinase, 3CLpro, is an attractive drug target for anti-SARS agents. We have employed molecular docking of a chemical database into the active site of 3CLpro to search for non-peptidyl inhibitors. One compound was identified to be the natural product sabadinine, isolated from a historical herbal remedy.