Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes.

The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive "LAMPshade" dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

Identification of Ebola Virus Inhibitors Targeting GP2 Using Principles of Molecular Mimicry.

A key step in the Ebola virus (EBOV) replication cycle involves conformational changes in viral glycoprotein 2 (GP2) which facilitate host-viral membrane fusion and subsequent release of the viral genome. Ebola GP2 plays a critical role in virus entry and has similarities in mechanism and structure to the HIV gp41 protein for which inhibitors have been successfully developed. In this work, a putative binding pocket for the C-terminal heptad repeat in the N-terminal heptad repeat trimer was targeted for identification of small molecules that arrest EBOV-host membrane fusion. Two computational structure-based virtual screens of ∼1.7 M compounds were performed (DOCK program) against a GP2 five-helix bundle, resulting in 165 commercially available compounds purchased for experimental testing. Based on assessment of inhibitory activity, cytotoxicity, and target specificity, four promising candidates emerged with 50% inhibitory concentration values in the 3 to 26 µM range. Molecular dynamics simulations of the two most potent candidates in their DOCK-predicted binding poses indicate that the majority of favorable interactions involve seven highly conserved residues that can be used to guide further inhibitor development and refinement targeting EBOV.IMPORTANCE The most recent Ebola virus disease outbreak, from 2014 to 2016, resulted in approximately 28,000 individuals becoming infected, which led to over 12,000 causalities worldwide. The particularly high pathogenicity of the virus makes paramount the identification and development of promising lead compounds to serve as inhibitors of Ebola infection. To limit viral load, the virus-host membrane fusion event can be targeted through the inhibition of the class I fusion glycoprotein of Ebolavirus In the current work, several promising small-molecule inhibitors that target the glycoprotein GP2 were identified through systematic application of structure-based computational and experimental drug design procedures.

Structure-based identification of inhibitors targeting obstruction of the HIVgp41 N-heptad repeat trimer.

The viral protein HIVgp41 is an attractive and validated drug target that proceeds through a sequence of conformational changes crucial for membrane fusion, which facilitates viral entry. Prior work has identified inhibitors that interfere with the formation of a required six-helix bundle, composed of trimeric C-heptad (CHR) and N-heptad (NHR) repeat elements, through blocking association of an outer CHR helix or obstructing formation of the inner NHR trimer itself. In this work, we employed similarity-based scoring to identify and experimentally characterize 113 compounds, related to 2 small-molecule inhibitors recently reported by Allen et al. (Bioorg. Med. Chem Lett.2015, 25 2853-59), proposed to act via the NHR trimer obstruction mechanism. The compounds were first tested in an HIV cell-cell fusion assay with the most promising evaluated in a second, more biologically relevant viral entry assay. Of the candidates, compound #11 emerged as the most promising hit (IC50=37.81µM), as a result of exhibiting activity in both assays with low cytotoxicity, as was similarly seen with the known control peptide inhibitor C34. The compound also showed no inhibition of VSV-G pseudotyped HIV entry compared to a control inhibitor suggesting it was specific for HIVgp41. Molecular dynamics simulations showed the predicted DOCK pose of #11 interacts with HIVgp41 in an energetic fashion (per-residue footprints) similar to the four native NHR residues (IQLT) which candidate inhibitors were intended to mimic.

Small molecule inhibitors of HIVgp41 N-heptad repeat trimer formation.

Identification of mechanistically novel anti-HIV fusion inhibitors was accomplished using a computer-aided structure-based design approach with the goal of blocking the formation of the N-heptad repeat (NHR) trimer of the viral protein gp41. A virtual screening strategy that included per-residue interaction patterns (footprints) was employed to identify small molecules compatible with putative binding pockets at the internal interface of the NHR helices at the core native viral six-helix bundle. From a screen of ∼2.8 million compounds using the DOCK program, 120 with favorable energetic and footprint overlap characteristics were purchased and experimentally tested leading to two compounds with favorable cell-cell fusion (IC50) and cytotoxicity profiles. Importantly, both hits were identified on the basis of scores containing footprint overlap terms and would not have been identified using the standard DOCK energy function alone. To our knowledge, these compounds represent the first reported small molecules that inhibit viral entry via the proposed NHR-trimer obstruction mechanism.

The membrane proximal external regions of gp41 from HIV-1 strains HXB2 and JRFL have different sensitivities to alanine mutation.

The transmembrane subunit (gp41) of the HIV envelope protein complex (Env) mediates the viral fusion step of HIV entry. The membrane proximal external region (MPER), one of the functional domains of gp41, has been the focus of a great deal of research because it is a target for neutralizing antibodies. In this study, we examined 23 amino acid residues in the MPER (660-683) in both a CXCR4 coreceptor-utilizing strain (HXB2) and a CCR5-utilizing strain (JRFL) by alanine scanning mutagenesis. Despite the high degree of gp41 sequence conservation, the effects of alanine mutation in the MPER were different between the two strains. Most mutations in HXB2 had fusogenicity and protein expression levels not less than 50% of that of the wild type in the case of cell-cell fusion. However, ∼30% of the mutants in HXB2 showed a severe defect in fusogenicity in viral entry. Mutations in the MPER of strain JRFL had more dramatic effects than that in HXB2 in cell-cell fusion and viral entry. The fact that there are large differences in the effects of mutation between two strains suggests the potential for the interaction of the MPER with nonconserved sequences such as the fusion peptide and/or other NHR domains as well as potential long-range structural effects on the conformational changes that occur with the Env complex during membrane fusion.

Development of a new cell culture-based method and optimized protocol for the detection of enteric viruses.

The development of rapid and effective methods to detect water- and food-borne enteric viruses is important for the prevention and control of mass infection. This study represents an attempt to develop a reliable cell culture-based detection system and optimize an effective and rapid protocol for the assaying of environmental samples for the presence of infectious enteric viruses. Six enteric viruses were used in this study: poliovirus, Coxsackie virus A9, Coxsackie virus B5, human rotavirus G1, hepatitis A virus, and adenovirus type 41. Among the cell lines from humans (A549, HeLa, HEK293, and HFF) and other primates (Vero, BS-C-1, FRhK-4, BGMK, and MA104), a cytopathic effect (CPE) analysis indicated that the MA104 cell line was the most optimal for use in the detection of infectious enteric viruses. Both the sensitivity and specificity of virus detection in MA104 cells were similar to or higher than those in standard BGMK cells. Next, a method was developed for the determination of the infectiousness of enteric viruses using the colorimetric thiazolyl blue (MTT) assay. This assay utilizes 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to yield % values based on colorimetric results. These results were compared with those from a conventional CPE-based TCID(50) assay, revealing no statistically significant difference between the two methods. The MTT% values in MA104 cells were comparable to those in BGMK cells. This MA104 cell-based MTT assay could substitute for the classical BGMK cell-based CPE assay for infectious enteric viruses.

Significant differences in cell-cell fusion and viral entry between strains revealed by scanning mutagenesis of the C-heptad repeat of HIV gp41.

The transmembrane subunit, gp41, of the HIV envelope mediates the viral fusion step of entry into the host cell. The protein consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular domain contains a fusion peptide, an N-terminal heptad repeat, a loop region, a C-terminal heptad repeat (CHR), and a membrane-proximal external region. For this study, we examined each amino acid in the CHR (residues 623-659) by alanine scanning mutagenesis in two HIV strains: one CCR5-utilizing strain (JRFL) and one CXCR4-utilizing strain (HXB2). We studied the functional importance of each amino acid residue by measuring mutational effects in both cell-cell fusion and viral entry and assessing envelope expression and gp120-gp41 proteolytic processing. The transmembrane subunit of the HIV envelope, gp41, is very sensitive to subtle changes, like alanine substitution, which severely affect envelope function at multiple sites. Two important general findings are apparent when the entire data set from this study is taken into account. (1) Strain HXB2 is much more stable to mutagenesis than strain JRFL, and (2) viral entry is much more stable to mutagenesis than cell-cell fusion. These findings strengthen our notion that gp41 is a vulnerable target for therapeutic and prophylactic intervention. Further structural studies aimed at gaining a full understanding of the intermediate states that drive HIV membrane fusion are imperative.

Inhibition of HIV entry by extracellular glucuronoxylomannan of Cryptococcus neoformans.

Cryptococcosis, caused by Cryptococcus neoformans, is the most common opportunistic fungal disease in HIV/AIDS patients. The prognosis of AIDS patients with Cryptococcus infection is very poor. One of the major characteristics in cryptococcosis patients is the presence of high concentrations of the cryptococcal capsule polysaccharide (CCP) in the serum and cerebrospinal fluid. CCP enhances HIV replication in H9 T-cells, but the mechanism is unknown. In this study, we tested whether extracellular glucuronoxylomannan (GXM), a major component of CCP, enhances HIV entry using replication-incompetent HIV and a cell line which expresses a stable amount of CD4 and both of the HIV co-receptors. Extracellular GXM had no effect on cell-cell fusion however; viral entry surprisingly was inhibited by GXM. Hence, any enhancement of replication must be due to an effect that occurs post-entry.

Permanent inhibition of viral entry by covalent entrapment of HIV gp41 on the virus surface.

HIV entry occurs by concerted conformational changes in the envelope protein complex on the surface of the virus. This complex is made up of a trimer of heterodimers of two subunits: surface subunit, gp120, and transmembrane subunit, gp41. Conformational changes in the envelope complex allow gp41 to mediate membrane fusion leading to exposure of two gp41 regions: N-heptad repeat (NHR) and C-heptad repeat (CHR). Peptides from the NHR or the CHR have been found to inhibit HIV entry. Herein we show that we can covalently inhibit HIV viral entry by permanently trapping the gp41 intermediate on the virus surface using a covalently reactive group on inhibitory peptides. This is evidence showing that vulnerable conformational intermediates exist transiently during HIV viral entry, and the details presented herein will facilitate development of envelope as a target for therapeutics and potential chemopreventive agents that could disable the virus before contact with the host cell.

Cellular signals involved in cyclooxygenase-2 expression induced by human cytomegalovirus.

Cyclooxygenase (COX)-2 plays an important role in inflammatory reaction and human cytomegalovirus (HCMV) is known to induce COX-2 gene expression. However, little is known for the mechanism of COX-2 gene expression by HCMV. In this study, three recently isolated HCMV strains including TB40/E and clinical isolates from Korean patients as well as highly laboratory adapted strain AD169 were used to stimulate COX-2 induction. Western blot analyses revealed that recently isolated HCMV strains induced COX-2 expression in human foreskin fibroblast (HFF) cells to higher levels than lab-adapted strain AD169. HCMV gene expression was not required for COX-2 induction since UV-inactivated virus was able to stimulate COX-2 gene expression during immediate early infection. Moreover, when HCMV-infected HFF cells were transfected with small interfering RNA for major immediate early (MIE) genes, HCMV-induced COX-2 expression was not abrogated, suggesting that HCMV MIE genes are not needed for COX-2 expression. In order to understand the signal pathways involved in COX-2 gene expression induced by HCMV, selective inhibitors were used. When HCMV-infected HFF cells were treated with epidermal growth factor receptor (EGFR) kinase inhibitors and c-Raf/mitogen-activated protein kinases (MAPK, MEK) 1/2 inhibitors, HCMV-induced COX-2 expression was diminished. However, COX-2 expression by HCMV infection was not inhibited if HCMV-infected HFF cells were treated with inhibitors of phosphatidylinositol-3-kinase, c-Jun N-terminal kinase, nuclear factor kappa B, p-38 MAPK and Rho-associated protein kinase. We concluded that EGFRK, Raf, MEK1/2 and extracellular signal-regulated kinases (ERK 1/2) pathway may participate in the COX-2 mediated inflammatory response to HCMV infection.

Analysis of substitution events in HIV-1 vif gene of the Korean clade.

Nucleotide and amino acid substitution pattern in vif gene of the Korean clade of HIV-1 isolated from Koreans were analyzed using consensus sequences. At nucleotide level, transition/transversion substitution ratio was 1.88, and nonsynonymous/synonymous substitution ratio was 2.67, suggesting a divergent evolution in the Korean clade. At amino acid level, there were 17 substitutions and G-->E substitution at position 37 may be responsible for change in predicted secondary structure.

Stimulation of interferon-beta gene expression by human cytomegalovirus via nuclear factor kappa B and phosphatidylinositol 3-kinase pathway.

Infection of human foreskin fibroblast (HFF) cells with human cytomegalovirus (HCMV) induces the secretion of soluble factors including interferon (IFN)-beta that stimulates human leukocyte antigen (HLA) class I expression. In this study, the mechanism of IFN-beta induction by HCMV was investigated. In HCMV-infected HFF cells, IFN-beta secretion increased at 6h post infection (h.p.i.). Reverse transcription polymerase chain reaction (RT-PCR) analysis using ultra violet (UV)-inactivated HCMV indicated that viral gene expression is not necessary for the stimulation of IFN-beta. Stimulation of IFN-beta by HCMV infection was not blocked by cycloheximide, an inhibitor of protein synthesis, further suggesting that the expression of HCMV genes is not required for the stimulation of IFN-beta gene transcription. IFN-beta may be produced from virus-infected cells as an inflammatory response and nuclear factor kappa B (NF-kappaB) plays a central role in inflammatory response. HCMV failed to induce the IFN-beta expression, when the virus-infected cells were treated with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, or LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI3-K). The result suggests that PI3-K and/or NF-kappaB may be related with the induction pathway of IFN-beta by HCMV.