Moonlighting chaperone activity of the enzyme PqsE contributes to RhlR-controlled virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals.

Peptide microarrays coupled to machine learning reveal individual epitopes from human antibody responses with neutralizing capabilities against SARS-CoV-2.

The coronavirus SARS-CoV-2 is the causative agent for the disease COVID-19. To capture the IgA, IgG, and IgM antibody response of patients infected with SARS-CoV-2 at individual epitope resolution, we constructed planar microarrays of 648 overlapping peptides that cover the four major structural proteins S(pike), N(ucleocapsid), M(embrane), and E(nvelope). The arrays were incubated with sera of 67 SARS-CoV-2 positive and 22 negative control samples. Specific responses to SARS-CoV-2 were detectable, and nine peptides were associated with a more severe course of the disease. A random forest model disclosed that antibody binding to 21 peptides, mostly localized in the S protein, was associated with higher neutralization values in cellular anti-SARS-CoV-2 assays. For antibodies addressing the N-terminus of M, or peptides close to the fusion region of S, protective effects were proven by antibody depletion and neutralization assays. The study pinpoints unusual viral binding epitopes that might be suited as vaccine candidates.

Labyrinthopeptins Exert Broad-Spectrum Antiviral Activity through Lipid-Binding-Mediated Virolysis.

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

Species-Specific Conservation of Linear Antigenic Sites on Vaccinia Virus A27 Protein Homologs of Orthopoxviruses.

The vaccinia virus (VACV) A27 protein and its homologs, which are found in a large number of members of the genus Orthopoxvirus (OPXV), are targets of viral neutralization by host antibodies. We have mapped six binding sites (epitopes #1A: aa 32-39, #1B: aa 28-33, #1C: aa 26-31, #1D: 28-34, #4: aa 9-14, and #5: aa 68-71) of A27 specific monoclonal antibodies (mAbs) using peptide arrays. MAbs recognizing epitopes #1A-D and #4 neutralized VACV Elstree in a complement dependent way (50% plaque-reduction: 12.5-200 µg/mL). Fusion of VACV at low pH was blocked through inhibition of epitope #1A. To determine the sequence variability of the six antigenic sites, 391 sequences of A27 protein homologs available were compared. Epitopes #4 and #5 were conserved among most of the OPXVs, while the sequential epitope complex #1A-D was more variable and, therefore, responsible for species-specific epitope characteristics. The accurate and reliable mapping of defined epitopes on immuno-protective proteins such as the A27 of VACV enables phylogenetic studies and insights into OPXV evolution as well as to pave the way to the development of safer vaccines and chemical or biological antivirals.

Diagnosing Zika virus infection against a background of other flaviviruses: Studies in high resolution serological analysis.

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.

Occupation-Associated Fatal Limbic Encephalitis Caused by Variegated Squirrel Bornavirus 1, Germany, 2013.

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost's squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost's squirrel. Antibodies against bornaviruses were detected in the patient's cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.

The Kaposi's sarcoma-associated herpesvirus (KSHV) non-structural membrane protein K15 is required for viral lytic replication and may represent a therapeutic target.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi's sarcoma (KS), which is characterized by proliferating spindle cells of endothelial origin, extensive neo-angiogenesis and inflammatory infiltrates. The KSHV K15 protein contributes to the angiogenic and invasive properties of KSHV-infected endothelial cells. Here, we asked whether K15 could also play a role in KSHV lytic replication. Deletion of the K15 gene from the viral genome or its depletion by siRNA lead to reduced virus reactivation, as evidenced by the decreased expression levels of KSHV lytic proteins RTA, K-bZIP, ORF 45 and K8.1 as well as reduced release of infectious virus. Similar results were found for a K1 deletion virus. Deleting either K15 or K1 from the viral genome also compromised the ability of KSHV to activate PLCγ1, Erk1/2 and Akt1. In infected primary lymphatic endothelial (LEC-rKSHV) cells, which have previously been shown to spontaneously display a viral lytic transcription pattern, transfection of siRNA against K15, but not K1, abolished viral lytic replication as well as KSHV-induced spindle cell formation. Using a newly generated monoclonal antibody to K15, we found an abundant K15 protein expression in KS tumor biopsies obtained from HIV positive patients, emphasizing the physiological relevance of our findings. Finally, we used a dominant negative inhibitor of the K15-PLCγ1 interaction to establish proof of principle that pharmacological intervention with K15-dependent pathways may represent a novel approach to block KSHV reactivation and thereby its pathogenesis.

Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.

Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1) is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any) in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV). 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs) were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.