Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers.

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD ] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

Blind esophageal brushing offers a safe and accurate method to monitor inflammation in children and young adults with eosinophilic esophagitis.

Patients with eosinophilic esophagitis (EoE) require frequent evaluation of mucosal inflammation via endoscopy. Instead of endoscopy, mucosal evaluation in adults with esophageal cancer and candidiasis is achieved using a cytology brush inserted through a nasogastric tube (NGT). We conducted a prospective cross-sectional study in children and young adults scheduled for routine esophagogastroduodenoscopy (EGD) where in Phase 1, we performed esophageal brushing through the endoscope under direct visualization and in Phase 2, we inserted the brush through a Cortrak® NGT prior to endoscopy. Eosinophil-derived neurotoxin (EDN) measured by ELISA in the samples extracted from brushes was validated as the sensitive biomarker. We collected 209 esophageal brushing samples from 94 patients and we found that EDN in brushing samples collected via EGD or NGT was significantly higher in patients having active EoE (n = 81, mean EDN 381 mcg/mL) compared with patients having gastroesophageal reflux disease (n = 31, mean EDN 1.9 mcg/mL, P = 0.003), EoE in remission (n = 47, mean EDN 3.7 mcg/mL, P = 0.003), or no disease (n = 50, mean EDN 1.1 mcg/mL, P = 0.003). EDN at a concentration of ≥10 mcg/mL of brushing sample was found to accurately detect active EoE. NGT brushing did not cause any significant adverse effects. We concluded that blind esophageal brushing using an NGT is a fast, less invasive, safe, and well-tolerated technique compared with EGD to detect and monitor EoE inflammation using EDN as the sensitive biomarker.

Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor.

Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

The role of viral genomic RNA and nucleocapsid protein in the autophagic clearance of hantavirus glycoprotein Gn.

Hantaviruses have tri-segmented negative sense RNA genome. The viral M-segment RNA encodes a glycoprotein precursor (GPC), which is cleaved into two glycoprotein molecules Gn and Gc that form spikes on the viral envelope. We previously reported that Gn is degraded shortly after synthesis by the host autophagy machinery. However, Gn being an important integral component of the virion, must escape degradation during the packaging and assembly stage of virus replication cycle. The mechanism regulating the intrinsic steady-state levels of Gn during the course of virus replication cycle is not clear. We transfected cells with plasmids expressing viral S-segment RNA, nucleocapsid protein and glycoproteins Gn and Gc and monitored their expression levels over time. These studies revealed that accumulation of nucleocapsid protein, glycoprotein Gc and viral S-segment RNA helped to stabilize Gn. These observations suggest that initiation of virus assembly may help Gn to escape autophagic degradation by yet unknown mechanism.

Autophagic clearance of Sin Nombre hantavirus glycoprotein Gn promotes virus replication in cells.

Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.