Noncoding RNome as Enabling Biomarkers for Precision Health.

Noncoding RNAs (ncRNAs), in the form of structural, catalytic or regulatory RNAs, have emerged to be critical effectors of many biological processes. With the advent of new technologies, we have begun to appreciate how intracellular and circulatory ncRNAs elegantly choreograph the regulation of gene expression and protein function(s) in the cell. Armed with this knowledge, the clinical utility of ncRNAs as biomarkers has been recently tested in a wide range of human diseases. In this review, we examine how critical factors govern the success of interrogating ncRNA biomarker expression in liquid biopsies and tissues to enhance our current clinical management of human diseases, particularly in the context of cancer. We also discuss strategies to overcome key challenges that preclude ncRNAs from becoming standard-of-care clinical biomarkers, including sample pre-analytics standardization, data cross-validation with closer attention to discordant findings, as well as correlation with clinical outcomes. Although harnessing multi-modal information from disease-associated noncoding RNome (ncRNome) in biofluids or in tissues using artificial intelligence or machine learning is at the nascent stage, it will undoubtedly fuel the community adoption of precision population health.

Molecular basis for specific viral RNA recognition and 2'-O-ribose methylation by the dengue virus nonstructural protein 5 (NS5).

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5' exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5' RNA sequence as substrate (GpppAG-RNA), leading to the formation of the 5' RNA cap: G0pppAG-RNA → (m7)G0pppAG-RNA ("cap-0")→(m7)G0pppAm2'-O-G-RNA ("cap-1"). To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5'-(m7)G0pppA1G2U3U4G5U6U7-3'), and S-adenosyl-l-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2'-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.

Antigenic Fingerprinting of Antibody Response in Humans following Exposure to Highly Pathogenic H7N7 Avian Influenza Virus: Evidence for Anti-PA-X Antibodies.

UNLABELLED: Infections with H7 highly pathogenic avian influenza (HPAI) viruses remain a major public health concern. Adaptation of low-pathogenic H7N7 to highly pathogenic H7N7 in Europe in 2015 raised further alarm for a potential pandemic. An in-depth understanding of antibody responses to HPAI H7 virus following infection in humans could provide important insight into virus gene expression as well as define key protective and serodiagnostic targets. Here we used whole-genome gene fragment phage display libraries (GFPDLs) expressing peptides of 15 to 350 amino acids across the complete genome of the HPAI H7N7 A/Netherlands/33/03 virus. The hemagglutinin (HA) antibody epitope repertoires of 15 H7N7-exposed humans identified clear differences between individuals with no hemagglutination inhibition (HI) titers (<1:10) and those with HI titers of >1:40. Several potentially protective H7N7 epitopes close to the HA receptor binding domain (RBD) and neuraminidase (NA) catalytic site were identified. Surface plasmon resonance (SPR) analysis identified a strong correlation between HA1 (but not HA2) binding antibodies and H7N7 HI titers. A proportion of HA1 binding in plasma was contributed by IgA antibodies. Antibodies against the N7 neuraminidase were less frequent but targeted sites close to the sialic acid binding site. Importantly, we identified strong antibody reactivity against PA-X, a putative virulence factor, in most H7N7-exposed individuals, providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human influenza A virus infection. This knowledge can help inform the development and selection of the most effective countermeasures for prophylactic as well as therapeutic treatments of HPAI H7N7 avian influenza virus. IMPORTANCE: An outbreak of pathogenic H7N7 virus occurred in poultry farms in The Netherlands in 2003. Severe outcome included conjunctivitis, influenza-like illness, and one lethal infection. In this study, we investigated convalescent-phase sera from H7N7-exposed individuals by using a whole-genome phage display library (H7N7-GFPDL) to explore the complete repertoire of post-H7N7-exposure antibodies. PA-X is a recently identified influenza virus virulence protein generated by ribosomal frameshifting in segment 3 of influenza virus coding for PA. However, PA-X expression during influenza virus infection in humans is unknown. We identified strong antibody reactivity against PA-X in most H7N7-exposed individuals (but not in unexposed adults), providing the first evidence for in vivo expression of PA-X and its recognition by the immune system during human infection with pathogenic H7N7 avian influenza virus.

High-Affinity H7 Head and Stalk Domain-Specific Antibody Responses to an Inactivated Influenza H7N7 Vaccine After Priming With Live Attenuated Influenza Vaccine.

Recent studies have shown that live attenuated influenza vaccines (LAIVs) expressing avian influenza virus hemagglutinins (HAs) prime for strong protective antibody responses to an inactivated influenza vaccine (IIV) containing the HA. To better understand this priming effect, we compared H7 HA head and stalk domain-specific B-cell responses in H7N7 LAIV-primed subjects and non-H7-primed controls after a single dose of H7N7 IIV. As previously reported, H7N7 LAIV-primed subjects but not control subjects generated strong hemagglutination-inhibiting and neutralizing antibody responses to the H7N7 IIV. Here, we found that the quantity, epitope diversity, and affinity of H7 head-specific antibodies increased rapidly in only H7N7 LAIV-primed subjects after receipt of the IIV. However, all cohorts generated a vigorous, high-affinity, stalk-specific antibody response. Consistent increases in circulating memory B-cell frequencies after receipt of the IIV reflected the specificity of high-affinity antibody production. Our findings emphasize the value of LAIVs as a vehicle for prepandemic vaccination.

Small molecule inhibitors that selectively block dengue virus methyltransferase.

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome.

Polymer-Based Precipitation of Extracellular Vesicular miRNAs from Serum Improve Gastric Cancer miRNA Biomarker Performance.

Circulating miRNAs are promising liquid biopsy biomarkers for noninvasive cancer detection. However, detection of subtle, but meaningful differences in circulating miRNA quantities between diseased and healthy samples remains a key challenge in clinical settings because biomarker signal/noise ratios are often low. Because extracellular vesicles (EVs) are key sources of circulating miRNAs in serum, it was hypothesized that isolating EVs would enrich miRNA biomarkers, leading to enhanced diagnostic ability and improved biomarker performance. This research assessed the performance of EV-miRNAs against serum miRNAs as biomarkers for gastric cancer (GC). It was first determined that polymer-based precipitation (PBP) gave the highest EV-miRNA recovery when compared with ultracentrifugation, column affinity, peptide affinity, and immunobead affinity EV purification. Four PBP reagents were used to isolate EV-miRNAs from 15 GC and 15 healthy controls and 133 GC-related miRNAs were profiled from EV fractions and total serum using real-time quantitative PCR. A PBP reagent that generated the most EV-miRNA biomarkers was selected and used to validate 11 EV-miRNAs in an independent set of 20 GC and 20 controls. Eight of these EV-miRNA biomarkers were found to give better GC detection accuracy (area under the curve, approximately 0.8). Overall, data suggest that EV miRNAs can improve GC detection performance compared with serum miRNAs and led to the identification of eight EV-miRNAs as potential noninvasive biomarkers for GC.

Trehalose significantly enhances the recovery of serum and serum exosomal miRNA from a paper-based matrix.

The preservation of nucleic acids from clinical samples is critical to facilitate accurate molecular diagnosis. The use of a paper matrix, Flinders Technology Associates (FTA) Elute cards, to archive DNA and viral RNA is well-documented. However, the feasibility of FTA Elute cards for archiving serum and serum exosomal microRNAs (miRNAs) remains unclear. Here, we performed a comprehensive evaluation of FTA Elute cards for miRNA storage and recovery in different pre-analytical conditions. The recovery of serum miRNA dry-spotted on FTA Elute cards by direct elution with water at high temperature was poor. However, serum miRNAs dry-spotted on the cards were isolated with about 40% yield when using QIAzol lysis reagent and recovery was improved remarkably (>80%) upon extraction from cards pre-treated with trehalose. miRNAs stored on the cards remained stable at room temperature and can be kept for prolonged periods. Furthermore, miRNAs could be similarly recovered from serum exosomes dry-spotted on the cards. Importantly, when using sera from gastric cancer (GC) patients, the miRNAs were efficiently recovered from trehalose pre-treated cards without affecting their representation. Collectively, we have demonstrated the potential of FTA Elute cards to archive serum and serum exosomal miRNAs, making it useful for biomarker discovery and diagnostics.

ISCOMATRIX™ adjuvant promotes epitope spreading and antibody affinity maturation of influenza A H7N9 virus like particle vaccine that correlate with virus neutralization in humans.

In a previously reported phase I clinical trial, subjects vaccinated with two doses of an unadjuvanted H7N9 virus like particle (VLP) vaccine responded poorly (15.6% seroconversion rates with 45µg hemagglutinin (HA) dose). In contrast, 80.6% of subjects receiving H7N9 VLP vaccine (5µg HA) with ISCOMATRIX™ adjuvant developed hemagglutination-inhibition (HI) responses. To better understand the role of adjuvant, complete antibody epitope repertoires of post-vaccination sera were investigated using Whole Genome Fragment Phage Display Library (GFPDL). In addition, antibody affinity maturation following vaccination was measured against HA1 and HA2 antigenic domains using real time Surface Plasmon Resonance (SPR) based kinetic assays. Unadjuvanted H7N9-VLP vaccine generated primarily antibodies targeting the C-terminus of the HA1 domain, predicted to be mostly buried on the native HA spikes, while adjuvanted VLP vaccine generated antibodies against large epitopes in the HA1 spanning the receptor binding domain (RBD). SPR analysis using a functional H7-HA1 domain demonstrated that sera from adjuvanted H7N9-VLP vaccine induced higher total binding antibodies and significantly higher antibody affinity maturation to HA1 compared to sera from unadjuvanted vaccine. Total antibody binding and affinity to the HA1 (but not HA2) domain correlated with HI and neutralization titers. This study demonstrates that ISCOMATRIX™ adjuvanted vaccine promotes higher quality antibody immune response against avian influenza in naïve humans.

Stabilization of dengue virus polymerase in de novo initiation assay provides advantages for compound screening.

Dengue virus (DENV) NS5 protein comprises an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain (RdRp). DENV RdRp is responsible for viral RNA synthesis via a de novo initiation mechanism and represents an attractive target for anti-viral therapy. Herein we describe the characterization of its de novo initiation activities by PAGE analyses and the knowledge gained was used to develop a fluorescent-based assay. A highly processive and robust assay was achieved by addition of cysteine in the assay buffer. This stabilized the apo-enzyme, and rendered optimal de novo initiation activity while balancing its intrinsic terminal transferase activity. Steady-state kinetic parameters of the NTP and RNA substrates under these optimal conditions were determined for DENV1-4 FL NS5. Heavy metal ions such as Zn(++) and Co(++) as well as high levels of monovalent salts, suppressed DENV polymerase de novo initiation activities. This assay was validated with nucleotide chain terminators and used to screen two diverse small library sets. The screen data obtained was further compared with concurrent screens performed with a DENV polymerase elongation fluorescent assay utilizing pre-complexed enzyme-RNA. A higher hit-rate was obtained for the de novo initiation assay compared to the elongation assay (∼2% versus ∼0.1%). All the hits from the latter assay are also identified in the de novo initiation assay, indicating that the de novo initiation assay performed with the stabilized apo-enzyme has the advantage of providing additional chemical starting entities for inhibiting this enzyme.

Crystal structure of the dengue virus methyltransferase bound to a 5'-capped octameric RNA.

The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2'-O positions of the viral RNA cap structure (GpppA→(7me)GpppA→(7me)GpppA(2'-O-me)). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5'-capped RNA octamer (G(ppp)AGAACCUG) at a resolution of 2.9 A. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5' end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.

Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2'-O methyltransferase activity in dengue virus.

Methyltransferases (MTases) from the genus Flavivirus encode both N-7 and 2'-O activities needed for type 1 (m(7)GpppNm) cap structure formation. We performed kinetic studies to understand the mechanisms of its progressive N-7 and 2'-O methylations. Sequential N-7 to 2'-O methylation occurred via a random bi bi and processive mechanism that does not involve enzyme-RNA dissociation. Analyses of steady state kinetic parameters showed that N-7 precedes 2'-O methylation as it turnovers RNA faster (k(cat)) resulting in 2.4-fold higher catalytic efficiency. Michaelis constants for S-adenosyl-methionine (AdoMet) in both reactions were about 10-fold lower than for their respective RNA substrates, suggesting that the rate-limiting steps in methylase reactions were associated with RNA templates. In the context of long viral RNA sequences, and compared to S-adenosyl-homocysteine, sinefungin was about 60- and 12-folds more potent against dengue N-7 and 2'-O MTase activity, exhibiting IC(50) values of 30 and 41nM, respectively.

Biochemical and genetic characterization of dengue virus methyltransferase.

We report that dengue virus (DENV) methyltransferase sequentially methylates the guanine N-7 and ribose 2'-O positions of viral RNA cap (GpppA-->m(7)GpppA-->m(7)GpppAm). The order of two methylations is determined by the preference of 2'-O methylation for substrate m(7)GpppA-RNA to GpppA-RNA, and the 2'-O methylation is not absolutely dependent on the prior N-7 methylation. A mutation that completely abolished the 2'-O methylation attenuated DENV replication in cell culture, whereas another mutation that abolished both methylations was lethal for viral replication, suggesting that N-7 methylation is more important than 2'-O methylation in viral replication. The latter mutant with lethal replication could be rescued by trans complementation using a wild-type DENV replicon. Furthermore, we found that chimeric DENVs containing the West Nile virus methyltransferase, polymerase, or full-length NS5 were nonreplicative, but the replication defect could also be rescued through trans complementation using the wild-type DENV replicon.