High Affinity Binding Sites Research Articles

Structural and Kinetic Characterization of an Acetoacetyl-Coenzyme A: Acetate Coenzyme A Transferase from the Extreme Thermophile Thermosipho melanesiensis.

CtfAB from the extremely thermophilic bacterium, Thermosipho melanesiensis, has been used for in vivo acetone production up to 70°C. This enzyme has tentatively been identified as the rate-limiting step, due to its relatively low binding affinity for acetate. However, existing kinetic and mechanistic studies on this enzyme are insufficient to evaluate this hypothesis. Here, kinetic analysis of purified recombinant T. melanesiensis CtfAB showed that it has a ping pong bi bi mechanism typical of CoA transferases with Km values for acetate and acetoacetyl-CoA of 85 mM and 135 mM, respectively. Product inhibition by acetyl-CoA was competitive with respect to acetoacetyl-CoA and non-competitive with respect to acetate. Crystal structures of wildtype and mutant T. melanesiensis CtfAB were solved in the presence of acetate and in the presence or absence of acetyl-CoA. These structures led to a proposed structural basis for the competitive and non-competitive inhibition of acetyl-CoA: acetate binds independently of acetyl-CoA in an apparent low affinity binding pocket in CtfA that is directly adjacent to a catalytic glutamate in CtfB. Similar to other CoA transferases, acetyl-CoA is bound in an apparent high affinity binding site in CtfB with most interactions occurring between the phospho-ADP of coenzyme A and CtfB residues far from the acetate binding pocket. This structural-based mechanism also explains the organic acid promiscuity of CtfAB. High affinity interactions are predominantly between the conserved phospho-ADP of coenzyme A and the variable organic acid binding site is a low affinity binding site with few specific interactions.

Enthalpy-Driven Interaction between Bovine Serum Albumin and Biomass-Derived Low-Melting Mixture Solvents (LoMMSs) for Efficient and Green Purification of Protein.

Green separation of protein (e.g., bovine serum albumin (BSA)) by low-melting mixture solvents (LoMMSs) depends on the underlying mechanism between BSA and LoMMSs. Here, we for the first time find that eco-friendly biomass-derived LoMMSs could be potentially used for the efficient and green purification of BSA protein by enthalpy-driven interactions. Biomass-derived LoMMSs possess the merits of high biocompatibility, high degradability, high abundance, and low cost. A single high-affinity binding site via hydrogen bonding and van der Waals forces is observed between BSA and LoMMSs by fluorescence and thermodynamic analysis. Experimental results from circular dichroism and infrared spectra demonstrate that the addition of LoMMSs stabilizes the secondary structure of the BSA protein. This work provides a valuable indication for the design of eco-friendly and cost-effective LoMMSs for the purification of protein.

Anchoring Sn Nanoparticles in Necklace-Like B,N,F-Doped Carbon Fibers Enables Anode-Less 5V-Class Li-Metal Batteries.

Li metal batteries (LMBs), particularly with a limited Li metal anode and a 5V-class cathode, offer significantly higher energy density compared to the state-of-the-art Li-ion batteries. However, the limited Li anode poses severe challenges to cycling stability due to low efficiency and large volume expansion issues associated with Li. Herein, we design a lightweight and functionalized host composed of Sn nanoparticles embedded into necklace-like B,N,F-doped carbon macroporous fibers (Sn@B/N/F-CMFs) toward anode-less 5V-class LMBs. The macroporous framework can decrease the local current density to homogenize Li deposition and release structural stress to realize high areal capacity of over 40 mAh cm-2. The lithiophilic B,N,F-doped carbon and Sn nanoparticles can function as high-affinity Li binding sites to uniformize Li nucleus growth on the internal and external surface of hollow fibers. Accordingly, the Sn@B/N/F-CMFs enable stable dendrite-free Li plating/stripping behaviors for 1700 h even in the carbonate-based electrolyte. When coupled with a 5V-class LiNi0.5Mn1.5O4cathode, the assembled anode-less pouch cell also displays stable cycling performance even under harsh conditions.

Properties of the major Zn2+-binding site of human alpha-fetoprotein, a potential foetal plasma zinc carrier.

The foetal plasma protein α-fetoprotein (AFP) harbours a high-affinity zinc binding site that is likely involved in transport and delivery of essential zinc during foetal development. Based on a recent electron microscopy structure of AFP and aided by biophysical studies on an AFP-derived peptide, we present a refined 5-coordinate model for this site.

In vitro spectroscopic studies of 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride with main carrier plasma proteins

Current methods of cancer treatment, particularly chemotherapy, are associated with harmful side effects. For this reason, it is significant to study new substances with anticancer potential with the highest possible efficacy and the lowest possible side effects. The aim of the study was the spectroscopic analysis of the interaction between 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride (Salt3) and main carrier proteins, such as human serum albumin (HSA), α1 acid glycoprotein (AGP), human γ globulin (HGG) and controlled normal serum (CNS).The association constants (Ka [mol·L-1]) and the number of binding site classes (n) for the binding of Salt3 with studied carrier proteins and controlled normal serum were calculated using the Klotz equation. To study HSA and AGP high affinity binding sites, the fluorescent markers were used. Spectral parameter A and the second derivative of differential absorption spectra were used to assess environmental changes around aromatic amino acids residues. The changes in HSA and AGP secondary structure in the complexes with Salt3 were evaluated using the analysis using circular dichroism.Salt3 slightly binds to HSA, AGP, HGG molecules and CNS. In addition, Salt3 affects the tertiary structure of the studied proteins, while it does not damage the secondary structure of the main carrier proteins responsible for Salt3 distribution in the bloodstream.Because Salt3 binds weakly to model carrier proteins and normal control serum, it can lead to both strong therapeutic and toxic effects. Considering these preliminary spectroscopic studies, additional tests as well as expanding research to include other techniques seem justified.

An ancient competition for the conserved branchpoint sequence influences physiological and evolutionary outcomes in splicing.

Recognition of the intron branchpoint during spliceosome assembly is a multistep process that defines both mRNA structure and amount. A branchpoint sequence motif UACUAAC is variably conserved in eukaryotic genomes, but in some organisms more than one protein can recognize it. Here we show that SF1 and Quaking (QKI) compete for a subset of intron branchpoints with the sequence ACUAA. SF1 activates exon inclusion through this sequence, but QKI represses the inclusion of alternatively spliced exons with this intron branchpoint sequence. Using mutant reporters derived from a natural intron with two branchpoint-like sequences, we find that when either branchpoint sequence is mutated, the other is used as a branchpoint, but when both are present, neither is used due to high affinity binding and strong splicing repression by QKI. QKI occupancy at the dual branchpoint site directly prevents SF1 binding and subsequent recruitment of spliceosome-associated factors. Finally, the ectopic expression of QKI in budding yeast (which lacks QKI) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of branchpoint sequences that closely mirror its high affinity binding site. This suggests that QKI and degenerate branchpoint sequences may have co-evolved as a means through which specific gene expression patterns could be maintained in QKI-expressing or non-expressing cells in metazoans, plants, and animals.

A suboptimal OCT4-SOX2 binding site facilitates the naïve-state specific function of a Klf4 enhancer.

Enhancers have critical functions in the precise, spatiotemporal control of transcription during development. It is thought that enhancer grammar, or the characteristics and arrangements of transcription factor binding sites, underlie the specific functions of developmental enhancers. In this study, we sought to identify grammatical constraints that direct enhancer activity in the naïve state of pluripotency, focusing on the enhancers for the naïve-state specific gene, Klf4. Using a combination of biochemical tests, reporter assays, and endogenous mutations in mouse embryonic stem cells, we have studied the binding sites for the transcription factors OCT4 and SOX2. We have found that the three Klf4 enhancers contain suboptimal OCT4-SOX2 composite binding sites. Substitution with a high-affinity OCT4-SOX2 binding site in Klf4 enhancer E2 rescued enhancer function and Klf4 expression upon loss of the ESRRB and STAT3 binding sites. We also observed that the low-affinity of the OCT4-SOX2 binding site is crucial to drive the naïve-state specific activities of Klf4 enhancer E2. Altogether, our work suggests that the affinity of OCT4-SOX2 binding sites could facilitate enhancer functions in specific states of pluripotency.

Expression of Growth Hormone-Releasing Hormone and Its Receptor Splice Variants in a Cohort of Hungarian Pediatric Patients with Hematological and Oncological Disorders: A Pilot Study.

Hematological and oncological diseases are still among the leading causes of childhood mortality. Expression of growth hormone-releasing hormone (GHRH) and its receptors (GHRH-R) has been previously demonstrated in various human tumors, but very limited findings are available about the presence and potential function of GHRH-Rs in oncological and hematological disorders of children. In this study, we aimed to investigate the expression of mRNA for GHRH and splice variant 1 (SV) of GHRH-R in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of GHRH-R protein were also studied by Western blot and ligand competition assays. Of the fifteen specimens studied, eleven pediatric samples (73%) showed the expression of mRNA for GHRH. These eleven samples also expressed mRNA for GHRH receptor SV1. GHRH-R protein was found to be expressed in two benign tumor samples and five malignant tumors examined by Western blot. The presence of specific, high affinity binding sites on GHRH-R was demonstrated in all of the seven human pediatric solid tumor samples investigated. Our results show that the expression of GHRH and SV1 of GHRH-R in hemato-oncological diseases in children can pave the way for further investigation of GHRH-Rs as potential molecular targets for diagnosis and therapy.

In vivo absorption, in vitro simulated digestion, and fecal fermentation properties of Imperata cylindrica polysaccharides and their effects on gut microbiota

Recently we have investigated polysaccharide from Imperata cylindrica (ICP) for its physicochemical structure and biological activities. However, the digestion characteristics have yet to be understood. This study investigated the digestion and metabolism characteristics of ICP through in vivo fluorescence tracking, in vitro simulated digestion, fecal fermentation experiments, and microbial sequencing. The results showed that ICP significant distribution in the gastrointestinal tract and kidneys. ICP underwent slight degradation during simulated gastric and intestinal digestion. During fecal fermentation, the utilization degree of ICP and the concentration of short-chain fatty acids (SCFAs) increased. ICP promoted the increase of beneficial microbial abundance. To understand the impact of ICP on the integrity and health of intestinal tissues, molecular docking was employed to preliminarily predict the interaction between ICP and key proteins. The results revealed that ICP could recognize and bind to key proteins through high-affinity targeting binding sites.

Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance

The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.

Structural basis for a highly conserved RNA-mediated enteroviral genome replication.

Enteroviruses contain conserved RNA structures at the extreme 5' end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3' spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3' spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform.

Peripheral gating of mechanosensation by glial diazepam binding inhibitor.

We report that diazepam binding inhibitor (DBI) is a glial messenger mediating crosstalk between satellite glial cells (SGCs) and sensory neurons in the dorsal root ganglion (DRG). DBI is highly expressed in SGCs of mice, rats, and humans, but not in sensory neurons or most other DRG-resident cells. Knockdown of DBI results in a robust mechanical hypersensitivity without major effects on other sensory modalities. In vivo overexpression of DBI in SGCs reduces sensitivity to mechanical stimulation and alleviates mechanical allodynia in neuropathic and inflammatory pain models. We further show that DBI acts as an unconventional agonist and positive allosteric modulator at the neuronal GABAA receptors, particularly strongly affecting those with a high-affinity benzodiazepine binding site. Such receptors are selectively expressed by a subpopulation of mechanosensitive DRG neurons, and these are also more enwrapped with DBI-expressing glia, as compared with other DRG neurons, suggesting a mechanism for a specific effect of DBI on mechanosensation. These findings identified a communication mechanism between peripheral neurons and SGCs. This communication modulates pain signaling and can be targeted therapeutically.

Bridging the gap between the evolutionary dynamics and the molecular mechanisms of meiosis: A model based exploration of the PRDM9 intra-genomic Red Queen.

Molecular dissection of meiotic recombination in mammals, combined with population-genetic and comparative studies, have revealed a complex evolutionary dynamic characterized by short-lived recombination hotspots. Hotspots are chromosome positions containing DNA sequences where the protein PRDM9 can bind and cause crossing-over. To explain these fast evolutionary dynamic, a so-called intra-genomic Red Queen model has been proposed, based on the interplay between two antagonistic forces: biased gene conversion, mediated by double-strand breaks, resulting in hotspot extinction (the hotspot conversion paradox), followed by positive selection favoring mutant PRDM9 alleles recognizing new sequence motifs. Although this model predicts many empirical observations, the exact causes of the positive selection acting on new PRDM9 alleles is still not well understood. In this direction, experiment on mouse hybrids have suggested that, in addition to targeting double strand breaks, PRDM9 has another role during meiosis. Specifically, PRDM9 symmetric binding (simultaneous binding at the same site on both homologues) would facilitate homology search and, as a result, the pairing of the homologues. Although discovered in hybrids, this second function of PRDM9 could also be involved in the evolutionary dynamic observed within populations. To address this point, here, we present a theoretical model of the evolutionary dynamic of meiotic recombination integrating current knowledge about the molecular function of PRDM9. Our modeling work gives important insights into the selective forces driving the turnover of recombination hotspots. Specifically, the reduced symmetrical binding of PRDM9 caused by the loss of high affinity binding sites induces a net positive selection eliciting new PRDM9 alleles recognizing new targets. The model also offers new insights about the influence of the gene dosage of PRDM9, which can paradoxically result in negative selection on new PRDM9 alleles entering the population, driving their eviction and thus reducing standing variation at this locus.

Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

Modulation of the photobehavior of gefitinib and its phenolic metabolites by human transport proteins.

The photobiological damage that certain drugs or their metabolites can photosensitize in proteins is generally associated with the nature of the excited species that are generated upon interaction with UVA light. In this regard, the photoinduced damage of the anticancer drug gefitinib (GFT) and its two main photoactive metabolites GFT-M1 and GFT-M2 in cellular milieu was recently investigated. With this background, the photophysical properties of both the drug and its metabolites have now been studied in the presence of the two main transport proteins of human plasma, i.e., serum albumin (HSA) and α1-acid glycoprotein (HAG) upon UVA light excitation. In general, the observed photobehavior was strongly affected by the confined environment provided by the protein. Thus, GFT-M1 (which exhibits the highest phototoxicity) showed the highest fluorescence yield arising from long-lived HSA-bound phenolate-like excited species. Conversely, locally excited (LE) states were formed within HAG, resulting in lower fluorescence yields. The reserve was true for GFT-M2, which despite being also a phenol, led mainly to formation of LE states within HSA, and phenolate-like species (with a minor contribution of LE) inside HAG. Finally, the parent drug GFT, which is known to form LE states within HSA, exhibited a parallel behavior in the two proteins. In addition, determination of the association constants by both absorption and emission spectroscopy revealed that the two metabolites bind stronger to HSA than the parent drug, whereas smaller differences were observed for HAG. This was further confirmed by studying the competing interactions between GFT or its metabolites with the two proteins using fluorescence measurements. These above experimental findings were satisfactorily correlated with the results obtained by means of molecular dynamics (MD) simulations, which revealed the high affinity binding sites, the strength of interactions and the involved amino acid residues. In general, the differences observed in the photobehavior of the drug and its two photoactive metabolites in protein media are consistent with their relative photosensitizing potentials.

Drugs Form Ternary Complexes with Human Liver Fatty Acid Binding Protein 1 (FABP1) and FABP1 Binding Alters Drug Metabolism.

Liver fatty acid binding protein 1 (FABP1) binds diverse endogenous lipids and is highly expressed in the human liver. Binding to FABP1 alters the metabolism and homeostasis of endogenous lipids in the liver. Drugs have also been shown to bind to rat FABP1, but limited data are available for human FABP1 (hFABP1). FABP1 has a large binding pocket, and up to two fatty acids can bind to FABP1 simultaneously. We hypothesized that drug binding to hFABP1 results in formation of ternary complexes and that FABP1 binding alters drug metabolism. To test these hypotheses, native protein mass spectrometry (MS) and fluorescent 11-(dansylamino)undecanoic acid (DAUDA) displacement assays were used to characterize drug binding to hFABP1, and diclofenac oxidation by cytochrome P450 2C9 (CYP2C9) was studied in the presence and absence of hFABP1. DAUDA binding to hFABP1 involved high (Kd,1 = 0.2 μM) and low (Kd,2 > 10 μM) affinity binding sites. Nine drugs bound to hFABP1 with equilibrium dissociation constant (Kd) values ranging from 1 to 20 μM. None of the tested drugs completely displaced DAUDA from hFABP1, and fluorescence spectra showed evidence of ternary complex formation. Formation of DAUDA-hFABP1-diclofenac ternary complex was verified with native MS. Docking predicted diclofenac binding in the portal region of FABP1 with DAUDA in the binding cavity. The catalytic rate constant of diclofenac hydroxylation by CYP2C9 was decreased by ∼50% (P < 0.01) in the presence of FABP1. Together, these results suggest that drugs form ternary complexes with hFABP1 and that hFABP1 binding in the liver will alter drug metabolism and clearance. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs bind fatty acid binding protein 1 (FABP1), forming ternary complexes with FABP1 and the fluorescent fatty acid 11-(dansylamino)undecanoic acid. These findings suggest that drugs will bind to apo-FABP1 and fatty acid-bound FABP1 in the human liver. The high expression of FABP1 in the liver, together with drug binding to FABP1, may alter drug disposition processes in vivo.

Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers

The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Oncogenic EGFR has been successfully targeted by tyrosine kinase inhibitors, but acquired drug resistance eventually overcomes the efficacy of these treatments. Attempts to surmount this therapeutic challenge are hindered by a poor understanding of how and why cancer mutations specifically amplify ligand-independent EGFR auto-phosphorylation signals to enhance cell survival and how this amplification is related to ligand-dependent cell proliferation. Here we show that drug-resistant EGFR mutations manipulate the assembly of ligand-free, kinase-active oligomers to promote and stabilize the assembly of oligomer-obligate active dimer sub-units and circumvent the need for ligand binding. We reveal the structure and assembly mechanisms of these ligand-free, kinase-active oligomers, uncovering oncogenic functions for hitherto orphan transmembrane and kinase interfaces, and for the ectodomain tethered conformation of EGFR. Importantly, we find that the active dimer sub-units within ligand-free oligomers are the high affinity binding sites competent to bind physiological ligand concentrations and thus drive tumor growth, revealing a link with tumor proliferation. Our findings provide a framework for future drug discovery directed at tackling oncogenic EGFR mutations by disabling oligomer-assembling interactions.

Molecular Deformation Is a Key Factor in Screening Aggregation Inhibitor for Intrinsically Disordered Protein Tau.

Direct inhibitor of tau aggregation has been extensively studied as potential therapeutic agents for Alzheimer's disease. However, the natively unfolded structure of tau complicates the structure-based ligand design, and the relatively large surface areas that mediate tau-tau interactions in aggregation limit the potential for identifying high-affinity ligand binding sites. Herein, a group of isatin-pyrrolidinylpyridine derivative isomers (IPP1-IPP4) were designed and synthesized. They are like different forms of molecular "transformers". These isatin isomers exhibit different inhibitory effects on tau self-aggregation or even possess a depolymerizing effect. Our results revealed for the first time that the direct inhibitor of tau protein aggregation is not only determined by the previously reported conjugated structure, substituent, hydrogen bond donor, etc. but also depends more importantly on the molecular shape. In combination with molecular docking and molecular dynamics simulations, a new inhibition mechanism was proposed: like a "molecular clip", IPP1 could noncovalently bind and fix a tau polypeptide chain at a multipoint to prevent the transition from the "natively unfolded conformation" to the "aggregation competent conformation" before nucleation. At the cellular and animal levels, the effectiveness of the inhibitor of the IPP1 has been confirmed, providing an innovative design strategy as well as a lead compound for Alzheimer's disease drug development.

Interaction of MLE with CLAMP zinc finger is involved in proper MSL proteins binding to chromosomes in Drosophila.

The Drosophila male-specific lethal (MSL) complex binds to the male X chromosome to activate transcription. It comprises five proteins (MSL1, MSL2, MSL3, male absent on the first (MOF), and maleless (MLE)) and two long noncoding RNAs (lncRNAs; roX1 and roX2). The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. MSL2 is expressed only in males and interacts with the N-terminal zinc finger of the transcription factor chromatin-linked adapter for MSL proteins (CLAMP), which is important for the specific recruitment of the MSL complex to the male X chromosome. Here, we found that MLE's unstructured C-terminal region interacts with thesixth zinc-finger domain of CLAMP. In vitro, 4-5 zinc fingers are critical for the specific DNA-binding of CLAMP with GArepeats, which constitute the core motif at the high affinity binding sites for MSL proteins. Deleting the CLAMP binding region in MLE decreases the association of MSL proteins with the male X chromosome and increases male lethality. These results suggest that interactions of unstructured regions in MSL2 and MLE with CLAMP zinc finger domains are important for the specific recruitment of the MSL complex to the male X chromosome.

Modulations in the host cell proteome by the hantavirus nucleocapsid protein.

Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell.