Lysine-rich Protein Research Articles

A Hydroxylamine‐Mediated Amidination of Lysine Residues That Retains the Protein's Positive Charge

AbstractLysine‐specific peptide and protein modification strategies are widely used to study charge‐related functions and applications. However, these strategies often result in the loss of the positive charge on lysine, significantly impacting the charge‐related properties of proteins. Herein, we report a strategy to preserve the positive charge and selectively convert amines in lysine side chains to amidines using nitriles and hydroxylamine under aqueous conditions. Various unprotected peptides and proteins were successfully modified with a high conversion rate. Moreover, the reactive amidine moiety and derived modification site enable subsequent secondary modifications. Notably, positive charges were retained during the modification. Therefore, positive charge‐related protein properties, such as liquid‐liquid phase separation behaviour of α‐synuclein, were not affected. This strategy was subsequently applied to a lysine rich protein to develop an amidine‐containing coacervate DNA complex with outstanding mechanical properties. Overall, our innovative strategy provides a new avenue to explore the characteristics of positively charged proteins.

A Hydroxylamine-Mediated Amidination of Lysine Residues That Retains the Protein's Positive Charge.

Lysine-specific peptide and protein modification strategies are widely used to study charge-related functions and applications. However, these strategies often result in the loss of the positive charge on lysine, significantly impacting the charge-related properties of proteins. Herein, we report a strategy to preserve the positive charge and selectively convert amines in lysine side chains to amidines using nitriles and hydroxylamine under aqueous conditions. Various unprotected peptides and proteins were successfully modified with a high conversion rate. Moreover, the reactive amidine moiety and derived modification site enable subsequent secondary modifications. Notably, positive charges were retained during the modification. Therefore, positive charge-related protein properties, such as liquid-liquid phase separation behaviour of α-synuclein, were not affected. This strategy was subsequently applied to a lysine rich protein to develop an amidine-containing coacervate DNA complex with outstanding mechanical properties. Overall, our innovative strategy provides a new avenue to explore the characteristics of positively charged proteins.

Imaging of adeno-associated viral capsids for purposes of gene editing using CEST NMR/MRI.

Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. NMR experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800-MHz vertical scanner. In vitro experiments were performed for testing of CEST-NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase-polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. CEST-NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

Role of Nitric Oxide-Derived Metabolites in Reactions of Methylglyoxal with Lysine and Lysine-Rich Protein Leghemoglobin.

Carbonyl stress occurs when reactive carbonyl compounds (RCC), such as reducing sugars, dicarbonyls etc., accumulate in the organism. The interaction of RCC carbonyl groups with amino groups of molecules is called the Maillard reaction. One of the most active RCCs is α-dicarbonyl methylglyoxal (MG) that modifies biomolecules forming non-enzymatic glycation products. Organic free radicals are formed in the reaction between MG and lysine or Nα-acetyllysine. S-nitrosothiols and nitric oxide (•NO) donor PAPA NONOate increased the yield of organic free radical intermediates, while other •NO-derived metabolites, namely, nitroxyl anion and dinitrosyl iron complexes (DNICs) decreased it. At the late stages of the Maillard reaction, S-nitrosoglutathione (GSNO) also inhibited the formation of glycation end products (AGEs). The formation of a new type of DNICs, bound with Maillard reaction products, was found. The results obtained were used to explain the glycation features of legume hemoglobin-leghemoglobin (Lb), which is a lysine-rich protein. In Lb, lysine residues can form fluorescent cross-linked AGEs, and •NO-derived metabolites slow down their formation. The knowledge of these processes can be used to increase the stability of Lb. It can help in better understanding the impact of stress factors on legume plants and contribute to the production of recombinant Lb for biotechnology.

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.

Germination, growth, yield and yield component of faba bean (Vicia faba L.) as influenced by seed size and sowing depth: A review

Faba bean (Vicia faba L.) is among the oldest crops in the world. Globally, it is third most important feed grain legume. Currently, 58 countries produce this bean on large scale. Probably faba beans are one of the best performing crops under global warming and climate change scenario because of its unique ability to excel under all most all type of climatic conditions coupled with its wide adoptability to range of soil environment. It is a good source of lysine rich protein and good source of levadopa (L-dopa), a precursor of dopamine, can be potentially used as medicine for the treatment of Parkinson’s disease. However, its production is very low due to a number of biotic and abiotic factors. Different agronomic practices also have an effect on germination, growth, yield and yield components of faba bean. Thus, the main objective of this seminar is to review the effect of seed size and sowing depth on faba bean production.

Redesigned reporter gene for improved proton exchange-based molecular MRI contrast

Reporter gene imaging allows for non-invasive monitoring of molecular processes in living cells, providing insights on the mechanisms underlying pathology and therapy. A lysine-rich protein (LRP) chemical exchange saturation transfer (CEST) MRI reporter gene has previously been developed and used to image tumor cells, cardiac viral gene transfer, and oncolytic virotherapy. However, the highly repetitive nature of the LRP reporter gene sequence leads to DNA recombination events and the expression of a range of truncated LRP protein fragments, thereby greatly limiting the CEST sensitivity. Here we report the use of a redesigned LRP reporter (rdLRP), aimed to provide excellent stability and CEST sensitivity. The rdLRP contains no DNA repeats or GC rich regions and 30% less positively charged amino-acids. RT-PCR of cell lysates transfected with rdLRP demonstrated a stable reporter gene with a single distinct band corresponding to full-length DNA. A distinct increase in CEST-MRI contrast was obtained in cell lysates of rdLRP transfected cells and in in vivo LRP expressing mouse brain tumors (p=0.0275, n = 10).

Penetration of phospholipid membranes by poly-l-lysine depends on cholesterol and phospholipid composition

Clusters of positively-charged basic amino acid residues, particularly lysine, are known to promote the interaction of many peripheral membrane proteins with the cytoplasmic surface of the plasma membrane via electrostatic interactions. In this work, cholesterol's effects on the interaction between lysine residues and membranes have been studied. Using poly-l-lysine (PLL) and vesicles as models to mimic the interaction between lysine-rich protein domains and the plasma membrane, light scattering measurements indicated cholesterol enhanced the electrostatic interaction through indirectly affecting the negatively charged phospholipid dioleoylphosphatidylserine, DOPS. Addition of PLL to lipid vesicles containing DOPS showed an initial increase in static light scattering (SLS), attributed to binding of PLL to the vesicle surface, followed by a slower continuously declining SLS signal, which, from comparison with fluorescent dye leakage studies could be attributed to vesicle lysis. Although electrostatic interactions between PLL and the membrane were not necessary for penetration to occur, cholesterol promoted membrane disruption of negatively charged vesicles, possibly by increasing the electrostatic interactions between PLL and the membrane. In contrast, cholesterol lowered the susceptibility of uncharged vesicles (formed using dioleoylphosphatidylcholine, DOPC) to PLL penetration. This can be explained by the absence of electrostatic interactions and cholesterol's known ability to increase membrane thickness and mechanical strength. Thus, the ability of cationic peptides to penetrate membranes including cholesterol is likely to depend on the membrane's PS:PC ratio.

Identification of Host Proteins Interacting with Toxoplasma gondii SAG1 by Yeast Two-Hybrid Assay

Toxoplasma gondii is one of the most successful human pathogens. To eliminate the infection, identification of receptors or binding partners from humans is indeed urgent. T. gondii surface antigen is the ultimate component involved during the attachment of parasite into host cell. However, mechanism of invasion between SAG and host-cell membrane remains unclear. Yeast two-hybrid experiment was used to identify the binding partners from cDNA human library by using T. gondii SAG1 as bait. Mated yeast cells were plated on DDO/X plates to confirm only prey plasmid that expressing interacting protein was selected. We detected 39 clones interacted with SAG1 based on a series of the selection procedures. After colony PCR, only 29 clones were positive and subsequently sent for sequencing. The yeast plasmids for true positive clones were rescued by transformation into E. coli TOP 10F' cells. Twenty-two clones were further examined by small-scale Y2H experiment. The results indicated that a strong interaction existed between Homo sapiens lysine-rich coil-coiled and SAG1 protein, which could activate the expressions of the reporter genes in diploid yeast. Co-immunoprecipitation experiment result indicated the binding between this prey and SAG1 protein was significant (Mann-Whitney U test, Z = - 1.964, P = 0.05). H. sapiens lysine-rich coil-coiled protein was found to be interacted with SAG1. This prey protein may serve as the potential drug target in vaccination study.

Expression and characterization of a lysine rich protein in cow milk

The lysine is considered as the most important essential amino acid, because it is the most limiting in the cereals grains. In this study, a lysine-rich (LR) gene, and the expression vector pcDNA3.1-LR and pBC1-LR were constructed. The LR was expressed in 293T cells driven by the vector pcDNA3.1-LR and checked by RT-PCR and WB. The mammary gland tissue-specific expression vector carrying the LR was injected directly into the lactating mammary glands of cows and the milk samples were checked by a complete amino acid analysis. The results showed that the LR protein was expressed successfully in cells and in cow milk; the expression of LR lasted for 6 d, and the lysine level of the injection group was significantly higher than that of negative controls (p Lass Than 0.05). This study provide a better understanding of how mammary gland expression systems increase the lysine content of milk that can be applied to transgenic dairy cow.

Non-invasive detection of adeno-associated viral gene transfer using a genetically encoded CEST-MRI reporter gene in the murine heart

Research into gene therapy for heart failure has gained renewed interest as a result of improved safety and availability of adeno-associated viral vectors (AAV). While magnetic resonance imaging (MRI) is standard for functional assessment of gene therapy outcomes, quantitation of gene transfer/expression relies upon tissue biopsy, fluorescence or nuclear imaging. Imaging of gene expression through the use of genetically encoded chemical exchange saturation transfer (CEST)-MRI reporter genes could be combined with clinical cardiac MRI methods to comprehensively probe therapeutic gene expression and subsequent outcomes. The CEST-MRI reporter gene Lysine Rich Protein (LRP) was cloned into an AAV9 vector and either administered systemically via tail vein injection or directly injected into the left ventricular free wall of mice. Longitudinal in vivo CEST-MRI performed at days 15 and 45 after direct injection or at 1, 60 and 90 days after systemic injection revealed robust CEST contrast in myocardium that was later confirmed to express LRP by immunostaining. Ventricular structure and function were not impacted by expression of LRP in either study arm. The ability to quantify and link therapeutic gene expression to functional outcomes can provide rich data for further development of gene therapy for heart failure.

Gene expression of an arabinogalactan lysine-rich protein CaAGP18 during vegetative and reproductive development of bell pepper (Capsicum annuum L.).

Lysine-rich (Lys-rich) proteins encoded by AGP17, AGP18, and AGP19 genes are cell wall-associated glycopeptides related to sexual reproduction in flowering plants. This subclass belongs to classical arabinogalactan proteins (AGPs) widely studied in model plants like Arabidopsis. In this study, we identified the CaAGP18 cDNA from bell pepper (Capsicum annuum L.), as well as its expression pattern during vegetative and reproductive development. The deduced amino acid sequence revealed a Lys-rich AGP18 protein of 238 amino acids residues in length with an estimated molecular mass of 22.85kDa and an isoelectric point of 9.7. The protein is predicted as canonical AGP due to the presence of a small Lys-rich region and a C-terminal sequence essential for posttranslational modification with a glycosylphosphatidylinositol (GPI). Phylogenetic analysis showed that CaAGP18 is clustered together with NtAGP18, SpAGP18, StAGP18 and NaAGP18 from Solanaceae species. CaAGP18 expression through plant phenological stages had the highest transcription level in leaves at the seedling stage, whereas in reproductive organs there was a significant up-regulation in pistils during anthesis, also in petals 2days post-anthesis (DPA), and in fruit at the expansion stage. Our results open future research for possible roles of CaAGP18 in cell expansion as a wall-associated plasticizer and reproductive processes like pistil interactions and petal cell death.

Cloning a lysine-rich protein gene from potato (Solanum tuberosum L.) cultivar Thuong Tin and construction of the expression vector

Lysine is one of the limiting essential amino acids because it is not synthesized in the body of animals and humans. They must obtain lysine from their diet. Recent results of gene transfer research showed the possibility of overexpression of genes encoding natural lysine-rich proteins in crops such as rice and corn, to improve protein quality by increasing the lysine content. However, there has been little report on cloning genes for lysine-rich proteins. In this article, we present the results of cloning the StTTLP gene encoding a lysine-rich protein from Thuong Tin potato cultivar. After successful cloning, we have constructed an expression vector to be used for gene transfer. The cloned gene had similarities of 94% and 99% to the SBgLR sequences that were registered in GenBank with the accession numbers KU987257.1 and AY377987.1, respectively. The deduced amino acid sequence of StTTLP protein has high lysine proportion of 16.9%. In addition, glutamic acid component was also high with the value of 22.8%. Thus, the cloned gene is considered as the gene encoding a lysine- and glutamic acid-rich protein. StTTLP gene was successfully cloned into the plant expression vector pCAMBIA2300 under the control of globulin 1 promoter (Glo1) from maize. These results provide a useful tool for gene engineering to improve the quality of protein in crop plants.

Development of high-lysine rice via endosperm-specific expression of a foreign LYSINE RICH PROTEIN gene.

BackgroundLysine (Lys) is considered to be the first limiting essential amino acid in rice. Although there have been extensive efforts to improve the Lys content of rice through traditional breeding and genetic engineering, no satisfactory products have been achieved to date.ResultsWe expressed a LYSINE-RICH PROTEIN gene (LRP) from Psophocarpus tetragonolobus (L.) DC using an endosperm-specific GLUTELIN1 promoter (GT1) in Peiai64S (PA64S), an elite photoperiod-thermo sensitive male sterility (PTSMS) line. The expression of the foreign LRP protein was confirmed by Western blot analysis. The Lys level in the transgenic rice seeds increased more than 30 %, the total amount of other amino acids also increased compared to wild-type. Persistent investigation of amino acids in 3 generations showed that the Lys content was significantly increased in seeds of transgenic rice. Furthermore, Lys content in the hybrid of the transgenic plants also had an approximate 20 % increase compared to hybrid control. At the grain-filling stage, we monitored the transcript abundance of many genes encoding key enzymes involved in amino acid metabolism, and the results suggested that reduced amino acid catabolism led to the accumulation of amino acids in the transgenic plants. The genetically engineered rice showed unfavorable grain phenotypes compared to wild-type, however, its hybrid displayed little negative effects on grain.ConclusionsEndosperm-specific expression of foreign LRP significantly increased the Lys content in the seeds of transgenic plant, and the the Lys increase was stably heritable with 3 generation investigation. The hybrid of the transgenic plants also showed significant increases of Lys content in the seeds. These results indicated that expression of LRP in rice seeds may have promising applications in improving Lys levels in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0837-x) contains supplementary material, which is available to authorized users.

Investigation of Novel Spectroscopic Features in the Near Ultraviolet Region Arising from Non-Aromatic Amino Acids in Peptides and Proteins

Absorption in the UV region has been dominated by aromatic amino acids in peptides and proteins due to the presence of a conjugated structure in their side chains. However, L-Lysine monohydrochloride displayed new near ultraviolet (270 nm) absorption spectra at high concentrations (0.5 M) in aqueous medium [1]. Similar spectral signatures have been reported for lysine rich proteins such as Human Serum Albumin [2]. The origin of these novel and intriguing spectral features in proteins can only be revealed in the absence of any aromatic moiety.We present here the experimental investigations on absorption spectra of amine containing non-aromatic compounds, lysine containing short peptides in aqueous medium and a lysine rich protein (Alpha3C) which lacks aromatic amino acids in order to elucidate the role of the NH2 moiety behind these spectral properties.We carried out excited state TDDFT electronic structure calculations on lysine residues sampled from classical atomistic molecular dynamics simulations of Alpha3C (PDB Code: 2LXY). The calculated UV-VIS spectra of lysine residues from Alpha3C indicate absorption features above 250 nm in agreement with experiments. The unique spectral signatures of lysine residues are shown to arise from charge transfer and pi-pi∗ transitions involving both, the NH3(+) head group and the peptide backbone. The long tail of the experimental absorption spectra above 250 nm is attributed to the sensitivity of the charge transfer and pi-pi∗ transitions to the lysine conformation and environment. Taken together, our joint experimental and theoretical investigation adds a new dimension towards the understanding of UV-VIS absorption in proteins and amino acids.

Pyramiding and evaluation of both a foreign Bacillus thuringiensis and a Lysine-rich protein gene in the elite indica rice 9311.

Gene pyramiding is an efficient approach for the genetic improvement of multiple agronomic traits simultaneously. In this study, we pyramided two foreign genes, cry1Ac driven by the rice Actin I promoter, and lysine-rich protein (LRP), driven by the endosperm-specific GLUTELIN1 (GT1) promoter, into the elite indica cultivar 9311. These two genes were chosen in an attempt to enhance insect-resistance and Lysine (Lys) content. In the pyramided line, the foreign gene cry1Ac was efficiently expressed in the leaves and stems, and exhibited highly efficient resistance to striped stem borer (SSB, Chilo suppressalis Walker) in the laboratory and rice leaf folder (RLF, Cnaphalocrocis medinalis Guenee) in the field. Furthermore, the LRP gene was highly expressed in the endosperm and produced a remarkable increase of Lys content in the seeds of the pyramided line. The data from field trials demonstrated that most of the agronomic traits including yield were well maintained in the pyramided line compared to the parental control. These results strongly suggest that the foreign cry1Ac and LRP genes have remarkable application potential in rice, and the resultant pyramided line serves as an ideal bridge material for the improvement of insect-resistance and high Lys rice in the future.

Microtubule-Associated Protein SBgLR Facilitates Storage Protein Deposition and Its Expression Leads to Lysine Content Increase in Transgenic Maize Endosperm.

Maize (Zea mays) seed is deficient in protein and lysine content. Many studies have been made to improve the nutritional quality of maize seeds. Previously, we reported the role of a natural lysine-rich protein gene SBgLR in increasing protein and lysine content. However, how the SBgLR improves lysine and protein content remains unclear. Here, the reasons and possible mechanism for SBgLR in protein and lysine improvement have been analyzed and discussed. Through seed-specific expression of SBgLR, we obtained transgenic maize with the simultaneously increased lysine and protein contents. High-protein and high-lysine characters were stably inherited across generations. The expression of SBgLR in maize kernels increased the accumulation of both zeins and non-zein proteins. Transmission electron microscopy showed that the number of protein bodies (PBs) was increased obviously in SBgLR transgenic immature endosperms with the morphology and structure of PBs unchanged. The proteinaceous matrix was more abundant in transgenic mature endosperms under scanning electron microscopy. The stabilities of zein and lysine-rich non-zein genes were also increased in transgenic endosperms. Finally, the potential application of SBgLR in maize nutrient improvement was evaluated. This study shows that a cytoskeleton-associated protein has potential applicable value in crop nutrient improving, and provided a feasible strategy for improvement of maize grain quality.

Seed-Specific Expression of the Arabidopsis AtMAP18 Gene Increases both Lysine and Total Protein Content in Maize.

Lysine is the most limiting essential amino acid for animal nutrition in maize grains. Expression of naturally lysine-rich protein genes can increase the lysine and protein contents in maize seeds. AtMAP18 from Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content was introduced into the maize genome with the seed-specific promoter F128. The protein and lysine contents of different transgenic offspring were increased prominently in the six continuous generations investigated. Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm. Compared with the wild type, more protein bodies were observed in the endosperm of transgenic maize. These results implied that, as a cytoskeleton binding protein, AtMAP18 facilitated the formation of protein bodies, which led to accumulation of both zein and non-zein proteins in the transgenic maize grains. Furthermore, F1 hybrid lines with high lysine, high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines. This article provides evidence supporting the use of cytoskeleton-associated proteins to improve the nutritional value of maize.

Tumor-specific expression and detection of a CEST reporter gene.

To develop an imaging tool that enables the detection of malignant tissue with enhanced specificity using the exquisite spatial resolution of MRI. Two mammalian gene expression vectors were created for the expression of the lysine-rich protein (LRP) under the control of the cytomegalovirus (CMV) promoter and the progression elevated gene-3 promoter (PEG-3 promoter) for constitutive and tumor-specific expression of LRP, respectively. Using those vectors, stable cell lines of rat 9L glioma, 9L(CMV-LRP) and 9L(PEG-LRP) , were established and tested for CEST contrast in vitro and in vivo. 9L(PEG-LRP) cells showed increased CEST contrast compared with 9L cells in vitro. Both 9L(CMV-LRP) and 9L(PEG-LRP) cells were capable of generating tumors in the brains of mice, with a similar growth rate to tumors derived from wild-type 9L cells. An increase in CEST contrast was clearly visible in tumors derived from both 9L(CMV-LRP) and 9L(PEG-LRP) cells at 3.4 ppm. The PEG-3 promoter:LRP system can be used as a cancer-specific, molecular-genetic imaging reporter system in vivo. Because of the ubiquity of MR imaging in clinical practice, sensors of this class can be used to translate molecular-genetic imaging rapidly.

Establishing the Lysine-rich Protein CEST Reporter Gene as a CEST MR Imaging Detector for Oncolytic Virotherapy.

To (a) evaluate whether the lysine-rich protein (LRP) magnetic resonance (MR) imaging reporter gene can be engineered into G47Δ, a herpes simplex-derived oncolytic virus that is currently being tested in clinical trials, without disrupting its therapeutic effectiveness and (b) establish the ability of chemical exchange saturation transfer (CEST) MR imaging to demonstrate G47Δ-LRP. The institutional subcommittee for research animal care approved all in vivo procedures. Oncolytic herpes simplex virus G47Δ, which carried the LRP gene, was constructed and tested for its capacity to replicate in cancer cells and express LRP in vitro. The LRP gene was detected through CEST imaging of lysates derived from cells infected with G47Δ-LRP or the control G47Δ-empty virus. G47Δ-LRP was then tested for its therapeutic effectiveness and detection with CEST MR imaging in vivo. Images of rat gliomas were acquired before and 8-10 hours after injection of G47Δ-LRP (n = 7) or G47Δ-empty virus (n = 6). Group comparisons were analyzed with a paired t test. No significant differences were observed in viral replication or therapeutic effectiveness between G47Δ-LRP and G47Δ-empty virus. An increase in CEST image contrast was observed in cell lysates (mean ± standard deviation, 0.52% ± 0.06; P = .01) and in tumors (1.1% ± 0.3, P = .02) after infection with G47Δ-LRP but not G47Δ-empty viruses. No histopathologic differences were observed between tumors infected with G47Δ-LRP and G47Δ-empty virus. This study has demonstrated the ability of CEST MR imaging to show G47Δ-LRP at acute stages of viral infection. The introduction of the LRP transgene had no effect on the viral replication or therapeutic effectiveness. This can aid in development of the LRP gene as a reporter for the real-time detection of viral spread. Online supplemental material is available for this article.