Abstract 393: S-nitrosylation of Atg7 by Thioredoxin-1 (trx1) Protects the Heart Against Myocardial Ischemia via Autophagy Regulation

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Narayani Nagarajan ◽  
Shinichi Oka ◽  
Gihoon NAH ◽  
Peiyong Zhai ◽  
Wataru Mizushima ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Glucose Deprivation ◽  
Wild Type ◽  
Area At Risk ◽  
Biochemical Assays ◽  
Coronary Ligation ◽  
Thioredoxin 1 ◽  
Lc3 Puncta ◽  
Cultured Cardiomyocytes

Thioredoxin 1 (Trx1) is an oxidoreductase that reduces proteins with disulfide bonds. Trx1 also functions as a transnitrosylase, but this occurs only when Trx1 is oxidized at Cys32 and Cys35. In cultured cardiomyocytes (CMs), glucose deprivation (GD) induces oxidation of Trx1 and Trx1 is transnitrosylated. Thus, we hypothesized that Trx1 promotes GD-induced autophagy through its function as a transnitrosylase rather than as an oxidoreductase. GD-induced autophagy, evaluated by counting GFP-LC3 puncta, was inhibited in the presence of the transnitrosylation-defective Trx1C73S mutant (GFP-LC3 puncta per cell under GD; control: 38; Trx1 knockdown: 2*; Trx1C73S: 17*; p<0.05 vs. control), suggesting that Cys73 in Trx1 plays an important role in mediating GD-induced autophagy. Mass spectrometric analyses and biochemical assays showed that Atg7, an essential autophagy regulator, is a Trx1 target and that Trx1 binds to Atg7 via Cys454 and Cys458 in Atg7, thereby transnitrosylating Atg7 at Cys294 and Cys402. Trx1C73S knock-in (Trx1C73S-KI) promoted coronary ligation (CL)-induced myocardial infarction (MI) (MI size/area at risk (AAR) (%); Wild type (WT): 21; Trx1C73S-KI: 42*; p<0.05 vs. WT), in association with reduced S-nitrosylation of Atg7. To test the role of S-nitrosylation of Atg7 in mediating autophagy, we transduced an S-nitrosylation defective Atg7 mutant (Atg7CC294/402SS) into adult cardiomyocytes isolated from cardiac-specific Atg7 knockout mice. Compared to intact Atg7, Atg7CC294/402SS was less able to induce autophagy, as evidenced by reduced LC-3II formation (relative LC3-II; intact Atg7: 1.0; Atg7CC294/402SS: 0.81*; p<0.05). Atg7C402S, but not Atg7C294S, knock-in exacerbated CL-induced MI (MI size/AAR (%); WT: 32; Atg7C402-KI: 42*; p<0.05 vs. WT). These results suggest that Trx1 protects the heart against MI by mediating autophagy via S-nitrosylation of Atg7 at Cys402.

scholarly journals Systematic Mutagenesis of the Saccharomyces cerevisiae MLH1 Gene Reveals Distinct Roles for Mlh1p in Meiotic Crossing Over and in Vegetative and Meiotic Mismatch Repair

2003 ◽  
Vol 23 (3) ◽  
pp. 873-886 ◽  
Author(s):  
Juan Lucas Argueso ◽  
Amanda Wraith Kijas ◽  
Sumeet Sarin ◽  
Julie Heck ◽  
Marc Waase ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Ternary Complex ◽  
Genetic Recombination ◽  
Crossing Over ◽  
Wild Type ◽  
Dna Mismatch ◽  
Biochemical Assays ◽  
Postmeiotic Segregation ◽  
New Alleles

ABSTRACT In eukaryotic cells, DNA mismatch repair is initiated by a conserved family of MutS (Msh) and MutL (Mlh) homolog proteins. Mlh1 is unique among Mlh proteins because it is required in mismatch repair and for wild-type levels of crossing over during meiosis. In this study, 60 new alleles of MLH1 were examined for defects in vegetative and meiotic mismatch repair as well as in meiotic crossing over. Four alleles predicted to disrupt the Mlh1p ATPase activity conferred defects in all functions assayed. Three mutations, mlh1-2, -29, and -31, caused defects in mismatch repair during vegetative growth but allowed nearly wild-type levels of meiotic crossing over and spore viability. Surprisingly, these mutants did not accumulate high levels of postmeiotic segregation at the ARG4 recombination hotspot. In biochemical assays, Pms1p failed to copurify with mlh1-2, and two-hybrid studies indicated that this allele did not interact with Pms1p and Mlh3p but maintained wild-type interactions with Exo1p and Sgs1p. mlh1-29 and mlh1-31 did not alter the ability of Mlh1p-Pms1p to form a ternary complex with a mismatch substrate and Msh2p-Msh6p, suggesting that the region mutated in these alleles could be responsible for signaling events that take place after ternary complex formation. These results indicate that mismatches formed during genetic recombination are processed differently than during replication and that, compared to mismatch repair functions, the meiotic crossing-over role of MLH1 appears to be more resistant to mutagenesis, perhaps indicating a structural role for Mlh1p during crossing over.

scholarly journals Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity

2000 ◽  
Vol 74 (23) ◽  
pp. 11388-11393 ◽  
Author(s):  
Peggy P. Li ◽  
Akira Nakanishi ◽  
Mary A. Tran ◽  
Adler M. Salazar ◽  
Robert C. Liddington ◽  
...  
Keyword(s):  
Life Cycle ◽  
Disulfide Bonds ◽  
Three Dimensional ◽  
Simian Virus 40 ◽  
Experimental System ◽  
Simian Virus ◽  
Dimensional Structure ◽  
Wild Type

ABSTRACT We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants—C9A, C49A, C87A, C104A, C207S, C254A, and C267L—retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca2+-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca2+ ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

scholarly journals Deletion of IGF-1 Receptors in Cardiomyocytes Attenuates Cardiac Aging in Male Mice

Endocrinology ◽  
2016 ◽  
Vol 157 (1) ◽  
pp. 336-345 ◽  
Author(s):  
Sangmi Ock ◽  
Wang Soo Lee ◽  
Jihyun Ahn ◽  
Hyun Min Kim ◽  
Hyun Kang ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Wild Type ◽  
Hypertrophic Response ◽  
Age Related ◽  
Receptor Activator ◽  
Phosphoinositide 3 Kinase ◽  
Cultured Cardiomyocytes

Abstract IGF-1 receptor (IGF-1R) signaling is implicated in cardiac hypertrophy and longevity. However, the role of IGF-1R in age-related cardiac remodeling is only partially understood. We therefore sought to determine whether the deletion of the IGF-1R in cardiomyocytes might delay the development of aging-associated myocardial pathologies by examining 2-year-old male cardiomyocyte-specific IGF-1R knockout (CIGF1RKO) mice. Aging was associated with the induction of IGF-1R expression in hearts. Cardiomyocytes hypertrophied with age in wild-type (WT) mice. In contrast, the cardiac hypertrophic response associated with aging was blunted in CIGF1RKO mice. Concomitantly, fibrosis was reduced in aged CIGF1RKO compared with aged WT hearts. Expression of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and receptor activator of nuclear factor-κB ligand was increased in aged WT hearts, but this increase was attenuated in aged CIGF1RKO hearts. Phosphorylation of Akt was increased in aged WT, but not in aged CIGF1RKO, hearts. In cultured cardiomyocytes, IGF-1 induced senescence as demonstrated by increased senescence-associated β-galactosidase staining, and a phosphoinositide 3-kinase inhibitor inhibited this effect. Furthermore, inhibition of phosphoinositide 3-kinase significantly prevented the increase in IL-1α, IL-1β, receptor activator of nuclear factor-κB ligand, and p21 protein expression by IGF-1. These data reveal an essential role for the IGF-1-IGF-1R-Akt pathway in mediating cardiomyocyte senescence.

scholarly journals RGG-motif protein Sbp1 is required for Processing body (P-body) disassembly

2021 ◽  
Author(s):  
Raju Roy ◽  
Ishwarya Achappa Kuttanda ◽  
Nupur Bhatter ◽  
Purusharth I Rajyaguru
Keyword(s):  
Mrna Decay ◽  
Glucose Deprivation ◽  
Low Complexity ◽  
Wild Type ◽  
Processing Bodies ◽  
P Bodies ◽  
Rna Granules ◽  
Processing Body ◽  
Mrna Fate

AbstractRNA granules are conserved mRNP complexes that play an important role in determining mRNA fate by affecting translation repression and mRNA decay. Processing bodies (P-bodies) harbor enzymes responsible for mRNA decay and proteins involved in modulating translation. Although many proteins have been identified to play a role in P-body assembly, a bonafide disassembly factor remains unknown. In this report, we identify RGG-motif translation repressor protein Sbp1 as a disassembly factor of P-bodies. Disassembly of Edc3 granules but not the Pab1 granules (a conserved stress granule marker) that arise upon sodium azide and glucose deprivation stress are defective in Δsbp1. Disassembly of other P-body proteins such as Dhh1 and Scd6 is also defective in Δsbp1. Complementation experiments suggest that the wild type Sbp1 but not an RGG-motif deletion mutant rescues the Edc3 granule disassembly defect in Δsbp1. We observe that purified Edc3 forms assemblies, which is promoted by the presence of RNA and NADH. Strikingly, addition of purified Sbp1 leads to significantly decreased Edc3 assemblies. Although low complexity sequences have been in general implicated in assembly, our results reveal the role of RGG-motif (a low-complexity sequence) in the disassembly of P-bodies.

Nampt Potentiates Antioxidant Defense in Diabetic Cardiomyopathy

2021 ◽  
Author(s):  
Shin-ichi Oka ◽  
Jaemin Byun ◽  
Chun-yang Huang ◽  
Nobushige Imai ◽  
Guersom Eduardo Ralda ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Diastolic Dysfunction ◽  
Diabetic Cardiomyopathy ◽  
Dependent Manner ◽  
Nad Kinase ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Thioredoxin 1 ◽  
Rate Limiting ◽  
Cultured Cardiomyocytes

Rationale: Diabetic cardiomyopathy is accompanied by increased production of NADH, predominantly through oxidation of fatty acids and consequent increases in oxidative stress. The role of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme of the salvage pathway of NAD + synthesis, in the development of diabetic cardiomyopathy is poorly understood. Objective: We investigated the role of endogenous and exogenous Nampt during the development of diabetic cardiomyopathy in response to high fat diet (HFD) consumption and in the context of oxidative stress. Methods and Results: HFD consumption upregulated endogenous Nampt, and HFD-induced cardiac diastolic dysfunction, fibrosis, apoptosis and pro-inflammatory signaling were alleviated in transgenic mice with cardiac-specific overexpression of Nampt. The alleviation of diastolic dysfunction observed in these mice was abolished by inhibition of NADP(H) production via NAD kinase (NADK) inhibition. Nampt overexpression decreased the GSSG/GSH ratio, oxidation of thioredoxin 1 (Trx1) targets, dityrosine, and the accumulation of toxic lipids, including ceramides and diglycerides, in the presence of HFD consumption. Nampt overexpression upregulated not only NAD + but also NADP + and NADPH in the heart and in cultured cardiomyocytes, which in turn stimulated the GSH and Trx1 systems and alleviated oxidative stress in the heart induced by HFD consumption. In cultured cardiomyocytes, Nampt-induced upregulation of NADPH was abolished in the presence of NADK knockdown, whereas that of NAD + was not. Nampt overexpression attenuated H 2 O 2 -induced oxidative inhibition of Prdx1 and mTOR in an NADK-dependent manner in cultured cardiomyocytes. Nampt overexpression also attenuated H 2 O 2 -induced cell death, an effect that was partly abolished by inhibition of NADK, Trx1 or GSH synthesis. In contrast, oxidative stress and the development of diabetic cardiomyopathy in response to HFD consumption were exacerbated in Nampt +/- mice. Conclusions: Nampt-mediated production of NAD + protects against oxidative stress in part through the NADPH-dependent reducing system, thereby alleviating the development of diabetic cardiomyopathy in response to HFD consumption.

Abstract 150: S-nitrosylation Of Thioredoxin1 At Cysteine 73 Promotes Trans-nitrosylation, Autophagy And Cell Survival During Glucose Deprivation.

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Narayani Nagarajan ◽  
Sebastiano Sciarretta ◽  
Junichi Sadoshima
Keyword(s):  
Cell Survival ◽  
Protein S ◽  
Glucose Deprivation ◽  
Protective Role ◽  
Redox Activity ◽  
Oxidoreductase Activity ◽  
Response To Stress ◽  
Thioredoxin 1 ◽  
Biotin Switch

Thioredoxin-1 (Trx1) is a key antioxidant protein that is known to play a protective role in the heart during oxidative stress mainly through its oxidoreductase activity. Trx1 can be S-nitrosylated and, in turn, can trans-nitrosylate other proteins. However, the role of Trx1-dependent S-nitrosylation in cardiomyocytes (CMs) is not known. Here, we investigated the role of Trx1-mediated protein S-nitrosylation in the regulation of CM survival in response to stress. Using the biotin-switch assay, we found that wild-type Trx1 (Trx1WT) was S-nitrosylated, whereas the extent of S-nitrosylation was attenuated in Trx1C73S, suggesting that Trx1 is S-nitrosylated at Cys73. Also, we observed that the redox activity of Trx1 was intact in the Trx1C73S mutant. Overall protein S-nitrosylation in rat neonatal CMs was increased in response to 4 hrs of glucose deprivation (GD). Using biotin-switch assay and immunocytochemistry (fluorescent staining of s-nitrosylated cysteines), we observed that overexpression of Trx1WT increased, whereas short-hairpin RNA-mediated knockdown of Trx1 (shTrx1) or overexpression of Trx1C73S decreased, total protein S-nitrosylation in response to GD. These results suggest that Trx1Cys73 plays a key role in the regulation of protein S-nitrosylation in CMs during GD. Overexpression of Trx1 increased CM survival after 24 hrs of GD (Trx1WT vs. LacZ: propidium iodide assay, 0.5 ± 0.08-fold, p<0.01). Conversely, shTrx1 or overexpression of Trx1C73S increased cell death during GD (Trx1C73S vs. LacZ: 1.7 ± 0.034-fold, p<0.05). Autophagy is a pro-survival mechanism during GD. Therefore, we tested the effect of Trx1 on autophagy. After 4 hrs of GD, knockdown of Trx1 or overexpression of Trx1C73S decreased autophagy compared to control cells (LC3-II/LC3-I, 0.7-fold; autophagosomes, 0.83 ± 0.16-fold; autolysosomes, 0.62 ± 0.13-fold, p<0.005). Taken altogether, our results suggest that Trx1 promotes autophagy during GD through a trans-nitrosylation dependent mechanism. S-nitrosylation of Trx1 at Cys73 is associated with an overall increase in protein S-nitrosylation in CMs and promotes autophagy and thus, cell survival during GD.

Accelerated LV remodeling after myocardial infarction in TIMP-1-deficient mice: effects of exogenous MMP inhibition

2005 ◽  
Vol 288 (1) ◽  
pp. H149-H158 ◽  
Author(s):  
John S. Ikonomidis ◽  
Jennifer W. Hendrick ◽  
Andrea M. Parkhurst ◽  
Amanda R. Herron ◽  
Patricia G. Escobar ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Ejection Fraction ◽  
Left Ventricular ◽  
Wild Type ◽  
End Diastolic Volume ◽  
Coronary Ligation ◽  
Lv Remodeling ◽  
And Function ◽  
Deficient Mice

Alterations in matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been implicated in adverse left ventricular (LV) remodeling after myocardial infarction (MI). However, the direct mechanistic role of TIMPs in the post-MI remodeling process has not been completely established. The goal of this project was to define the effects of altering endogenous MMP inhibitory control through combined genetic and pharmacological approaches on post-MI remodeling in mice. This study examined the effects of MMP inhibition (MMPi) with PD-166793 (30 mg·kg−1·day−1) on LV geometry and function (conductance volumetry) after MI in wild-type (WT) mice and mice deficient in the TIMP-1 gene [TIMP-1 knockout (TIMP1-KO)]. At 3 days after MI (coronary ligation), mice were randomized into four groups: WT-MI/MMPi ( n = 10), TIMP1-KO-MI/MMPi ( n = 10), WT-MI ( n = 22), and TIMP1-KO-MI ( n = 23). LV end-diastolic volume (EDV) and ejection fraction were determined 14 days after MI. Age-matched WT ( n = 20) and TIMP1-KO ( n = 28) mice served as reference controls. LVEDV was similar under control conditions in WT and TIMP1-KO mice (36 ± 2 and 40 ± 2 μl, respectively) but was greater in TIMP1-KO-MI than in WT-MI mice (48 ± 2 vs. 61 ± 5 μl, P < 0.05). LVEDV was reduced from MI-only values in WT-MI/MMPi and TIMP1-KO-MI/MMPi mice (42 ± 2 and 36 ± 2 μl, respectively, P < 0.05) but was reduced to the greatest degree in TIMP1-KO mice ( P < 0.05). LV ejection fraction was reduced in both groups after MI and increased in TIMP1-KO-MI/MMPi, but not in WT-MI/MMPi, mice. These unique results demonstrated that myocardial TIMP-1 plays a regulatory role in post-MI remodeling and that the accelerated myocardial remodeling induced by TIMP-1 gene deletion can be pharmacologically “rescued” by MMP inhibition. These results define the importance of local endogenous control of MMP activity with respect to regulating LV structure and function after MI.

Is dimerization a common feature in thioredoxins? The case of thioredoxin fromLitopenaeus vannamei

2017 ◽  
Vol 73 (4) ◽  
pp. 326-339 ◽  
Author(s):  
Adam A. Campos-Acevedo ◽  
Rogerio R. Sotelo-Mundo ◽  
Javier Pérez ◽  
Enrique Rudiño-Piñera
Keyword(s):  
Quaternary Structure ◽  
Hydrophobic Interactions ◽  
Cysteine Residue ◽  
Redox Activity ◽  
Regulatory Function ◽  
Wild Type ◽  
Biochemical Assays

The quaternary structure of the redox protein thioredoxin (Trx) has been debated. For bacterial Trx, there is no question regarding its monomeric state. In humans and other eukaryotes, the presence of a cysteine residue at the crystallographic symmetry axis points to the relevance of dimer formation in solution andin vivo. Crystallographic data for shrimp thioredoxin (LvTrx) obtained under different redox conditions reveal a dimeric arrangement mediated by a disulfide bond through residue Cys73 and other hydrophobic interactions located in the crystallographic interface, as reported for human Trx. Through the analysis of five mutants located at the crystallographic interface, this study provides structural and biochemical evidence for the existence in solution of monomeric and dimeric populations of wild-typeLvTrx and five mutants. Based on the results of biochemical assays, SAXS studies and the crystallographic structures of three of the studied mutants (Cys73Ser, Asp60Ser and Trp31Ala), it is clear that the Cys73 residue is essential for dimerization. However, its mutation to Ser produces an enzyme which has similar redox activityin vitroto the wild type. A putative regulatory function of dimerization is proposed based on structural analysis. Nonetheless, the biological role ofLvTrx dimerization needs to be experimentally unveiled. Additionally, the findings of this work reopen the discussion regarding the existence of similar behaviour in human thioredoxin, which shares a Cys at position 73 withLvTrx, a structural feature that is also present in some Trxs from vertebrates and crustaceans.

scholarly journals Ablation of the FACIT collagen XII disturbs musculoskeletal ECM organization and causes patella dislocation and myopathy

2021 ◽  
Author(s):  
Mengjie Zhu ◽  
Fabian Metzen ◽  
Janina Betz ◽  
Mark Hopkinson ◽  
Andrew A Pitsillides ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Triple Helix ◽  
Early Stage ◽  
Local Effect ◽  
Patella Dislocation ◽  
Wild Type ◽  
Collagen Triple Helix ◽  
C Terminus ◽  
Gastrocnemius Muscles

Collagen XII, belonging to the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The trimeric molecule consists of three N-terminal noncollagenous NC3 domains joined by disulfide bonds followed by a short interrupted collagen triple helix at the C-terminus. Collagen XII is expressed widely in the musculoskeletal system and mutations in the COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome, which is associated with skeletal abnormalities and muscle weakness. Our study defines the role of collagen XII in patella development using the Col12a1-/- mouse model. Deficiency in Col12a1 expression causes malformed facies patellaris femoris grooves at an early stage, which leads to patella subluxation and growth retardation. Due to the patella subluxation, more muscle fibers with centralized nuclei occur in the quadriceps than in the gastrocnemius muscles indicating a local effect. To further understand the role of collagen XII in the skeletal tissues single cell RNAseq (scRNA-seq) was performed. Comparison of the gene expression in the tenocyte cell sub-population of wild type and Col12a1-/- mice showed that several matrix genes are altered. Finally, we reinvestigated collagen XII deficient patients and observed a patella instability.

scholarly journals Role of Conserved Cysteines in the Alphavirus E3 Protein

2008 ◽  
Vol 83 (6) ◽  
pp. 2584-2591 ◽  
Author(s):  
Megan M. Parrott ◽  
Sarah A. Sitarski ◽  
Randy J. Arnold ◽  
Lora K. Picton ◽  
R. Blake Hill ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Viral Entry ◽  
Disulfide Bonds ◽  
Functional Role ◽  
Sindbis Virus ◽  
Virus Assembly ◽  
Wild Type ◽  
Virus Particles ◽  
Extracellular Glycoprotein

ABSTRACT Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.

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