The double-stranded RNA-binding protein, Staufen1, is an IRES-transacting factor regulating HIV-1 cap-independent translation initiation.

Translation initiation of the viral genomic mRNA (vRNA) of human immunodeficiency virus-type 1 (HIV-1) can be mediated by a cap- or an internal ribosome entry site (IRES)-dependent mechanism. A previous report shows that Staufen1, a cellular double-stranded (ds) RNA-binding protein (RBP), binds to the 5'untranslated region (5'UTR) of the HIV-1 vRNA and promotes its cap-dependent translation. In this study, we now evaluate the role of Staufen1 as an HIV-1 IRES-transacting factor (ITAF). We first confirm that Staufen1 associates with both the HIV-1 vRNA and the Gag protein during HIV-1 replication. We found that in HIV-1-expressing cells, siRNA-mediated depletion of Staufen1 reduces HIV-1 vRNA translation. Using dual-luciferase bicistronic mRNAs, we show that the siRNA-mediated depletion and cDNA-mediated overexpression of Staufen1 acutely regulates HIV-1 IRES activity. Furthermore, we show that Staufen1-vRNA interaction is required for the enhancement of HIV-1 IRES activity. Interestingly, we find that only Staufen1 harboring an intact dsRNA-binding domain 3 (dsRBD3) rescues HIV-1 IRES activity in Staufen1 CRISPR-Cas9 gene edited cells. Finally, we show that the expression of Staufen1-dsRBD3 alone enhances HIV-1 IRES activity. This study provides evidence of a novel role for Staufen1 as an ITAF promoting HIV-1 vRNA IRES activity.

LDL particle size and antioxidant HDL function improve after sustained virological response in patients with chronic HCV.

HCV infection is associated with an increased incidence of cardiovascular (CV) events. Mechanisms underlying this association remain unknown. In our study, twenty HCV patients (median age 60.5 years, 65% male and 80% with cirrhosis) were evaluated prior, during and after direct-acting antiviral treatment. Ninety percent of patients achieved sustained virological response (SVR). Significant changes were observed in LDL particle size index, measured by LDL-C/apoB ratio, which increased after treatment (p = 0.023). In addition, HDL antioxidant capacity improved gradually from 34.4% at baseline to 42.4% at 4 weeks (p = 0.011), 65.9% at end of treatment EOT (p = 0.002) and remained elevated at 12-week (p = 0.001) after EOT compared to baseline values. Our findings suggest that a shift to a less atherogenic lipid profile may be a possible mechanism associated with CV risk reduction in patients with HCV infection achieving SVR.

The internal ribosome entry site of the Dengue virus mRNA is active when cap-dependent translation initiation is inhibited.

Dengue virus (DENV) is an enveloped, positive-sense, single-stranded RNA virus belonging to the Flaviviridae family. Translation initiation of the DENV mRNA can occur following a cap-dependent or a cap-independent mechanism. Two non-mutually exclusive cap-independent mechanisms of translation initiation have been described for the DENV mRNA. The first corresponds to a 5'end-dependent internal ribosome entry site (IRES)-independent mechanism, while the second relies on IRES-dependent initiation. In this report, we study the recently discovered DENV IRES. Results show that the DENV IRES is functional in the rabbit reticulocyte (RRL) in vitro translation system. In accordance, the activity of DENV IRES was resistant to the cleavage of eIF4G by the Foot-and-mouth disease virus leader protease in RRL. In cells, the DENV IRES exhibited only a marginal activity under standard culture conditions. The DENV IRES showed weak activity in HEK 293T cells; however, the DENV IRES activity was significantly enhanced in HEK 293T cells expressing the Human rhinovirus 2A protease. These findings suggest that the DENV IRES enables viral protein synthesis under conditions that suppress canonical translation initiation.IMPORTANCE Dengue virus (DENV), the etiological agent of Dengue, a febrile and hemorrhagic disease, infects millions of people per year in tropical and subtropical countries. When infecting cells, DENV induces stress conditions known to inhibit canonical protein synthesis. Under these conditions, DENV mRNA thrives using non-canonical modes of translation initiation. In this study, we characterize the mechanism dependent upon an internal ribosome entry site (IRES). Herein, we describe the activity of the DENV IRES in vitro and cells. We show that in cells, DENV IRES enables the viral mRNA to translate under conditions that suppress canonical translation initiation.

Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation.

The full-length mRNAs of the human immunodeficiency virus type-1 (HIV-1), the human T-cell lymphotropic virus type-1 (HTLV-1), and the mouse mammary tumor virus (MMTV) harbor IRESs. The activity of the retroviral-IRESs requires IRES-transacting factors (ITAFs), being hnRNP A1, a known ITAF for the HIV-1 IRES. In this study, we show that hnRNP A1 is also an ITAF for the HTLV-1 and MMTV IRESs. The MMTV IRES proved to be more responsive to hnRNP A1 than either the HTLV-1 or the HIV-1 IRESs. The impact of post-translational modifications of hnRNP A1 on HIV-1, HTLV-1 and MMTV IRES activity was also assessed. Results show that the HIV-1 and HTLV-1 IRESs were equally responsive to hnRNP A1 and its phosphorylation mutants S4A/S6A, S4D/S6D and S199A/D. However, the S4D/S6D mutant stimulated the activity from the MMTV-IRES to levels significantly higher than the wild type hnRNP A1. PRMT5-induced symmetrical di-methylation of arginine residues of hnRNP A1 enabled the ITAF to stimulate the HIV-1 and HTLV-1 IRESs while reducing the stimulatory ability of the ITAF over the MMTV IRES. We conclude that retroviral IRES activity is not only dependent on the recruited ITAFs but also relies on how these proteins are modified at the post-translational level.

The Andes Orthohantavirus NSs Protein Antagonizes the Type I Interferon Response by Inhibiting MAVS Signaling.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-ß, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Correlation between female sex, IL28B genotype, and the clinical severity of bronchiolitis in pediatric patients.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) that impact on the differential expression of interleukin 28B (IL28B) are implicated in the progression of viral-induced diseases. In this prospective longitudinal cohort study, we evaluated the association between IL28B SNPs rs12979860 and rs8099917 and the clinical outcome of bronchiolitis in pediatric patients. METHODS: A total of 682 infants suffering from bronchiolitis, categorized based on the final clinical outcome as mild or severe, were genotyped for IL28B SNPs rs12979860 and rs8099917. RESULTS: When infants were categorized exclusively based on the final clinical outcome, no association was established between IL28B SNPs and the severity of bronchiolitis. However, when stratified by sex, the homozygotes for the minor alleles of rs12979860 (T) and rs8099917 (G) were associated with a mild disease in girls but not in boys. CONCLUSION: SNPs rs12979860 and rs8099917 correlate with the severity of bronchiolitis and display a sex bias, where GG rs8099917 and TT rs12979860 genotypes are associated with a mild disease in girls but not in boys. These findings suggest that innate immunity and female sex links with the outcome of the diseases induced by respiratory viruses, such as RSV.

Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper protein (HBZ) is expressed in all cases of ATL and is directly associated with virus pathogenicity. The two isoforms of the HBZ protein are synthesized from antisense messenger RNAs (mRNAs) that are either spliced (sHBZ) or unspliced (usHBZ) versions of the HBZ transcript. The sHBZ and usHBZ mRNAs have entirely different 5'untranslated regions (5'UTR) and are differentially expressed in cells, with the sHBZ protein being more abundant. Here, we show that differential expression of the HBZ isoforms is regulated at the translational level. Translation initiation of the usHBZ mRNA relies on a cap-dependent mechanism, while the sHBZ mRNA uses internal initiation. Based on the structural data for the sHBZ 5'UTR generated by SHAPE in combination with 5' and 3' deletion mutants, the minimal region harboring IRES activity was mapped to the 5'end of the sHBZ mRNA. In addition, the sHBZ IRES recruited the 40S ribosomal subunit upstream of the initiation codon, and IRES activity was found to be dependent on the ribosomal protein eS25 and eIF5A.

Hepatitis A outbreak since November 2016 affecting men who have sex with men (MSM) in Chile connected to the current outbreak in MSM in Europe, situation up to October 2017.

A hepatitis A outbreak has occurred in Chile since November 2016. Men are predominantly affected, with a large proportion of men who have sex with men (MSM). We describe 12 consecutive unrelated confirmed cases who presented at our healthcare institution in Santiago Metropolitan Area. Nine were men, all reporting having had sex with men. Ten viral sequences, genotyped as IA, clustered with the V16-25801 strain causing outbreaks mostly in MSM in Europe since mid-2016.

Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection / Detección de concentraciones víricas elevadas en la bilis y en las heces de pacientes con infección crónica por el virus de la hepatitis C

BACKGROUND: The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. OBJECTIVES: We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. MATERIALS AND METHODS: Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. RESULTS: The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). CONCLUSIONS: HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread

INTRODUCCIÓN: El ciclo de vida del virus de la hepatitis C (VHC) está estrechamente ligado al metabolismo lipídico. Recientemente, se ha descrito que el NPC1L1 (un transportador del colesterol) actúa como un receptor del VHC. Este receptor se expresa en la membrana canalicular de los hepatocitos y en el intestino, y actúa como uno de los principales transportadores durante la circulación enterohepática del colesterol. OBJETIVOS: Planteamos la hipótesis de que el VHC podría tener un ciclo similar, por lo que nuestro objetivo fue estudiar la presencia del VHC en la bilis y en las heces de pacientes infectados. MATERIALES Y MÉTODOS: Se obtuvieron muestras de sangre, heces y bilis duodenal de pacientes infectados por el VHC. La concentración vírica en la bilis se normalizó respecto a la concentración de sales biliares de cada muestra y también se evaluó la presencia de la proteína central del VHC. Se reclutaron un total de 12 pacientes. Se detectó el ARN del VHC en la bilis de 10 pacientes. RESULTADOS: La media de la concentración vírica fue 2,5log10UI/60mg de sales biliares. En las muestras de heces, se detectó el ARN del VHC en 10 pacientes (media de la concentración 2,7log10UI/g de heces). CONCLUSIONES: El ARN del VHC es fácilmente detectable y está presente en concentraciones relativamente elevadas en las muestras de bilis y heces de pacientes infectados. Esto puede tener importancia como foco de infección en varones que mantienen relaciones sexuales con otros varones. Es posible que la secreción biliar del VHC pueda desempeñar un papel en la persistencia de la virosis a través de la circulación enterohepática del virus o la propagación intrahepática

Detection of high biliary and fecal viral loads in patients with chronic hepatitis C virus infection.

BACKGROUND: The life cycle of the hepatitis C virus (HCV) is closely associated with lipid metabolism. Recently, NPC1L1 (a cholesterol transporter) has been reported to function as an HCV receptor. This receptor is expressed in the hepatocyte canalicular membrane and in the intestine; serving as a key transporter for the cholesterol enterohepatic cycle. OBJECTIVES: We hypothesized that HCV might have a similar cycle, so we aimed to study the presence of HCV in bile and stools of infected patients. MATERIALS AND METHODS: Blood, feces, and duodenal bile samples were collected from patients infected with HCV. The biliary viral load was normalized to the bile salt concentration of each sample and the presence of HCV core protein was also evaluated. A total of 12 patients were recruited. HCV RNA was detected in the bile from ten patients. RESULTS: The mean viral load was 2.5log10IU/60mg bile salt. In the stool samples, HCV RNA was detected in ten patients (mean concentration 2.7log10IU/g of feces). CONCLUSIONS: HCV RNA is readily detectable and is present at relatively high concentrations in the bile and stool samples of infected patients. This may be relevant as a source of infection in men who have sex with men. Biliary HCV secretion may perhaps play a role in the persistence of viral infection via an enterohepatic cycle of the virus or intrahepatic spread.

Targeting deoxyhypusine hydroxylase activity impairs cap-independent translation initiation driven by the 5'untranslated region of the HIV-1, HTLV-1, and MMTV mRNAs.

Replication of the human immunodeficiency virus type 1 (HIV-1) is dependent on eIF5A hypusination. Hypusine is formed post-translationally on the eIF5A precursor by two consecutive enzymatic steps; a reversible reaction involving the enzyme deoxyhypusine synthase (DHS) and an irreversible step involving the enzyme deoxyhypusine hydroxylase (DOHH). In this study we explored the effect of inhibiting DOHH activity and therefore eIF5A hypusination, on HIV-1 gene expression. Results show that the expression of proteins from an HIV-1 molecular clone is reduced when DOHH activity is inhibited by Deferiprone (DFP) or Ciclopirox (CPX). Next we evaluated the requirement of DOHH activity for internal ribosome entry site (IRES)-mediated translation initiation driven by the 5'untranslated region (5'UTR) of the full length HIV-1 mRNA. Results show that HIV-1 IRES activity relies on DOHH protein concentration and enzymatic activity. Similar results were obtained for IRES-dependent translation initiation mediated by 5'UTR of the human T-cell lymphotropic virus type 1 (HTLV-1) and the mouse mammary tumor virus (MMTV) mRNAs. Interestingly, activity of the poliovirus IRES, was less sensitive to the targeting of DOHH suggesting that not all viral IRESs are equally dependent on the cellular concentration or the activity of DOHH. In summary we present evidence indicating that the cellular concentration of DOHH and its enzymatic activity play a role in HIV-1, HTLV-1 and MMTV IRES-mediated translation initiation.

Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis.

Polypyrimidine tract-binding protein binds to the 5' untranslated region of the mouse mammary tumor virus mRNA and stimulates cap-independent translation initiation.

The 5' untranslated region (UTR) of the full-length mRNA of the mouse mammary tumor virus (MMTV) harbors an internal ribosomal entry site (IRES). In this study, we show that the polypyrimidine tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the MMTV 5' UTR stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Results show that PTB1 and PTB4, but not PTB2, stimulate MMTV-IRES activity. PTB1 promotes MMTV-IRES-mediated initiation more strongly than PTB4. When expressed in combination, PTB1 further enhanced PTB4 stimulation of the MMTV-IRES, while PTB2 fully abrogates PTB4-induced stimulation. PTB1-induced stimulation of MMTV-IRES was not altered in the presence of PTB4 or PTB2. Mutational analysis reveals that stimulation of MMTV-IRES activity is abrogated when PTB1 is mutated either in RRM1/RRM2 or RRM3/RRM4. In contrast, a PTB4 RRM1/RRM2 mutant has reduced effect over MMTV-IRES activity, while stimulation of the MMTV-IRES activity is still observed when the PTB4 RRM3/RMM4 mutant is used. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity. In contrast, PTB2 acts as a negative modulator of PTB4-induced stimulation of MMTV-IRES. We conclude that PTB1 and PTB4 act as IRES trans-acting factors of the MMTV-IRES.

Association of Single-Nucleotide Polymorphisms in IL28B, but Not TNF-α, With Severity of Disease Caused by Andes Virus.

BACKGROUND: Andes virus (ANDV) is the sole etiologic agent of hantavirus cardiopulmonary syndrome (HCPS) in Chile, with a fatality rate of about 35%. Individual host factors affecting ANDV infection outcome are poorly understood. In this case-control genetic association analysis, we explored the link between single-nucleotide polymorphisms (SNPs) rs12979860, rs8099917 and rs1800629 and the clinical outcome of ANDV-induced disease. The SNPs rs12979860 and rs8099917 are known to play a role in the differential expression of the interleukin 28B gene (IL28B), whereas SNP rs1800629 is implicated in the expression of tumor necrosis factor α gene (TNF-α). METHODS: A total of 238 samples from confirmed ANDV-infected patients collected between 2006 and 2014, and categorized according to the severity of the disease, were genotyped for SNPs rs12979860, rs8099917, and rs1800629. RESULTS: Analysis of IL28B SNPs rs12979860 and rs8099917 revealed a link between homozygosity of the minor alleles (TT and GG, respectively), displaying a mild disease progression, whereas heterozygosity or homozygosity for the major alleles (CT/CC and TG/TT, respectively) in both IL28B SNPs is associated with severe disease. No association with the clinical outcome of HCPS was observed for TNF-α SNP rs1800629 (TNF -308G>A). CONCLUSIONS: The IL28B SNPs rs12979860 and rs8099917, but not TNF-α SNP rs1800629, are associated with the clinical outcome of ANDV-induced disease, suggesting a possible link between IL28B expression and ANDV pathogenesis.

The 5' untranslated region of the human T-cell lymphotropic virus type 1 mRNA enables cap-independent translation initiation.

UNLABELLED: The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5'-untranslated region (5'UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5'UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE: The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene expression.

A cis-acting element present within the Gag open reading frame negatively impacts on the activity of the HIV-1 IRES.

Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1.

Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1.

The 5'untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5'UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5'UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5'UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5'UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5'UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5'UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed.