Reovirus µ2 protein modulates host cell alternative splicing by reducing protein levels of U5 snRNP core components.

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV's ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that the M1 gene segment, encoding the µ2 protein, is the primary determinant of MRV's ability to alter AS, and that the amino acid at position 208 in µ2 is critical to induce these changes. Moreover, we showed that the expression of µ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of µ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and µ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS.

Reovirus µ2 Protein Impairs Translation to Reduce U5 snRNP Protein Levels.

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family that infects a large range of mammals, including humans. Recently, studies have shown that MRV alters cellular alternative splicing (AS) during viral infection. The structural protein µ2 appears to be the main determinant of these AS modifications by decreasing the levels of U5 core components EFTUD2, PRPF8, and SNRNP200 during infection. In the present study, we investigated the mechanism by which µ2 exerts this effect on the U5 components. Our results revealed that µ2 has no impact on steady-state mRNA levels, RNA export, and protein stability of these U5 snRNP proteins. However, polysome profiling and metabolic labeling of newly synthesized proteins revealed that µ2 exerts an inhibitory effect on global translation. Moreover, we showed that µ2 mutants unable to accumulate in the nucleus retain most of the ability to reduce PRPF8 protein levels, indicating that the effect of µ2 on U5 snRNP components mainly occurs in the cytoplasm. Finally, co-expression experiments demonstrated that µ2 suppresses the expression of U5 snRNP proteins in a dose-dependent manner, and that the expression of specific U5 snRNP core components have different sensitivities to µ2's presence. Altogether, these results suggest a novel mechanism by which the µ2 protein reduces the levels of U5 core components through translation inhibition, allowing this viral protein to alter cellular AS during infection.

The Epstein-Barr virus EBNA1 protein modulates the alternative splicing of cellular genes.

BACKGROUND: Alternative splicing (AS) is an important mRNA maturation step that allows increased variability and diversity of proteins in eukaryotes. AS is dysregulated in numerous diseases, and its implication in the carcinogenic process is well known. However, progress in understanding how oncogenic viruses modulate splicing, and how this modulation is involved in viral oncogenicity has been limited. Epstein-Barr virus (EBV) is involved in various cancers, and its EBNA1 oncoprotein is the only viral protein expressed in all EBV malignancies. METHODS: In the present study, the ability of EBNA1 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in 1238 cancer-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by EBNA1. RESULTS: Upon EBNA1 expression, we detected modifications to the AS profiles of 89 genes involved in cancer. Moreover, we show that EBNA1 modulates the expression levels of various splicing factors such as hnRNPA1, FOX-2, and SF1. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that EBNA1 immunoprecipitates specific cellular mRNAs, but not the ones that are spliced differently in EBNA1-expressing cells. CONCLUSION: The EBNA1 protein can modulate the AS profiles of numerous cellular genes. Interestingly, this modulation protein does not require the RNA binding activity of EBNA1. Overall, these findings underline the novel role of EBNA1 as a cellular splicing modulator.

Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma.

BACKGROUND: Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. RESULTS: Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. CONCLUSIONS: This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.

U5 snRNP Core Proteins Are Key Components of the Defense Response against Viral Infection through Their Roles in Programmed Cell Death and Interferon Induction.

The spliceosome is a massive ribonucleoprotein structure composed of five small nuclear ribonucleoprotein (snRNP) complexes that catalyze the removal of introns from pre-mature RNA during constitutive and alternative splicing. EFTUD2, PRPF8, and SNRNP200 are core components of the U5 snRNP, which is crucial for spliceosome function as it coordinates and performs the last steps of the splicing reaction. Several studies have demonstrated U5 snRNP proteins as targeted during viral infection, with a limited understanding of their involvement in virus-host interactions. In the present study, we deciphered the respective impact of EFTUD2, PRPF8, and SNRNP200 on viral replication using mammalian reovirus as a model. Using a combination of RNA silencing, real-time cell analysis, cell death and viral replication assays, we discovered distinct and partially overlapping novel roles for EFTUD2, PRPF8, and SNRNP200 in cell survival, apoptosis, necroptosis, and the induction of the interferon response pathway. For instance, we demonstrated that EFTUD2 and SNRNP200 are required for both apoptosis and necroptosis, whereas EFTUD2 and PRPF8 are required for optimal interferon response against viral infection. Moreover, we demonstrated that EFTUD2 restricts viral replication, both in a single cycle and multiple cycles of viral replication. Altogether, these results establish U5 snRNP core components as key elements of the cellular antiviral response.

Cellulosic copper nanoparticles and a hydrogen peroxide-based disinfectant trigger rapid inactivation of pseudoviral particles expressing the Spike protein of SARS-CoV-2, SARS-CoV, and MERS-CoV.

Severe acute respiratory syndrome (SARS) is a viral respiratory infection caused by human coronaviruses that include SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). Although their primary mode of transmission is through contaminated respiratory droplets from infected carriers, the deposition of expelled virus particles onto surfaces and fomites could contribute to viral transmission. Here, we use replication-deficient murine leukemia virus (MLV) pseudoviral particles expressing SARS-CoV-2, SARS-CoV, or MERS-CoV Spike (S) protein on their surface. These surrogates of native coronavirus counterparts serve as a model to analyze the S-mediated entry into target cells. Carboxymethyl cellulose (CMC) nanofibers that are combined with copper (Cu) exhibit strong antimicrobial properties. S-pseudovirions that are exposed to CMC-Cu nanoparticles (30 s) display a dramatic reduction in their ability to infect target Vero E6 cells, with ∼97% less infectivity as compared to untreated pseudovirions. In contrast, addition of the Cu chelator tetrathiomolybdate protects S-pseudovirions from CMC-Cu-mediated inactivation. When S-pseudovirions were treated with a hydrogen peroxide-based disinfectant (denoted SaberTM) used at 1:250 dilution, their infectivity was dramatically reduced by ∼98%. However, the combined use of SaberTM and CMC-Cu is the most effective approach to restrict infectivity of SARS-CoV-2-S, SARS-CoV-S, and MERS-CoV-S pseudovirions in Vero E6 cell assays. Together, these results show that cellulosic Cu nanoparticles enhance the effectiveness of diluted SaberTM sanitizer, setting up an improved strategy to lower the risk of surface- and fomite-mediated transmission of enveloped respiratory viruses.

Viral modulation of cellular RNA alternative splicing: A new key player in virus-host interactions?

Upon viral infection, a tug of war is triggered between host cells and viruses to maintain/gain control of vital cellular functions, the result of which will ultimately dictate the fate of the host cell. Among these essential cellular functions, alternative splicing (AS) is an important RNA maturation step that allows exons, or parts of exons, and introns to be retained in mature transcripts, thereby expanding proteome diversity and function. AS is widespread in higher eukaryotes, as it is estimated that nearly all genes in humans are alternatively spliced. Recent evidence has shown that upon infection by numerous viruses, the AS landscape of host-cells is affected. In this review, we summarize recent advances in our understanding of how virus infection impacts the AS of cellular transcripts. We also present various molecular mechanisms allowing viruses to modulate cellular AS. Finally, the functional consequences of these changes in the RNA splicing signatures during virus-host interactions are discussed. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing.

How Many Mammalian Reovirus Proteins are involved in the Control of the Interferon Response?

As with most viruses, mammalian reovirus can be recognized and attacked by the host-cell interferon response network. Similarly, many viruses have developed resistance mechanisms to counteract the host-cell response at different points of this response. Reflecting the complexity of the interferon signaling pathways as well as the resulting antiviral response, viruses can-and often have-evolved many determinants to interfere with this innate immune response and allow viral replication. In the last few years, it has been evidenced that mammalian reovirus encodes many different determinants that are involved in regulating the induction of the interferon response or in interfering with the action of interferon-stimulated gene products. In this brief review, we present our current understanding of the different reovirus proteins known to be involved, introduce their postulated modes of action, and raise current questions that may lead to further investigations.

Proteomes of the aphid Macrosiphum euphorbiae in its resistance and susceptibility responses to differently compatible parasitoids.

Host insects are either susceptible or resistant to parasitoids, where resistant hosts express immunity factors and compatible parasitoids express virulence factors that may reveal the manipulation of susceptible hosts. Using proteomics we compared responses of the same host, the aphid Macrosiphum euphorbiae, challenged by a well-adapted parasitoid Aphidius nigripes or by a less adapted relative, Aphidius ervi. The host was found to be equally acceptable to both parasitoids, but while A. nigripes normally developed and killed hosts (high susceptibility), development of the incompatible A. ervi was arrested at the primary egg stage (high resistance). Two-dimensional gels at two stages of parasitism revealed divergence in patterns of protein regulation of the M. euphorbiae host, responding to A. ervi or A. nigripes, with the greatest number of protein modulations in the host resistance response. In A. ervi-resistant hosts, proPO was strongly up-regulated, as were also three cuticle proteins, suggesting a PO basis and exoskeleton reinforcement as early and late responses of M. euphorbiae to the risk of parasitism. Resistance also correlated with up-regulation of antioxidative, energy-related, cytoskeleton and heat shock proteins. In A. nigripes-susceptible hosts, various proteins implicated in host and bacterial symbiont metabolism were significantly altered, suggesting complex host nutritional modulation. Over-expression of energy-related proteins also increased when A. nigripes established and developed. Aphid proteomes of compatible and incompatible Aphidius parasitism provide an integrative basis for consolidating our knowledge of host-parasitoid interactions.

Conformational and orientational transformation of silk proteins in the major ampullate gland of Nephila clavipes spiders.

The orientational and conformational transformation of the native liquid silk into a solid fiber in the major ampullate gland of the spider Nephila clavipes has been studied by Raman spectromicroscopy. The spectra show that the conformation of silk proteins in the glandular sac contains several secondary structure elements, which is consistent with intrinsically unfolded proteins. A few alpha-helices are also present and involve some alanine residues located in the polyalanine segments of the spidroin sequence. The conversion of the silk solution in the major ampullate gland appears to be a two-state process without intermediate states. In the first and second limbs of the duct, silk is isotropic and spidroins are generally native-like. beta-Sheets start to develop between the second and the third limb of the duct, suggesting that early beta-sheets are generated by shear forces. However, most of the beta-sheets are formed between the draw down taper and the valve. The early beta-sheets formed upward of the draw down taper might play the role of nucleation sites for the subsequent beta-sheet aggregation. The alignment of the polypeptides chains occurs near the valve, revealing that orientational and conformational changes do not occur simultaneously. Extensional flow seems to be the driving force to produce the orientational order, which in turn is associated with the formation of the major part of the beta-sheets. The slow evolution of the spidroin conformation up to the draw down taper followed by the rapid transformation between the drawn down taper and the valve may be important to achieve the optimal structure of the final fiber.

Correction: 2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO.

[This corrects the article DOI: 10.1371/journal.pone.0193804.].

2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO.

The 5' RNA cap structure (m7GpppRNA) is a key feature of eukaryotic mRNAs with important roles in stability, splicing, polyadenylation, mRNA export, and translation. Higher eukaryotes can further modify this minimal cap structure with the addition of a methyl group on the ribose 2'-O position of the first transcribed nucleotide (m7GpppNmpRNA) and sometimes on the adjoining nucleotide (m7GpppNmpNmpRNA). In higher eukaryotes, the DXO protein was previously shown to be responsible for both decapping and degradation of RNA transcripts harboring aberrant 5' ends such as pRNA, pppRNA, GpppRNA, and surprisingly, m7GpppRNA. It was proposed that the interaction of the cap binding complex with the methylated cap would prevent degradation of m7GpppRNAs by DXO. However, the critical role of the 2'-O-methylation found in higher eukaryotic cap structures was not previously addressed. In the present study, we demonstrate that DXO possesses both decapping and exoribonuclease activities toward incompletely capped RNAs, only sparing RNAs with a 2'-O-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2'-O-methylation on the cap structure drastically reduces the affinity of DXO for RNA. Moreover, immunofluorescence and structure-function assays also revealed that a nuclear localisation signal is located in the amino-terminus region of DXO. Overall, these results are consistent with a quality control mechanism in which DXO degrades incompletely capped RNAs.

Temperature-mediated relationship between western flower thrips (Thysanoptera: Thripidae) and chrysanthemum.

Modeling the effect of temperature on the sustainability of insect-plant interactions requires assessment of both insect and plant performance. We examined the effect of temperature on western flower thrips, Frankliniella occidentalis (Pergande), a generalist herbivore with a high reproductive rate, and chrysanthemum inflorescences, a high quality but relatively fixed, ephemeral resource for thrips population growth. We hypothesized that different thrips versus plant responses to temperature result in significant statistical interaction of temperature with thrips abundance and flower damage attributes over time. Experiments were conducted at five temperatures between 20.7 and 35.3 degrees C, with thrips infestation and time after infestation as main effects. Only minor, uncontrolled variations in relative humidity and light intensity may otherwise have influenced the results. High temperatures lead to an initially rapid increase in density of thrips followed by abrupt declines in abundance. The rate of floral senescence increased with temperature and thrips infestation, as indicated by a reduced fresh biomass and greater leaching of yellow pigments. Multiple regression indicated that indices of plant damage responded more directly to thrips density at low than high temperature, supporting the conclusion that temperature affected the outcome beyond what was predictable simply from differential plant and insect optima. The relative intensity of damage caused by individual thrips decreased with increasing temperature, likely caused by thrips competition and reduced survival, growth, and fecundity on depleted inflorescences. Reduced per capita damage at high temperature may be common in insects exploiting fixed plant resources that exhibit an accelerated rate of deterioration at high temperatures.

Transcriptome-wide analysis of alternative RNA splicing events in Epstein-Barr virus-associated gastric carcinomas.

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

Aphid clonal resistance to a parasitoid fails under heat stress.

Parasitoid virulence and host resistance are complex interactions depending on metabolic rate and cellular activity, which in aphids additionally implicate heritable secondary symbionts among the Enterobacteriaceae. As performance of the parasitoid, the aphid host and its symbionts may differentially respond to temperature, the success or failure of aphid parasitism is difficult to predict when temperature varies. We tested the hypothesis that resistance of the pea aphid Acyrthosiphon pisum to the parasitoid Aphidius ervi, which is linked to aphid secondary symbionts, may depend on temperature in several resistant and non-resistant aphid clonal lineages of different geographic origin and of known bacterial symbiosis, using experiments in controlled environments. Complete immunity to A. ervi at 20 degrees C in three different aphid clones whose symbiosis is characterized by the possession of Hamiltonella defensa reversed to high susceptibility at 25 degrees C and especially 30 degrees C, suggesting that the aphid's immune responses to the establishment and early development of the parasitoid is strongly reduced at moderately high temperatures. There was no evidence that a pea aphid control genotype that was susceptible to A. ervi at 20 degrees C could become more resistant as temperature increases, as has been suggested for insect fungal pathogens. By contrast, our results suggest that aphid clonal resistance to A. ervi and related parasitoids is characteristic of cool temperature conditions, similar to various other fitness attributes of aphids. Based on evidence that H. defensa symbionts characterized all three A. ervi resistant pea aphid clones studied, but was absent in control aphids that remained susceptible at all temperatures, we suggest that secondary symbiosis plays a key role in the heat sensitivity of aphid clonal resistance. Our study may also indicate that aphid natural control of variably susceptible host populations by aphid parasitoids is more likely at moderate to high temperatures.

Global Profiling of the Cellular Alternative RNA Splicing Landscape during Virus-Host Interactions.

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.

Diversity of molecular transformations involved in the formation of spider silks.

Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Taking advantage of the efficiency of Raman spectromicroscopy in investigating micrometer-sized biological samples, we have determined the secondary structure of proteins in the complete set of glands of the orb-weaving spider Nephila clavipes. The major and minor ampullate silks in the sac of their glands have identical secondary structures typical of natively unfolded proteins. Spidroins are converted into fibers containing highly oriented ß-sheets. The capture spiral represents a distinct structural singleton. The proteins are highly disordered prior to spinning and undergo no molecular change or alignment upon spinning. The cylindrical, aciniform, and piriform proteins are folded in their initial state with a predominance of α-helices, but whereas the cylindrical gland forms a fiber similar to the major ampullate thread, the aciniform and piriform glands produce fibers dominated by moderately oriented ß-sheets and α-helices. The conformation of the proteins before spinning is related to intrinsic characteristics of their primary structure. Proteins that are unfolded in the gland have repeat sequences composed of submotifs and display no sequence regions with aggregation propensity. By contrast, the folded proteins have neither submotifs nor aggregation-prone sequence regions. Taken together, the Raman data show a remarkable diversity of molecular transformations occurring upon spinning.

Quantitative determination of band distortions in diamond attenuated total reflectance infrared spectra.

Due to its unmatched hardness and chemical inertia, diamond offers many advantages over other materials for extreme conditions and routine analysis by attenuated total reflection (ATR) infrared spectroscopy. Its low refractive index can offer up to a 6-fold absorbance increase compared to germanium. Unfortunately, it also results for strong bands in spectral distortions compared to transmission experiments. The aim of this paper is to present a methodological approach to determine quantitatively the degree of the spectral distortions in ATR spectra. This approach requires the determination of the optical constants (refractive index and extinction coefficient) of the investigated sample. As a typical example, the optical constants of the fibroin protein of the silk worm Bombyx mori have been determined from the polarized ATR spectra obtained using both diamond and germanium internal reflection elements. The positions found for the amide I band by germanium and diamond ATR are respectively 6 and 17 cm(-1) lower than the true value dtermined from the k(nu) spectrum, which is calculated to be 1659 cm(-1). To determine quantitatively the effect of relevant parameters such as the film thickness and the protein concentration, various spectral simulations have also been performed. The use of a thinner film probed by light polarized in the plane of incidence and diluting the protein sample can help in obtaining ATR spectra that are closer to their transmittance counterparts. To extend this study to any system, the ATR distortion amplitude has been evaluated using spectral simulations performed for bands of various intensities and widths. From these simulations, a simple empirical relationship has been found to estimate the band shift from the experimental band height and width that could be of practical use for ATR users. This paper shows that the determination of optical constants provides an efficient way to recover the true spectrum shape and band frequencies of distorted ATR spectra.

Impact of environmental stress on aphid clonal resistance to parasitoids: Role of Hamiltonella defensa bacterial symbiosis in association with a new facultative symbiont of the pea aphid.

Resistance to endoparasitoids in aphids involves complex interactions between insect and microbial players. It is now generally accepted that the facultative bacterial symbiont Hamiltonella defensa of the pea aphid Acyrthosiphon pisum is implicated in its resistance to the parasitoid Aphidius ervi. It has also been shown that heat negatively affects pea aphid resistance, suggesting the thermosensitivity of its defensive symbiosis. Here we examined the effects of heat and UV-B on the resistance of A. pisum to A. ervi and we relate its stability under heat stress to different facultative bacterial symbionts hosted by the aphid. For six A. pisum clones harboring four different facultative symbiont associations, the impact of heat and UV-B was measured on their ability to resist A. ervi parasitism under controlled conditions. The results revealed that temperature strongly affected resistance, while UV-B did not. As previously shown, highly resistant A. pisum clones singly infected with H. defensa became more susceptible to parasitism after exposure to heat. Interestingly, clones that were superinfected with H. defensa in association with a newly discovered facultative symbiont, referred to as PAXS (pea aphid X-type symbiont), not only remained highly resistant under heat stress, but also expressed previously unknown, very precocious resistance to A. ervi compared to clones with H. defensa alone. The prevalence of dual symbiosis involving PAXS and H. defensa in local aphid populations suggests its importance in protecting aphid immunity to parasitoids under abiotic stress.

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution.

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.