O Vírus Respiratório Sincicial Humano (hRSV) é o patógeno mais comumente associado à doença do trato respiratório inferior em lactentes e crianças. Altas taxas de admissão hospitalar, freqüência de casos e severidade da doenças foi demonstrado em crianças abaixo de dois anos de vida. A freqüência de casos positivos durante o ano de 2004 foi de 43% (188/435) das amostras coletadas no Hospital Universitário/USP na cidade de São Paulo e os isolados brasileiros do grupo A e B agruparam-se nos genótipos previamente caracterizados: GA2, GA5, SAB1, SAB4 e BA like, respectivamente. Palivizumab (PZ) é atualmente o único anticorpo monoclonal disponível para uso em humanos para infecções causadas pelo HRSV. Foi observado o surgimento de escapes mutantes ao PZ in vivo e in vitro, sendo que estas mutações no gene F determinam resistência ao palivizumab. Nós avaliamos através de seqüenciamento da região F1 a ocorrência de escapes mutantes em aspirados de nasofaringe. Realizamos RT-PCR para amplificação de fragmentos do gene F e as seqüências de nucleotídeos foram determinadas. As 30 seqüências analisadas não revelaram mutações ao PZ e através desses dados podemos aferir que o PZ usado profilaticamente em grupos específicos da população é eficaz. / The Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract disease in infants and young children. RSV has high rates of hospital admission and the frequency and severity of infections caused by RSV were assessed in children 2 years of age. In our study the frequency of RSV detection during 2004 was 43% (188/435) of the samples collected from Hospital University/USP in São Paulo city. Partial sequences of G protein gene of 45 isolates from group antigenic A and 8 isolates from antigenic group B were clustered into previously characterized genotypes: GA2,GA5,SAB1, SAB4 e BA like, respectively. Palivizumab (PZ) is the only monoclonal antibody currently available for uses in humans against RSV infectious disease. Here we evaluated the potential for PZ-resistant RSV mutants to arise in clinical samples. Samples from aspirates nasopharyngeal, reverse-PCR-amplified F gene fragments, and the nucleotide sequences were determined. In thirty sequences no revealed F gene mutations. This work shows that palivizumab prophylaxis is safe and efficacious.

Positive-stranded (+)RNA viruses replicate inside cells and depend on many cellular factors to complete their infection cycle. In the meanwhile, (+)RNA viruses face the host innate immunity, such as cell-intrinsic restriction factors that could block virus replication. Firstly, I have established that the plant DDX17-like RH30 DEAD-box helicase conducts strong inhibitory function on tombusvirus replication when expressed in plants and yeast surrogate host. This study demonstrates that RH30 blocks the assembly of viral replicase complex, the activation of RNA-dependent RNA polymerase function of p92pol and viral RNA template recruitment. In addition, the features rendering the abundant plant DEAD-box helicases either antiviral or pro-viral functions in tombusvirus replication are intriguing. I found the reversion of the antiviral function of DDX17-like RH30 DEAD-box helicase and the coopted pro-viral DDX3-like RH20 helicase due to deletion of unique N-terminal domains. The discovery of the sequence plasticity of DEAD-box helicases that can alter recognition of different cis-acting elements in the viral genome illustrates the evolutionary potential of RNA helicases in the arms race between viruses and their hosts. Moreover, I discovered that Xpo1 possesses an anti-viral function and exports previously characterized cell-intrinsic restriction factors (CIRFs) from the nucleus to the replication compartment of tombusviruses. Altogether, in my PhD studies, I found plant RH30 DEAD-box helicase is a potent host restriction factor inhibiting multiple steps of the tombusvirus replication. In addition, I provided the evidence supporting that the Nterminal domain determines the functions of antiviral DDX17-like RH30 DEAD-box helicase and pro-viral DDX3-like RH20 DEAD-box helicase in tombusvirus replication. Moreover, I discovered the emerging significance of the Xpo1-dependent nuclear export pathway in tombusvirus replication.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Influenza virus belongs to the Orthomyxovirus family of viruses that causes respiratory infection in humans, leading to morbidity and mortality. The mature influenza A virion has an envelope that contains two major surface glycoproteins proteins – hemagglutinin (HA) and neuraminidase (NA). HA is a highly antigenic molecules and is responsible binding to host cell surface receptors (Sialic acid), and membrane fusion between the viral membrane and the host endosomal membrane. Most of the antibody response generated against influenza virus either by vaccination or by natural infection is directed against HA. Influenza virus has segmented negative–sense RNA genome which gives the virus the ability to evade the host immune response by incorporating mutations (antigenic drift) and/or by reassotment with other subtypes of influenza A viruses (antigenic shift). Currently licensed vaccines which include an inactivated vaccine, a live attenuated vaccine, and recombinant subunit vaccine are beneficial for providing protection against seasonal influenza viruses that are closely related to the vaccine strain but fail to provide protection against drifted strains. This limits their breadth of protection and thus requires annual revaccination with reformulated vaccines. Also, because selection of a vaccine strain for the next season is purely based on surveillance and prediction, sometimes mismatches do happen between the selected vaccine strains and circulating viruses, resulting in a drastic decrease in vaccine efficacy and thus high morbidity and mortality. Furthermore, the production of these seasonal vaccines takes 6-8 months on an average, and does not guarantee protection against infection with novel reassortant viruses which can cause pandemics. To overcome the draw-backs of seasonal influenza virus vaccines and to enhance our pandemic preparedness, there is an increasing need for game-changing influenza virus vaccines that can confer robust, long-lasting protection against a broad spectrum of influenza virus isolates. Influenza hemagglutinin (HA) is highly immunogenic and thus a major target for vaccine design. HA is synthesized as a precursor polypeptide (HA0), assembles into a trimer, matures by proteolytic cleavage along the secretory pathway and is transported to the cell surface. Mature HA has a globular head domain, primarily composed of the HA1 subunit, which mediates receptor binding, while the stem domain, predominantly comprises of the HA2 subunit, and houses the fusion peptide. At neutral pH, the HA stem is trapped in a metastable state but undergoes an extensive conformational rearrangement at low pH in the late endosome (host-cell endosome) to trigger the fusion of virus and host membranes. Clusters of ‘antigenic sites’ have been identified in the head domain of HA, indicating that it harbors an almost continuous carpet of epitopes that are targeted by antibodies. However, these immunodominant sites constantly accumulate mutations to escape immune pressure, and thereby narrow the breadth of head-directed neutralizing antibodies (nAbs). In contrast to the highly-variable head domain, the membrane-proximal HA stem subdomain has much less sequence variability and, thus, is a desirable target for influenza vaccine development. In the recent past, several broadly neutralizing antibodies (bnAbs) targeting this subdomain with neutralizing activity against diverse influenza A virus subtypes have been isolated from infected people, further proving that this subdomain of HA can be targeted as a vaccine candidate. Steering the immune response towards this conserved, subimmunodominant stem subdomain in the presence of the variable immunodominant head domain of HA has been quite challenging. Alternatively, mimicking the epitome of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogenic capable of eliciting a broadly protective immune response has been difficult because of the metastable nature of HA. Addressing the aforementioned challenges, here we describe the design, stabilization and characterization of novel stem derived immunogens from HA of influenza A viruses using a protein minimization approach. Chapter 1 gives an overview of the influenza virus life cycle, nomenclature and classification of influenza virus; outlines the structural organization and functional properties of different viral proteins. An introduction to the kind of immune responses generated during vaccination or natural infection with the virus is discussed. The conventional vaccines that are currently used and their limitations, recent progress in the field of novel vaccine developmental approaches targeting the conserved epitopes on HA, is also described in this chapter. This chapter also gives a broad overview of bnAbs that have been isolated in the recent past, which target the novel antigenic signatures on HA. The design of a stem domain construct from an H3N2 virus (A/HK/68) is described in Chapter 2. In order to ensure that HA2 folds into the neutral pH conformation, regions of HA1 interacting with it were included in the design. Additionally, two Asp mutations were introduced in the B loop of HA2 to destabilize the low pH conformation and stabilize the desired native, neutral pH conformation. Studies using small peptides (57-98 of HA2) indicated that Asp mutations at positions 63 and 73 destabilized the low pH conformation. Studies on mutants with additional pairs of introduced Cys residues showed that the designed protein H3HA6 was folded into the neutral pH form. Immunization studies using mice showed that the protein was highly immunogenic and provided complete protection against a lethal dose of a homologous virus. Two constructs H3HA6a and H3HA6b, designed from the stem region of drifted H3N2 viruses (A/Phil/2/82 and A/Bris/10/07) were tested for protection against HK/68 to determine the extent of cross-strain protection provided by HA6. While HA6a (from A/Phil/2/82) provided near complete protection against HK/68, HA6b could protect against challenge only partially, possibly because of lower titers of antibodies elicited by this antigen. Studies using FcRγ chain knockout mice indicated that majority of the protection mediated by anti-HA6 antibodies was because of antibody mediated effectors functions, although neutralization as a mechanism of protection was also likely to contribute. In all the 18 subtypes of HA, the B loop contains residues that form the hydrophobic core of the extended coiled coil of the low pH form. As in the case of H3HA6, we suggest that these residues could be mutated to Asp to destabilize the low pH conformation. Two circularly permuted stem domain constructs from an H1N1 virus (A/PR/8/34) and an H5N1 virus (A/Viet/1203/04) were made. The design and characterization of these proteins is described in Chapter 3. H1HA6, H1HA0HA6 and H5HA6 were purified from inclusion bodies and refolded. The proteins H1HA6 and H1HA0HA6 were highly immunogenic and provided protection against a lethal challenge with homologous PR/8/34 virus. Anti-H1HA6 sera had higher titres of antibodies against heterogonous HAs as compared to convalescent sera. Stem derived immunogens from drifted H1N1 viruses (A/NC/20/99 and A/Cal/7/09) have been made and tested for cross-protection with PR/8/34 challenge. While H5HA6 also elicited high titers of antibodies, it could only protect partially against PR/8/34 challenge probably because high enough titers of cross-reactive protective antibodies were not elicited by this protein. These stem immunogens conferred robust subtype specific and modest heterosubtypic protection in vivo against lethal virus challenge. However, the immunogens, especially H1HA6, a stem immunogen from group 1 (PR8) virus is aggregation prone when expressed in E.coli. The strategy used to improve the biophysical and biochemical properties and thus the immunogenicity of these stem derived immunogens is discussed in Chapter 4. A random mutagenesis library of H1HA6 was constructed by error prone PCR using modified nucleotide analogues. The library was displayed on the yeast cell surface to isolate mutants showing better surface expression and improvement in binding to the broadly neutralizing antibody CR6261 compared to the wild-type protein. We isolated few clones, of which one mutant (H1HA6P2) dominated the enriched population. The other mutants differed slightly from H1HA6P2. This mutant differs from the wild-type by two mutations K314E and M317T (H1 numbering) which are close to the CR6261 binding site but outside the antibody foot-print (epitope). This mutant showed improved binding to CR6261 and exhibited significant improvement in surface expression. Improvement was also observed in binding of this mutant to F16v3-ScFv (another broadly neutralizing antibody). Two cysteine mutations were also introduced to further stabilize the trimeric form of the protein. Chapter 5 describes the biophysical and biochemical characterization of the high affinity isolated mutant at the protein level. We expressed this affinity matured mutant gene in E.coli and purified the protein from inclusion bodies. The stabilized mutant protein showed remarkable improvement in biophysical and biochemical properties and was recognized by stem directed conformation sensitive broadly neutralizing antibodies CR6261, F10 and F16v3 with affinity comparable to the full-length HA ectodomain. These results clearly suggest that this mutant protein is properly folded in its native pre-fusion conformation and thus can be an excellent candidate for eliciting stem directed broadly neutralizing antibodies. All these stabilized versions of stem derived immunogens will be tested for immunogenicity and cross-protection with different viral challenges. Chapter 6 describes the development of a method for mapping antibody epitopes (especially conformational epitopes) down to the residue level. Using a panel of single cysteine mutants, displayed on the yeast cell surface, this bypasses the need for laborious and time consuming protein purifications steps used in conventional methods for epitope mapping. We made a panel of single cysteine mutants, covering the entire surface of the antigen (CcdB, a bacterial toxin protein), displayed each mutant individually as well as in a pool, representing all mutants together on the yeast cell surface, and covalently labeled the cysteine with biotin-PEG2-maleimide to mask the area. The effect on antibody binding was monitored to identify the residues and relative positions important for antibody interactions with the displayed antigen by flow cytometry. By using this method we were able to map the conformational as well as linear epitopes of a panel of monoclonal antibodies down to the residue level with ease, and also identify the regions on the antigen which contribute to the antigen city during immunization in different animals. Since, this method is quite easy, rapid and gives in-depth information about antigenic epitopes, it can be useful in rational design of epitomes specific vaccines and other antibody therapeutics. It can easily be extended to other display systems and is a general approach to probe macromolecular interfaces.

O estudo dos protozoários é importante por diversos motivos, entre eles sua diversidade, sua importância evolucionária e impacto na saúde pública. Muitos aspectos tornam o estudo desses seres vivo ainda mais fascinantes, como por exemplo, fenômenos como o trans-splincing e a presença de um vírus em algumas espécies de leishmanias. A proteína U5-15k é considerada de grande importância fazendo parte do spliciossoma. Neste trabalho tivemos o objetivo de iniciar a caracterização desta proteína empregando ferramentas de bioinformática para analisar sua sequencia de aminoácidos, sua estrutura secundária e modelar sua estrutura por homologia, também foi possível otimizar sua expressão, purificação, caracterizar sua auto clivagem e fazer estudos biofísicos de Espalhamento Dinâmico de Luz e Espectroscopia de Dicroísmo Circular. O Leishmania RNA vírus 1-4 (LRV1-4) é um vírus da família Totiviridae de capsídeo icosaédrico que codifica duas proteínas (proteína capsidial e RNA polimerase). Sendo pouco estudado tivemos o objetivo de iniciar a caracterização molecular deste vírus com objetivos futuros de empreender estudos estruturais e funcionais. Não foi possível obter expressão recombinante em nenhuma das duas proteínas virais, por isso se optou por se estabelecer um novo protocolo de lise celular e purificação com o qual se obteve imagens inéditas de Microscopia Eletrônica de Varredura, na qual o vírus é purificado ainda envolto em material genético. / The study of protozoa is important for several reasons, including their diversity, their evolutionary importance and public health impact. Many aspects make the protozoas study even more exciting, for example, phenomena such as trans-splincing and the presence of virus in some species of Leishmania. The U5-15k protein is considered of great importance as part of the spliciossome machinery. In this work we intended to characterize this protein , this using bioinformatics tools to analyze its amino acid sequence, its secondary structure and its structure by homology modeling, it was possible to optimize expression, purification, characterization and make biophysical studies of their self cleavage of Dynamic Light Scattering and Spectroscopy of Circular Dichroism. The Leishmania RNA virus 1-4 (LRV1-4) is a virus belonging to the Totiviridae family with an icosahedral capsid structure that encodes two proteins (a capsid major protein and an RNA polymerase). It was not possible to obtain the recombinant expression of any of the two viral proteins, so it was decided to establish a new protocol for cell lysis and purification with which it was obtained unprecedented images of Transmission electron microscopy, in which the virus is further purified wrap on genetic material.

O Vírus Respiratório Sincicial Humano (hRSV) é o patógeno mais comumente associado à doença do trato respiratório inferior em lactentes e crianças. Altas taxas de admissão hospitalar, freqüência de casos e severidade da doenças foi demonstrado em crianças abaixo de dois anos de vida. A freqüência de casos positivos durante o ano de 2004 foi de 43% (188/435) das amostras coletadas no Hospital Universitário/USP na cidade de São Paulo e os isolados brasileiros do grupo A e B agruparam-se nos genótipos previamente caracterizados: GA2, GA5, SAB1, SAB4 e BA like, respectivamente. Palivizumab (PZ) é atualmente o único anticorpo monoclonal disponível para uso em humanos para infecções causadas pelo HRSV. Foi observado o surgimento de escapes mutantes ao PZ in vivo e in vitro, sendo que estas mutações no gene F determinam resistência ao palivizumab. Nós avaliamos através de seqüenciamento da região F1 a ocorrência de escapes mutantes em aspirados de nasofaringe. Realizamos RT-PCR para amplificação de fragmentos do gene F e as seqüências de nucleotídeos foram determinadas. As 30 seqüências analisadas não revelaram mutações ao PZ e através desses dados podemos aferir que o PZ usado profilaticamente em grupos específicos da população é eficaz. / The Respiratory Syncytial Virus (RSV) is the most common cause of lower respiratory tract disease in infants and young children. RSV has high rates of hospital admission and the frequency and severity of infections caused by RSV were assessed in children 2 years of age. In our study the frequency of RSV detection during 2004 was 43% (188/435) of the samples collected from Hospital University/USP in São Paulo city. Partial sequences of G protein gene of 45 isolates from group antigenic A and 8 isolates from antigenic group B were clustered into previously characterized genotypes: GA2,GA5,SAB1, SAB4 e BA like, respectively. Palivizumab (PZ) is the only monoclonal antibody currently available for uses in humans against RSV infectious disease. Here we evaluated the potential for PZ-resistant RSV mutants to arise in clinical samples. Samples from aspirates nasopharyngeal, reverse-PCR-amplified F gene fragments, and the nucleotide sequences were determined. In thirty sequences no revealed F gene mutations. This work shows that palivizumab prophylaxis is safe and efficacious.

This thesis is devoted to designing and applying advanced algorithmical and statistical tools for analysis of NGS data related to cancer and infection diseases. NGS data under investigation are obtained either from host samples or viral variants. Recently, random peptide phage display libraries (RPPDL) were applied to studies of host's antibody response to different diseases. We study human antibody response to breast cancer and mouse antibody response to Lyme disease by sequencing of the whole antibody repertoire profiles which are represented by RPPDL. Alternatively, instead of sequencing immune response NGS can be applied directly to a viral population within an infected host. Specifically, we analyze the following RNA viruses: the human immunodeficiency virus (HIV) and the infectious bronchitis virus (IBV). Sequencing of RNA viruses is challenging because there are many variants inside population due to high mutation rate. Our results show that NGS helps to understand RNA viruses and explore their interaction with infected hosts. NGS also helps to analyze immune response to different diseases, trace changing of immune response at different disease stages.

Le virus de la stomatite vésiculaire (VSV) et le virus Nipah (NiV) appartiennent respectivement aux familles des Rhabdoviridae et des Paramyxoviridae. VSV est un modèle du virus de la rage tandis que NiV est un virus émergeant, appartenant à la sous-famille des Paramyxovirinae, pour lequel les données moléculaires et structurales sont limitées. Ces sont des virus enveloppés dont le génome code pour cinq à neuf protéines. Le complexe de réplication de ces virus est constitué de trois protéines : la phosphoprotéine (P), la nucléoprotéine (N) et la polymérase virale (L). La N encapside le génome viral et l'ensemble N-ARN sert de matrice pour la transcription et la réplication. La P joue deux rôles : elle sert de cofacteur pour la polymérase et forme le complexe N0-P qui maintient la N sous une forme soluble, compétente pour l'encapsidation des génomes néo-synthétisés. Un premier objectif de mon travail de thèse consistait à étudier la structure et la dynamique des protéines P de VSV et de NiV. Ce sont des protéines modulaires qui contiennent des domaines structurés, séparés par des régions flexibles. A mon arrivée au laboratoire un travail important avait été déjà réalisé sur la P de VSV et j'ai participé à l'achèvement de cette étude. Je me suis ensuite intéressé à la protéine P de NiV. J'ai cristallisé et résolu par diffraction des rayons X les structures du domaine C-terminal et du domaine central (codes PDB : 4F9X et 4GJW). La combinaison de ces modèles cristallographiques avec des données de SAXS sur la P entière et des données de résonance magnétique nucléaire (RMN, collaboration IBS) va permettre d'obtenir un modèle atomique de la P entière sous la forme d'un ensemble de conformères. Un deuxième objectif était d'étudier les complexes N0-P. J'ai activement participé au développement de la méthode de reconstitution et à la caractérisation structurale du complexe N0-P de VSV, entre un mutant de la N (NΔ21) et un peptide N-terminal de la P (code PDB : 3PMK). J'ai ensuite reconstitué, cristallisé et résolu la structure de complexe N0-P de NiV entre la N (tronquée de son domaine C-terminal) et la partie N-terminale de la P. Ces structures montrent par quel mécanismes moléculaires la P maintien la N sous forme monomérique, en empêchant sa polymérisation et son interaction avec l'ARN. Les résultats présentés ici ont permis de générer de nouvelles hypothèses pour expliquer les mécanismes d'encapsidation et d'initiation de la synthèse d'ARN chez ces virus. Le complexe N0-P étant essentiel pour la réplication du virus, l'information structurale obtenue au cours de ce travail devrait permettre d'envisager l'utilisation de ce complexe comme cible pour le développement de composés antiviraux. / Abstract Vesicular stomatitis virus (VSV) and Nipah virus (NiV) belong to the Rhabdoviridae and Paramyxoviridae families, respectively. VSV serves as model system for rabies virus while NiV is an emerging pathogen of the Paramyxovirinae subfamily, for which molecular and structural data are scarce. Both viruses are enveloped and their genomes encode five to nine proteins. Three proteins form their replication complex: the phosphoprotein (P), the nucleoprotein (N) and the viral polymerase (L). N encapsidates the viral genome and this N-RNA complex serves as template for transcription and replication. P has two functions: it serves as a polymerase cofactor and forms an N0-P complex, which keeps the N protein in a soluble and monomeric state, competent for the encapsidation of the newly synthesized genomes. The first goal during the PhD work was to study the structure and dynamics of the VSV and NiV P proteins. These proteins are modular, containing structured domains separated by flexible regions. Before my arrival, a large amount of work was already done on the VSV P protein in the lab and I was involved in the final stages of this work. Then this I studied the NiV P protein, crystallizing and solving the structures of its Central and C-terminal domains by X-ray crystallography (PDB codes: 4F9X and 4GJW). Combining these structures with small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR, collaboration with IBS group) data obtained for the entire protein will allow the construction of an atomic model of the phosphoprotein in the form of a conformational ensemble. The second goal was to study the N0-P complex. I actively participated in the development of the method which permitted the reconstruction of the VSV N0-P complex, using a truncation mutant of the N protein (NΔ21) and an N-terminal peptide from P, and to its structural determination (PDB code: 3PMK). Then I reconstructed, crystallized and solved the structure of the NiV N0-P complex using a C-terminally truncated N protein and the N-terminal region of the P protein. Both structures yielded insights into the molecular mechanisms used by the phosphoproteins in order to maintain the corresponding nucleoproteins in their monomeric state, thus inhibiting their polymerization and interaction with RNA. The results presented here also offered new hypothesis about mechanisms of encapsidation and of RNA synthesis initiation. Given that the N0-P complex is an essential component of the replication complex, the structural information gained from this work allow us to consider this complex as a potential antiviral target.

Le virus de la mosaïque jaune du navet (TYMV) possède un génome monopartite constitué d’ARN de polarité positive codant pour trois protéines, dont seule la polyprotéine 206K est indispensable à la réplication virale.Elle subit une maturation protéolytique, générant les protéines 140K et 66K, localisées au niveau de l’enveloppe des chloroplastes, siège de la réplication virale.Adressée aux chloroplastes, la protéine 140K y recrute la 66K et se comporte comme une protéine intégrale membranaire.Le domaine d’adressage aux chloroplastes (DAC) de la protéine 140K a été défini grâce à la transfection et à des protoplastes d’Arabidopsis thaliana par différentes constructions codantpour des versions délétées de la protéine fusionnées à l’EGFP, et à leur observation en microscopie confocale. Le DAC comprend deux hélices alpha amphipathiques dont la présence a été attestée par dichroïsme circulaire. Leur nécessité pour la localisation aux chloroplastes, l’association aux membranes et la réplication virale, a été étudiée. Différents patterns de distribution subcellulaire de la protéine 140K ont été observés. Ils sont corrélés au taux d’expression de la protéine. Sa dimérisation a également été démontrée.L’implication d’autres résidus du DAC dans la localisation subcellulaire, la dimérisation et la réplication virale, a également été recherchée. / Turnip yellow mosaic virus (TYMV) is a positive single-stranded RNA virus. Among the three ORFs encoded by the TYMV genome, 206K is the only protein required for viral replication. It is cleaved into 140K and 66K, which are both present at the chloroplast envelope membrane, where viral replication takes place.The 140K protein is targeted to chloroplasts, where it recruits 66K, and behaves as an integral membrane protein. The chloroplast targeting domain (DAC) of the 140K protein was defined using Arabidopsis thaliana protoplasts transfected by various constructs encoding deleted versions of 140Kfused to EGFP and subsequent confocal microscopy. The DAC comprises two amphipathic alpha helices, as confirmed by circular dichroism. Their involvement in chloroplast localisation and membrane association has been assessed, as well as their contribution to viral replication.We observed different subcellular distribution patterns of 140K protein, which correlate with the expression level of the protein. Its capability to dimerize has also been demonstrated.The involvement of other DAC residues in subcellular localisation, dimerization and viral replication has been studied.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.