Differential patterns of acquired virulence genes distinguish Salmonella strains

Analysis of several Salmonella typhimurium in vivo-induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity. Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence. Some of these regions distinguish broad host range from host-adapted Salmonella serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation. Maintenance of this archipelago of acquired sequence by selection in specific hosts reveals a fossil record of the evolution of pathogenic species.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4+ T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4+ cells during gene therapy of AIDS.

A system for functional analysis of Ebola virus glycoprotein

Ebola virus causes hemorrhagic fever in humans and nonhuman primates, resulting in mortality rates of up to 90%. Studies of this virus have been hampered by its extraordinary pathogenicity, which requires biosafety level 4 containment. To circumvent this problem, we developed a novel complementation system for functional analysis of Ebola virus glycoproteins. It relies on a recombinant vesicular stomatitis virus (VSV) that contains the green fluorescent protein gene instead of the receptor-binding G protein gene (VSVΔG*). Herein we show that Ebola Reston virus glycoprotein (ResGP) is efficiently incorporated into VSV particles. This recombinant VSV with integrated ResGP (VSVΔG*-ResGP) infected primate cells more efficiently than any of the other mammalian or avian cells examined, in a manner consistent with the host range tropism of Ebola virus, whereas VSVΔG* complemented with VSV G protein (VSVΔG*-G) efficiently infected the majority of the cells tested. We also tested the utility of this system for investigating the cellular receptors for Ebola virus. Chemical modification of cells to alter their surface proteins markedly reduced their susceptibility to VSVΔG*-ResGP but not to VSVΔG*-G. These findings suggest that cell surface glycoproteins with N-linked oligosaccharide chains contribute to the entry of Ebola viruses, presumably acting as a specific receptor and/or cofactor for virus entry. Thus, our VSV system should be useful for investigating the functions of glycoproteins from highly pathogenic viruses or those incapable of being cultured in vitro.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Mapping the parameters of prion-induced neuropathology

We present a theoretical framework that enables us to dissect out the parametric dependencies of the pathogenesis of prion diseases. We are able to determine the influence of both host-dependent factors (connectivity, cell density, protein synthesis rate, and cell death) and strain-dependent factors (cell tropism, virulence, and replication rate). We use a model based on a linked system of differential equations on a lattice to explore how the regional distribution of central nervous system pathology in Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia relates to each of these factors. The model then is used to make qualitative predictions about the pathology for two possible hypothetical triggers of neuronal loss in prion diseases. Pathological progression in overexpressing mouse models has been shown to depend on the site of initial infection. The model allows us to compare the pathologies resulting from different inoculation routes.

Ribozymes that cleave reovirus genome segment S1 also protect cells from pathogenesis caused by reovirus infection

Reovirus genome segment S1 encodes protein σ1, which is the receptor binding protein, modulates tissue tropism, and specifies the nature of the antiviral immune response. It makes up less than 2% of reovirus particles and is synthesized in very small amounts in infected cells. Any antiviral strategy aimed at reducing specifically the expression of this genome segment should, in principle, reduce the infectivity of the virus. To test this hypothesis, we have assembled two hammer-head motif-containing ribozymes (Rzs) targeted to cleave at the conserved B and C domains of the reovirus s1 RNA. Protein-independent but Mg2+-dependent sequence-specific cleavage of s1 RNA was achieved by both the Rzs in trans. Cells that transiently express these Rzs, when challenged with reovirus, were protected against the cytopathic effects caused by the virus. This protection correlated with the specific intracellular reduction of s1 transcripts that was due to their cleavage by the Rzs. Rz-treated cells that were challenged with reovirus showed almost complete disappearance of protein σ1 without significantly altering the levels of the other reovirus structural proteins. Thus, Rzs, besides acting as antiviral agents, could be exploited as biological tools to delineate specific functions of target genes.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.

Cell-surface receptors for retroviruses and implications for gene transfer.

Retroviruses can utilize a variety of cell-surface proteins for binding and entry into cells, and the cloning of several of these viral receptors has allowed refinement of models to explain retrovirus tropism. A single receptor appears to be necessary and sufficient for entry of many retroviruses, but exceptions to this simple model are accumulating. For example, HIV requires two proteins for cell entry, neither of which alone is sufficient; 10A1 murine leukemia virus can enter cells by using either of two distinct receptors; two retroviruses can use different receptors in some cells but use the same receptor for entry into other cells; and posttranslational protein modifications and secreted factors can dramatically influence virus entry. These findings greatly complicate the rules governing retrovirus tropism. The mechanism underlying retrovirus evolution to use many receptors for cell entry is not clear, although some evidence supports a mutational model for the evolution of new receptor specificities. Further study of factors that govern retrovirus entry into cells are important for achieving high-efficiency gene transduction to specific cells and for the design of retroviral vectors to target additional receptors for cell entry.

Propagation of an attenuated virus by design: engineering a novel receptor for a noninfectious foot-and-mouth disease virus.

To gain entry into cells, viruses utilize a variety of different cell-surface molecules. Foot-and-mouth disease virus (FMDV) binds to cell-surface integrin molecules via an arginine-glycine-aspartic acid (RGD) sequence in capsid protein VP1. Binding to this particular cell-surface molecule influences FMDV tropism, and virus/receptor interactions appear to be responsible, in part, for selection of antigenic variants. To study early events of virus-cell interaction, we engineered an alternative and novel receptor for FMDV. Specifically, we generated a new receptor by fusing a virus-binding, single-chain antibody (scAb) to intracellular adhesion molecule 1 (ICAM1). Cells that are normally not susceptible to FMDV infection became susceptible after being transfected with DNA encoding the scAb/ICAM1 protein. An escape mutant (B2PD.3), derived with the mAb used to generate the genetically engineered receptor, was restricted for growth on the scAb/ICAM1 cells, but a variant of B2PD.3 selected by propagation on scAb/ICAM1 cells grew well on these cells. This variant partially regained wild-type sequence in the epitope recognized by the mAb and also regained the ability to be neutralize by the mAb. Moreover, RGD-deleted virions that are noninfectious in animals and other cell types grew to high titers and were able to form plaques on scAb/ ICAM1 cells. These studies demonstrate the first production of a totally synthetic cell-surface receptor for a virus. This novel approach will be useful for studying virus reception and for the development of safer vaccines against viral pathogens of animals and humans.

Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Contribuição das células-tronco mesenquimais para as propriedades tumorigênicas de células de glioblastoma humano

Células-tronco mesenquimais (CTM) apresentam tropismo a tumores, sendo importantes componentes do estroma tumoral. No cérebro, o nicho perivascular é uma importante fonte de CTM, as quais podem contribuir direta e/ou indiretamente para o desenvolvimento de tumores, embora os mecanismos envolvidos sejam pouco conhecidos. No presente trabalho, investigou-se a influência de CTM sobre a proliferação, capacidade invasiva e tumorigenicidade de células de Glioblastoma (GBM) humano. Sabe-se que CTM produzem TGFB1, uma citocina multifuncional envolvida em imunomodulação, proliferação, migração e transição epitelial-mesenquimal de células tumorais. Experimentos in vitro, realizados com meios condicionados de CTM de cordão umbilical humano com silenciamento permanente do gene TGFB1, demonstraram que o TGFB1 secretado por CTM é capaz de aumentar significativamente a proliferação e viabilidade de células de GBM humano da linhagem U87FP635. Esses resultados revelam uma importante ação parácrina dessa citocina regulatória, quando produzida por outros tipos celulares contidos no microambiente tumoral. Entretanto, sob condições experimentais que melhor mimetizam o microambiente tumoral, detectou-se que CTM também afetam o comportamento de células tumorais por um mecanismo alternativo, dependente de contato celular, mas independente dos níveis de TGFB1 secretados pelas CTM. Sob condições de cocultivo celular, envolvendo contato físico entre CTM e células de GBM U87FP635, detectou-se um aumento significativo na quantidade de células tumorais viáveis. Quando cultivadas na forma de esferoides tumorais, o contato com CTM aumentou a capacidade invasiva das células U87FP635. Finalmente, em modelo in vivo ectópico de GBM, células U87FP635 geraram tumores mais desenvolvidos quando coinjetadas com CTM. Esses efeitos pró-tumorigênicos foram observados tanto em contato com CTM controles, quanto com CTM contendo o gene TGFB1 permanentemente silenciado. Assim, esses achados indicam que CTM podem exercer efeitos pró-tumorigênicos por dois mecanismos alternativos e independentes: ação parácrina de TGFB1 secretado por CTM e ação mediada por contato célula-célula. Nas condições experimentais testadas, o mecanismo dependente de contato célula-célula demonstrou ser predominante. O estudo proteômico do secretoma dessas células identificou 126 proteínas diferencialmente expressas além de 10 proteínas exclusivamente detectadas em meios condicionados de cocultivos de CTM com células de GBM U87FP635. Cerca de 80% dessas proteínas exclusivamente secretadas pelo contato célula-célula são componentes de exossomos e estão envolvidas em proliferação celular e desenvolvimento tecidual. Esses resultados apontam uma interação dinâmica de comunicação entre CTM e células tumorais, e revelam algumas proteínas interessantes potencialmente envolvidas em uma ação pró-tumorigênica de CTM mediada por contato celular

Rôle des cellules endothéliales dans l’immunité innée précoce induite lors d’infections par des coronavirus murins

Les cellules endothéliales (EC) constituent une première barrière physique à la dissémination de virus pléiotropiques circulant par voie hématogène mais leur contribution à la défense innée anti-virale est peu connue. Des dysfonctions des EC de la barrière hémato-encéphalique (BMEC) et des sinusoïdes hépatiques (LSEC) ont été rapportées dans des neuropathologies et des hépatites aiguës ou chroniques d’origine virale, suggérant que des atteintes à leur intégrité contribuent à la pathogenèse. Les sérotypes de coronavirus de l’hépatite murine (MHV), se différenciant par leur capacité à induire des hépatites et des maladies neurologiques de sévérité variable et/ou leur tropisme pour les EC, représentent des modèles viraux privilégiés pour déterminer les conséquences de l’infection des EC sur la pathogenèse virale. Lors d’infection par voie hématogène, le sérotype MHV3, le plus virulent des MHV, induit une hépatite fulminante, caractérisée par une réponse inflammatoire sévère, et des lésions neurologiques secondaires alors que le sérotype moins virulent, MHV-A59, induit une hépatite modérée sans atteintes secondaires du système nerveux central (SNC). Par ailleurs, le sérotype MHV3, à la différence du MHV-A59, démontre une capacité à stimuler la production de cytokines par la voie TLR2. Les variants atténués du MHV3, les virus 51.6-MHV3 et YAC-MHV3, sont caractérisés par un faible tropisme pour les LSEC et induisent respectivement une hépatite modérée et subclinique. Compte tenu de l’importance des LSEC dans le maintien de la tolérance hépatique et de l’élimination des pathogènes circulants, il a été postulé que la sévérité de l’hépatite et de la réponse inflammatoire lors d’infections par les MHV est associée à la réplication virale et à l’altération des propriétés tolérogéniques et vasculaires des LSEC. Les désordres inflammatoires hépatiques pourraient résulter d’une activation différentielle du TLR2, plutôt que des autres TLR et des hélicases, selon les sérotypes. D’autre part, compte tenu du rôle des BMEC dans la prévention des infections du SNC, il a été postulé que l’invasion cérébrale secondaire par les coronavirus est reliée à l’infection des BMEC et le bris subséquent de la barrière hémato-encéphalique (BHE). À l’aide d’infections in vivo et in vitro par les différents sérotypes MHV, chez des souris ou des cultures de BMEC et de LSEC, nous avons démontré, d’une part, que l’infection in vitro des LSEC par le sétotype MHV3, à la différence des variants 51.6- et YAC-MHV3, altérait la production du facteur vasodilatant NO et renversait leur phénotype tolérogénique en favorisant la production de cytokines et de chimiokines inflammatoires. Ces dysfonctions se traduisaient in vivo par une réponse inflammatoire incontrôlée et une dérégulation du recrutement intrahépatique de leucocytes, favorisant la réplication virale et les dommages hépatiques. Nous avons aussi démontré, à l’aide de souris TLR2 KO et de LSEC dont l’expression du TLR2 a été abrogée par des siRNA, que la sévérité de l’hépatite et de la réponse inflammatoire induite par le sérotype MHV3, dépendait en partie de l’induction et de l’activation préférentielle du TLR2 par le virus dans le foie. D’autre part, la sévérité de la réplication virale au foie et des désordres dans le recrutement leucocytaire intrahépatique induits par le MHV3, et non par le MHV-A59 et le 51.6-MHV3, corrélaient avec une invasion virale subséquente du SNC, au niveau de la BHE. Nous avons démontré que l’invasion cérébrale du MHV3 était associée à une infection productive des BMEC et l’altération subséquente des protéines de jonctions serrées occludine, VE-cadhérine et ZO-1 se traduisant par une augmentation de la perméabilité de la BHE et l’entrée consécutive du virus dans le cerveau. Dans l’ensemble, les résultats de cette étude mettent en lumière l’importance du maintien de l’intégrité structurale et fonctionnelle des LSEC et des BMEC lors d’infections virales aigües par des MHV afin de limiter les dommages hépatiques associés à l’induction d’une réponse inflammatoire exagérée et de prévenir le passage des virus au cerveau suite à une dissémination par voie hématogène. Ils révèlent en outre un nouveau rôle aggravant pour le TLR2 dans l’évolution de l’hépatite virale aigüe ouvrant la voie à de nouvelles avenues thérapeutiques visant à moduler l’activité inflammatoire du TLR2.

Host insulin stimulates Echinococcus multilocularis insulin signalling pathways and larval development.

BACKGROUND The metacestode of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a lethal zoonosis. Infections are initiated through establishment of parasite larvae within the intermediate host's liver, where high concentrations of insulin are present, followed by tumour-like growth of the metacestode in host organs. The molecular mechanisms determining the organ tropism of E. multilocularis or the influences of host hormones on parasite proliferation are poorly understood. RESULTS Using in vitro cultivation systems for parasite larvae we show that physiological concentrations (10 nM) of human insulin significantly stimulate the formation of metacestode larvae from parasite stem cells and promote asexual growth of the metacestode. Addition of human insulin to parasite larvae led to increased glucose uptake and enhanced phosphorylation of Echinococcus insulin signalling components, including an insulin receptor-like kinase, EmIR1, for which we demonstrate predominant expression in the parasite's glycogen storage cells. We also characterized a second insulin receptor family member, EmIR2, and demonstrated interaction of its ligand binding domain with human insulin in the yeast two-hybrid system. Addition of an insulin receptor inhibitor resulted in metacestode killing, prevented metacestode development from parasite stem cells, and impaired the activation of insulin signalling pathways through host insulin. CONCLUSIONS Our data indicate that host insulin acts as a stimulant for parasite development within the host liver and that E. multilocularis senses the host hormone through an evolutionarily conserved insulin signalling pathway. Hormonal host-parasite cross-communication, facilitated by the relatively close phylogenetic relationship between E. multilocularis and its mammalian hosts, thus appears to be important in the pathology of alveolar echinococcosis. This contributes to a closer understanding of organ tropism and parasite persistence in larval cestode infections. Furthermore, our data show that Echinococcus insulin signalling pathways are promising targets for the development of novel drugs.