María Teresa Rejas

Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain

The cellular distribution of insulin-like growth factor I (IGF-I) immunoreactivity was examined in the rat brain from embryonic day 15 to maturity. IGF-I immunoreactivity was found in the perikarya of neurons distributed along the entire extension of the neuronal tube in all the embryonic ages studied (E15, E17, E19 and E21). In E21 animals, the majority of immunoreactive neurons was located in the olfactory bulb, cerebral cortex, hippocampus, striatum, diencephalon, mesencephalic colliculi, trigeminal nuclei, trigeminal ganglion and in motoneurons of the brainstem. In 10- and 20-day-old rats, in addition to the above areas, IGF-I immunoreactivity was also observed in capillary walls, ependymal cells, choroid plexus, glial cells and most fiber paths. In postnatal ages, immunoreactivity in neuronal somas was mainly restricted to the cell nuclei. However, IGF-I immunoreactivity in the neuronal cytoplasm was observed in 20-day-old rats treated with colchicine while fiber paths and neuronal cell nuclei were negative in these animals. In the telencephalon of 20-day-old rats injected with colchicine, the most intense immunoreactive neurons were observed in the olfactory bulb, cerebral cortex, tenia tecta, hippocampus, islands of Calleja, septal nuclei, striatum, endopyriform nucleus and amygdala. Most diencephalic nuclei, the substantia nigra, the mesencephalic colliculi, Purkinje cells in the cerebellar cortex and several nuclei in mesencephalon, pons and medulla oblongata were also immunoreactive. In adult rats injected with colchicine, IGF-I immunoreactivity was located in the same areas as in 20-day-old rats. The number of immunoreactive cells and the intensity of the staining was reduced in adult rats as compared to that found in young postnatal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

A Severe Acute Respiratory Syndrome Coronavirus That Lacks the E Gene Is Attenuated In Vitro and In Vivo

ABSTRACT A deletion mutant of severe acute respiratory syndrome coronavirus (SARS-CoV) has been engineered by deleting the structural E gene in an infectious cDNA clone that was constructed as a bacterial artificial chromosome (BAC). The recombinant virus lacking the E gene (rSARS-CoV-ΔE) was rescued in Vero E6 cells. The recovered deletion mutant grew in Vero E6, Huh-7, and CaCo-2 cells to titers 20-, 200-, and 200-fold lower than the recombinant wild-type virus, respectively, indicating that although the E protein has an effect on growth, it is not essential for virus replication. No differences in virion stability under a wide range of pH and temperature were detected between the deletion mutant and recombinant wild-type viruses. Although both viruses showed the same morphology by electron microscopy, the process of morphogenesis seemed to be less efficient with the defective virus than with the recombinant wild-type one. The rSARS-CoV-ΔE virus replicated to titers 100- to 1,000-fold lower than the recombinant wild-type virus in the upper and lower respiratory tract of hamsters, and the lower viral load was accompanied by less inflammation in the lungs of hamsters infected with rSARS-CoV-ΔE virus than with the recombinant wild-type virus. Therefore, the SARS-CoV that lacks the E gene is attenuated in hamsters, might be a safer research tool, and may be a good candidate for the development of a live attenuated SARS-CoV vaccine.

Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice

Abstract Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-Δ[6–9b]), the structural gene E (rSARS-CoV-ΔE), and a combination of both sets of genes (rSARS-CoV-Δ[E,6–9b]) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.1 and CaCo-2) cell lines and for transgenic (Tg) mice expressing the SARS-CoV receptor human angiotensin converting enzyme-2 (hACE-2), indicating that none of these proteins is essential for the viral cycle. Furthermore, in Vero E6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the final morphology of the virions. Nevertheless, in the absence of E protein, release of virus particles efficacy was reduced. Viruses lacking E protein grew about 100-fold lower than the wt virus in lungs of Tg infected mice but did not grow in the brains of the same animals, in contrast to the rSARS-CoV-Δ[6–9b] virus, which grew almost as well as the wt in both tissues. Viruses lacking E protein were highly attenuated in the highly sensitive hACE-2 Tg mice, in contrast to the minimal rSARS-CoV-Δ[6–9b] and wt viruses. These data indicate that E gene might be a virulence factor influencing replication level, tissue tropism and pathogenicity of SARS-CoV, suggesting that ΔE attenuated viruses are promising vaccine candidates.

Infectious Bursal Disease Virus: Ribonucleoprotein Complexes of a Double-Stranded RNA Virus

Abstract Genome-binding proteins with scaffolding and/or regulatory functions are common in living organisms and include histones in eukaryotic cells, histone-like proteins in some double-stranded DNA (dsDNA) viruses, and the nucleocapsid proteins of single-stranded RNA viruses. dsRNA viruses nevertheless lack these ribonucleoprotein (RNP) complexes and are characterized by sharing an icosahedral T =2 core involved in the metabolism and insulation of the dsRNA genome. The birnaviruses, with a bipartite dsRNA genome, constitute a well-established exception and have a single-shelled T =13 capsid only. Moreover, as in many negative single-stranded RNA viruses, the genomic dsRNA is bound to a nucleocapsid protein (VP3) and the RNA-dependent RNA polymerase (VPg). We used electron microscopy and functional analysis to characterize these RNP complexes of infectious bursal disease virus, the best characterized member of the Birnaviridae family. Mild disruption of viral particles revealed that VP3, the most abundant core protein, present at ∼450 copies per virion, is found in filamentous material tightly associated with the dsRNA. We developed a method to purify RNP and VPg–dsRNA complexes. Analysis of these complexes showed that they are linear molecules containing a constant amount of protein. Sensitivity assays to nucleases indicated that VP3 renders the genomic dsRNA less accessible for RNase III without introducing genome compaction. Additionally, we found that these RNP complexes are functionally competent for RNA synthesis in a capsid-independent manner, in contrast to most dsRNA viruses.

The ESCRT machinery is not required for human cytomegalovirus envelopment

The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold‐labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV‐infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag‐derived virus‐like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.

Interaction of PLP with GFP-MAL2 in the Human Oligodendroglial Cell Line HOG

The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP), the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP)-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested.

Megakaryocytes promote hepatoepithelial liver cell development in E11.5 mouse embryos by cell‐to‐cell contact and by vascular endothelial growth factor A signaling

In the mouse embryo, hematopoietic progenitor cells migrate to the fetal liver (FL) between gestational days (E) 9.5 and 10.5, where they rapidly expand to form the main fetal reservoir of hematopoietic cells. The embryonic megakaryocyte progenitors (MKPs) in the E11.5 FL were identified as CD49fHCD41H (and c‐KitDKDR+CD42+CD9++CD31+) cells, expressing several hepato‐specific proteins. Unlike adult bone marrow megakaryocytes (MKs), embryonic MKPs were CD45− and represent an abundant population in the FL. The CD49fHCD41H MKPs purified by cytometry differentiated in vitro to produce proplatelets, independent of thrombopoietin stimulation, and they responded to stimulation with adenosine diphosphate, thrombin, and the PAR4 thrombin receptor‐activating peptide. Moreover, after removing CD49fHCD41H MKPs from purified E11.5 FL hepatoepithelial‐enriched cell preparations (c‐KitDCD45−Ter119−), the remaining CD49fD cells neither differentiated nor survived in vitro. Indeed, direct cell‐to‐cell contact between the CD49fHCD41H and CD49fD populations was required to promote the hepatocyte differentiation of CD49fD cells. The addition of vascular endothelial growth factor A (VEGF‐A) and medium conditioned by E11.5 CD49fHCD41H MKPs produced a partial effect on CD49fD cells, inducing the formation of hepatoepithelial layers. This effect was abolished by anti‐VEGF‐A antibodies. Together, these findings strongly suggest that CD49fHCD41H MKPs are fundamental to promote FL development, as proposed in adult liver regeneration. Conclusion: The cells of the MK lineage present in the developing mouse embryo liver promote the growth of hepatoepithelial cells in vitro through VEGF‐A signaling and may play a role in liver development in vivo. (HEPATOLOGY 2012;56:1934–1945)

Ultrastructural characterization of membranous torovirus replication factories

Plus‐stranded RNA viruses replicate in the cytosol of infected cells, in membrane‐bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double‐membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV‐infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double‐stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.