Lymphotoxin-mediated crosstalk between B cells and splenic stroma promotes the initial type I interferon response to cytomegalovirus

Toll-like receptor (TLR)-dependent pathways control the production of IFNalphabeta, a key cytokine in innate immune control of viruses including mouse cytomegalovirus (MCMV). The lymphotoxin (LT) alphabeta-LTbeta receptor signaling pathway is also critical for defense against MCMV and thought to aid in the IFNbeta response. We find that upon MCMV infection, mice deficient for lymphotoxin (LT)alphabeta signaling cannot mount the initial part of a biphasic IFNalphabeta response, but show normal levels of IFNalphabeta during the sustained phase of infection. Significantly, the LTalphabeta-dependent, IFNalphabeta response is independent of TLR signaling. B, but not T, cells expressing LTbeta are essential for promoting the initial IFNalphabeta response. LTbetaR expression is required strictly in splenic stromal cells for initial IFNalphabeta production to MCMV and is dependent upon the NF-kappaB-inducing kinase (NIK). These results reveal a TLR-independent innate host defense strategy directed by B cells in communication with stromal cells via the LTalphabeta cytokine system.     

Ultrastructural studies of H-1 parvovirus replication. IV. Crystal development and structure with the temperature-sensitive mutant ts1.

Electron microscopy was used to study the development and structure of viral crystals of ts1, a temperature-sensitive mutant of H-1 parvovirus. At early times postinfection, at the restrictive temperature, empty H-1 capsids aggregated to form conspicuous noncrystalline conglomerates in human NB cell nuclei; these particles did not associate with euchromatin as in wild-type H-1 infections. Later on, the capsid aggregated appeared to form polycrystals exhibiting rod-like, hexagonal, and cubic patterns that were interconvertible using a goniometer specimen stage. The unit cell of this crystal was cubic, consisted of 16 empty particles, and measured 50 nm on each side. Full particles made at the permissive temperature were never observed under restrictive conditions. Experiments in which cultures were shifted form the permissive to the restrictive temperature showed that full virions were not incorporated into crystals. The crystals dissociated into individual particles when changes were made from restrictive to permissive conditions. Correlations between the formation of crystals at the restrictive temperature, their dissociation into capsid components after shifting from the restrictive to the permissive state, and the extent of host cell damage were also observed. Possible roles of cellular functions in regulating ts1 H-1 polycrystal assembly and dissociation are discussed.     

Greenhouse bumblebees (<Emphasis Type="Italic">Bombus terrestris</Emphasis>) spread their genes into the wild

Bumblebees (Bombus spp.) are commonly used for greenhouse pollination of tomatoes and other crop plants. The colonies used for this purpose are provided by commercial bumblebee breeders, which by now operate at a professional company level. As a result of this practice commercially bred bumblebee colonies are transported and used over large distances and national borders, introducing subspecies into non-endemic regions. The question whether and to what extends gene flow between such managed greenhouse and wild bumblebee populations exists, so far has not been addressed. Here we used samples from three greenhouses in Poland and the surrounding populations to address this question. Using microsatellite DNA data we found strong genetic introgression from the sampled greenhouse populations into the adjacent populations. Depending on the analysed population, the number of individuals assigned to the greenhouse populations ranged from 0.08 to 0.47. We also found that more distant populations were much less affected by genetic introgression from the greenhouses.     

SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.     

DNA double-strand break repair functions defend against parvovirus infection.

We measured parvovirus replication and sensitivity to X-ray damage in nine CHO cell lines representing a variety of DNA repair deficiencies. We found that parvovirus replication efficiency increases with radiosensitivity. Parvovirus replication is disrupted at an early stage of infection in DNA repair-proficient cells, before conversion of the single-stranded viral DNA genome into the double-stranded replicative form. Thus, status of the DNA repair machinery inversely correlates with parvovirus replication and is proportional to the host's ability to repair X-ray-induced damage.     

Parvovirus genome: nucleotide sequence of H-1 and mapping of its genes by hybrid-arrested translation.

The nucleotide sequence of the parvovirus H-1 has been determined by the chain-terminating method of Sanger. The sequence is 5,176 nucleotides long. Two large open reading frames (1 and 2) and two smaller open reading frames (3 and 4) of potential importance were identified in the plus-strand sequence. Promoter sequences are located at map positions 4 and 38 when map positions are expressed as percent of genome length from the 3' end of the virion minus strand. The locations for the genes for the parvovirus capsid proteins and a 76,000-dalton noncapsid protein (NCVP1) were mapped by hybrid-arrested translation. The gene for the capsid proteins VP1 and VP2' is located in the 5' half of the virus genome. The gene for NCVP1 is located in the 3' half of the viral DNA.     

Defective interfering particles of parvovirus H-1.

Defective interfering particles of the parvovirus H-1 were produced by serial propagation at high multiplicities of infection. Such particles interfere with the synthesis of capsid proteins and infectious virus of standard H-1. The interference is sensitive to UV irradiation, dependent on the multiplicity of the challenge virus, and is active in heterotypic infections against parvovirus H-3 or LuIII. Defective interfering particle genomes have alterations characterized by integral numbers (1 to 10 or more) of a 60-base-pair addition in the neighborhood of the origin of replicative-form DNA replication and deletions that are located primarily within two regions, 32 to 44 or 80 to 90 on the genome map. Some of the implications of these findings are discussed.     

Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells.

The cotransfection of selectable marker genes and the gene for the nonstructural proteins NS1 and NS2 of the autonomous parvovirus H-1 failed to produce cell lines that constitutively expressed NS1. A plasmid, pP38NS1cat, was constructed that expressed the NS1-NS2 gene from the H-1 P38 coat protein promoter in place of the natural P4 promoter. The P38 promoter is constitutively weak and is trans-activated by NS1. Stable cell lines were isolated that contained pP38NS1cat that was constitutively silent, but inducible with exogenous NS1 by superinfection or by treatment with sodium butyrate. The cells that were induced for this self-stimulatory genetic circuit did not remain in the culture, suggesting that expression of NS1-NS2 is cytotoxic or that the expression is not sustained. The properties of these cell lines and an example of the construction of a cell line inducible for expression of the viral coat protein gene and the bacterial gene for chloramphenicol acetyltransferase (cat) are described.