Persistent infection with primate foamy virus type 1 increases human immunodeficiency virus type 1 cell binding via a Bet-independent mechanism

We report that human T cells persistently infected with primate foamy virus type 1 (PFV-1) display an increased capacity to bind human immunodeficiency virus type 1 (HIV-1), resulting in increased cell permissiveness to HIV-1 infection and enhanced cell-to-cell virus transmission. This phenomenon is independent of HIV-1 receptor, CD4, and it is not related to PFV-1 Bet protein expression. Increased virus attachment is specifically inhibited by heparin, indicating that it should be mediated by interactions with heparan sulfate glycosaminoglycans expressed on the target cells. Given that both viruses infect similar animal species, the issue of whether coinfection with primate foamy viruses interferes with the natural course of lentivirus infections in nonhuman primates should be considered.     

An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement

Adult epithelial stem cells are thought to reside in specific niches, where they are maintained by adhesion to stromal cells and by intercellular signals. In niches that harbor multiple adjacent stem cells, such as those maintaining Drosophila germ cells, lost stem cells are replaced by division of neighboring stem cells or reversion of transit cells. We have characterized the Drosophila follicle stem cell (FSC) niche as a model of the epithelial niche to learn whether nonneighboring cells can also generate stem cell replacements. Exactly two stroma-free FSC niches holding single FSCs are located in fixed locations on opposite edges of the Drosophila ovariole. FSC daughters regularly migrate across the width of the ovariole to the other niche before proliferating and contributing to the follicle cell monolayer. Crossmigrating FSC daughters compete with the resident FSC for niche occupancy and are the source of replacement FSCs. The ability of stem cell daughters to target a distant niche and displace its resident stem cell suggests that precancerous mutations might spread from niche to niche within stem cell-based tissues.     

Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy viruses using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocol, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (M. Mühle, A. Bleiholder, S. Kolb, J. Hübner, M. Löchelt, J. Denner, Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening, Virology 412 (2011) 333–340), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1.     

Replication of hepatitis C virus subgenomes in nonhepatic epithelial and mouse hepatoma cells

The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.     

The gastric epithelial progenitor cell niche and differentiation of the zymogenic (chief) cell lineage

In the mammalian gastrointestinal tract, the cell fate decisions that specify the development of multiple, diverse lineages are governed in large part by interactions of stem and early lineage progenitor cells with their microenvironment, or niche. Here, we show that the gastric parietal cell (PC) is a key cellular component of the previously undescribed niche for the gastric epithelial neck cell, the progenitor of the digestive enzyme secreting zymogenic (chief) cell (ZC). Genetic ablation of PCs led to failed patterning of the entire zymogenic lineage: progenitors showed premature expression of differentiated cell markers, and fully differentiated ZCs failed to develop. We developed a separate mouse model in which PCs localized not only to the progenitor niche, but also ectopically to the gastric unit base, which is normally occupied by terminally differentiated ZCs. Surprisingly, these mislocalized PCs did not maintain adjacent zymogenic lineage cells in the progenitor state, demonstrating that PCs, though necessary, are not sufficient to define the progenitor niche. We induced this PC mislocalization by knocking out the cytoskeleton-regulating gene Cd2ap in Mist1^−/− mice, which led to aberrant E-cadherin localization in ZCs, irregular ZC-ZC junctions, and disruption of the ZC monolayer by PCs. Thus, the characteristic histology of the gastric unit, with PCs in the middle and ZCs in the base, may depend on establishment of an ordered adherens junction network in ZCs as they migrate into the base.     

Interspecies transmission of simian foamy virus in a natural predator-prey system

Simian foamy viruses (SFV) are ancient retroviruses of primates and have coevolved with their host species for as many as 30 million years. Although humans are not naturally infected with foamy virus, infection is occasionally acquired through interspecies transmission from nonhuman primates. We show that interspecies transmissions occur in a natural hunter-prey system, i.e., between wild chimpanzees and colobus monkeys, both of which harbor their own species-specific strains of SFV. Chimpanzees infected with chimpanzee SFV strains were shown to be coinfected with SFV from colobus monkeys, indicating that apes are susceptible to SFV superinfection, including highly divergent strains from other primate species.     

Characterization of the genome of feline foamy virus and its proteins shows distinct features different from those of primate spumaviruses.

The genome of the feline foamy virus (FeFV) isolate FUV was characterized by molecular cloning and nucleotide sequence analysis of subgenomic proviral DNA. The overall genetic organization of FeFV and protein sequence comparisons of different FeFV genes with their counterparts from other known foamy viruses confirm that FeFV is a complex foamy virus. However, significant differences exist when FeFV is compared with primate foamy viruses. The FeFV Gag protein is smaller than that of the primate spumaviruses, mainly due to additional MA/CA sequences characteristic of the primate viruses only. Gag protein sequence motifs of the NC domain of primate foamy viruses assumed to be involved in genome encapsidation are not conserved in FeFV. FeFV Gag and Pol proteins were detected with monospecific antisera directed against Gag and Pol domains of the human foamy virus and with antisera from naturally infected cats. Proteolytic processing of the FeFV Gag precursor was incomplete, whereas more efficient proteolytic cleavage of the pre125Pro-Pol protein was observed. The active center of the FeFV protease contains a Gln that replaces an invariant Gly residue at this position in other retroviral proteases. Functional studies on FeFV gene expression directed by the promoter of the long terminal repeat showed that FeFV gene expression was strongly activated by the Bell/Tas transactivator protein. The FeFV Bell/Tas transactivator is about one-third smaller than its counterpart of primate spumaviruses. This difference is also reflected by a limited sequence similarity and only a moderate conservation of structural motifs of the different foamy virus transactivators analyzed.     

Replication of human cytomegalovirus DNA: lack of dependence on cell DNA synthesis.

Human cytomegalovirus (CMV) DNA synthesis was studied in 5-fluorouracil (FU)-treated and untreated human embryonic lung cells, which differ greatly with respect to the number of cells in the culture synthesizing cellular DNA. CMV DNA synthesis proceeded at the same rate in FU-treated and in untreated cells. CMV infection also reversed the inhibitory effects of FU and activated cellular DNA synthesis in some of the cells in the FU-treated culture. Autoradiographic studies showed that more than 20% of the cells in the infected FU-treated culture synthesized viral DNA when less than 1% had synthesized cellular DNA, indicating that the synthesis of viral macromolecules proceeds in cells that do not synthesize cellular DNA from the time of infection, and that viral DNA synthesis proceeds independently of the host cell DNA synthesis. Combined autoradiographic and immunofluorescence studies of both the FU-treated and untreated infected cells showed that, whereas 20% of the cells in the cultures synthesize viral DNA and viral antigens, only about 3 to 6% of those cells that synthesize cellular DNA also synthesize viral antigen. Thus, productive infection was delayed or inhibited in those cells that were stimulated by CMV infection to synthesize cellular DNA.     

Multicellular cuddling in a stem cell niche

Haematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate in any blood cell type throughout life and thereby sustain the entire blood system. To do so, HSPCs had been shown to seed, in a multi-step process, intermediate haematopoietic niches before colonizing the adult marrow. While HSPC birth had been thoroughly characterized in the past, both in mammals and in zebrafish, how perivascular niches could host HSPCs and sustain their expansion was poorly understood. In an article published in the last issue of Cell, Tamplin et al.¹ elegantly exploited the many advantages provided by the zebrafish embryo to describe how endothelium remodeling in the perivascular niche, referred to as “cuddling,” favors HSPCs colonization and expansion.     

Nervous activity in a stem cell niche

In this issue of Cell, report a new regulatory axis for the mobilization of hematopoietic stem cells that links these cells to the nervous system and bone in an unanticipated way. The new findings suggest that the nervous system, which has the inherent ability to integrate information from throughout the organism, may govern the local relationship between stem cells and their niches.     

Palindromic sequence plays a critical role in human foamy virus dimerization

The retroviral RNA genome is dimeric, consisting of two identical strands of RNA linked near their 5' ends by a dimer linkage structure. Previously it was shown that human foamy virus (HFV) RNA transcribed in vitro contained three sites, designated SI, SII, and SIII, which contributed to the dimerization process (O. Erlwein, D. Cain, N. Fischer, A. Rethwilm, and M. O. McClure, Virology 229:251-258, 1997). To characterize these sites further, a series of mutants were designed and tested for their ability to dimerize in vitro. The primer binding site and a G tetrad in SI were dispensable for dimerization. However, a mutant that changed the 3' end of SI migrated slower on nondenaturing gels than wild-type RNA dimers. The sequence composition of the SII palindrome, consisting of 10 nucleotides, proved to be critical for in vitro dimerization, since mutations within this sequence or replacement of the sequence with a different palindrome of equal length impaired in vitro dimerization. The length of the palindrome also seems to play an important role. A moderate extension to 12 nucleotides was tolerated, whereas an extension to 16 nucleotides or more impaired dimerization. When nucleotides flanking the palindrome were mutated in a random fashion, dimerization was unaffected. Changing the SIII sequence also led to decreased dimer formation, confirming its contribution to the dimerization process. Interesting mutants were cloned into the infectious molecular clone of HFV, HSRV-2, and were transfected into BHK-21 cells. Mutations in SII that reduced dimerization in vitro also abolished virus replication. In contrast, constructs containing mutations in SI and SIII replicated to some extent in cell culture after an initial drop in viral replication. Analysis of the SIM1 mutant revealed reversion to the wild type but with the insertion of an additional two nucleotides. Analysis of cell-free virions demonstrated that both replication-competent and replication-defective mutants packaged nucleic acid. Thus, efficient dimerization is a critical step for HFV to generate infectious virus, but HFV RNA dimerization is not a prerequisite for packaging.