Plasma membrane receptors for ecotropic murine retroviruses require a limiting accessory factor.

A retroviral vector was used to express various amounts of the receptor (ecoR) for ecotropic host range murine retroviruses on naturally barren hamster, mink, and human cells. These cells and murine cells were then incubated for 2 h with dilutions of a helper-free ecotropic retrovirus that encodes human growth hormone, and the number of infected cells was later determined by growth hormone-specific immunofluorescence. For all cells under the conditions of these studies, virus adsorption was the limiting step of infection and the cellular capacities for infection were unsaturated either at cell surfaces or at intracellular sites. Thus, infections occurred at low multiplicities of infection per cell and were directly proportional to virus and cell concentrations, and only a small percentage (ca. 5%) of the infectious virions became adsorbed from the medium during the 2-h incubations. Although increasing the adsorption by raising virus or cell concentrations results in more infections in the cultures, increasing adsorption by raising the number of ecoR above a low threshold had no effect on infections. Thus, cells with a low number of ecoR were infected as efficiently as highly adsorbing cells that contained many times more ecoR. To reconcile these results, we conclude that only a small, set number of cell surface ecoR can be functional for infection and that all excess ecoR can only bind virus into an unsalvageable pool. Therefore, retroviral receptors on single cells are functionally diverse. Our results suggest that activity of ecoR in infection requires a limiting second cellular component.     

Enhancer detection in the zebrafish using pseudotyped murine retroviruses

Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.     

Transplacental transmission of a leukemogenic murine leukemia virus.

Recombinant inbred BXH-2 mice spontaneously produce a B-tropic murine leukemia virus (MuLV) beginning early in life and have a high incidence of spontaneous myeloid leukemia. These traits are not characteristic of the progenitor strains (C57BL/6J and C3H/HeJ) or of 11 other recombinant inbred BXH strains. Genetic analysis has shown that the virus is not transmitted through the germ line, suggesting that the virus is passed from one generation to the next by horizontal transmission. An additional ecotropic proviral locus was detected in some mice of this strain after several generations of inbreeding. We show that BXH ecotropic virus was transmitted to other strains when fostered on viremic BXH-2 mice and that these mice go on to develop tumors of hematopoietic origin. Our earlier finding that virus is expressed early in gestation suggested that the ecotropic MuLV is also transmitted in utero. In order to determine the stage at which the ecotropic MuLV is transmitted in utero, preimplantation stage embryos were transferred to the uteri of recipient ecotropic virus-negative mice. These mice were found to be negative for the presence of the ecotropic MuLV, suggesting that transplacental transmission of the ecotropic virus readily occurs in BXH-2 mice. Although other viruses, including human lentiviruses, are transmitted across the placental barrier, transplacental transmission of MuLV is a rare event. Thus, the BXH-2 mouse strain may contribute to our understanding of the mechanism of transplacental transmission and pathogenesis and offers a potential new model for use in drug therapy of exogenously transmitted viruses related to lentiviruses.     

Transfer of Fv-1 locus-specific resistance to murine N-tropic and B-tropic retroviruses by cytoplasmic RNA.

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.     

Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.

Expression of cell surface CD4 influences susceptibility of cells to human immunodeficiency virus (HIV) infection; however, some CD4-positive human and mouse cells are still resistant to HIV infection. To search for mechanisms of resistance to HIV independent of CD4 expression, HIV expression was studied in human and mouse cells normally resistant to HIV infection by introducing infectious virus by transfection of HIV DNA or infection with HIV pseudotyped with amphotropic or polytropic murine leukemia viruses. The results indicated that even when barriers to viral entry were bypassed, mouse NIH 3T3 cells and Dunni cells still showed a marked reduction in number of cells expressing HIV compared with the human cells studied, although the intensity of immunostaining of individual positive mouse cells was indistinguishable from that seen on permissive human cell lines. CD4 expression in mouse cells or human brain or skin cells did not influence the number of HIV foci observed after transfection with HIV DNA or infection with pseudotyped HIV. These results suggested that in addition to a block in the usual HIV fusion and entry process, CD4-positive mouse cells differed from human cells in exhibiting partial resistance to HIV infection which acted at a postpenetration step in the infection cycle. This resistance was partially overcome when mouse cells were infected by direct exposure to human lymphocytes producing HIV pseudotyped by amphotropic murine leukemia virus.     

Horizontal versus familial transmission of Helicobacter pylori

Transmission of Helicobacter pylori is thought to occur mainly during childhood, and predominantly within families. However, due to the difficulty of obtaining H. pylori isolates from large population samples and to the extensive genetic diversity between isolates, the transmission and spread of H. pylori remain poorly understood. We studied the genetic relationships of H. pylori isolated from 52 individuals of two large families living in a rural community in South Africa and from 43 individuals of 11 families living in urban settings in the United Kingdom, the United States, Korea, and Colombia. A 3,406 bp multilocus sequence haplotype was determined for a total of 142 H. pylori isolates. Isolates were assigned to biogeographic populations, and recent transmission was measured as the occurrence of non-unique isolates, i.e., isolates whose sequences were identical to those of other isolates. Members of urban families were almost always infected with isolates from the biogeographic population that is common in their location. Non-unique isolates were frequent in urban families, consistent with familial transmission between parents and children or between siblings. In contrast, the diversity of H. pylori in the South African families was much more extensive, and four distinct biogeographic populations circulated in this area. Non-unique isolates were less frequent in South African families, and there was no significant correlation between kinship and similarity of H. pylori sequences. However, individuals who lived in the same household did have an increased probability of carrying the same non-unique isolates of H. pylori, independent of kinship. We conclude that patterns of spread of H. pylori under conditions of high prevalence, such as the rural South African families, differ from those in developed countries. Horizontal transmission occurs frequently between persons who do not belong to a core family, blurring the pattern of familial transmission that is typical of developed countries. Predominantly familial transmission in urban societies is likely a result of modern living conditions with good sanitation and where physical contact between persons outside the core family is limited and regulated by societal rules. The patterns observed in rural South African families may be representative of large parts of the developing world.     

Interleukin-7 retroviruses transform pre-B cells by an autocrine mechanism not evident in Abelson murine.

In this study, we have constructed retroviral vectors expressing the interleukin-7 (IL-7) cDNA and have used infection with these retroviruses to express this cytokine endogenously in an IL-7-dependent pre-B-cell line. Infection with IL-7 retroviruses, but not with a control retrovirus, resulted in the conversion of the cells to IL-7 independence. The frequency at which this occurred, together with data on vector expression levels, indicated that secondary events were required for factor independence in this system. Southern analysis showed that the IL-7-dependent clones harbored unrearranged copies of the vector proviruses. The factor-independent cells produced variable quantities of IL-7 as measured by an IL-7-specific bioassay, and their proliferation could be substantially inhibited by a neutralizing antibody directed against IL-7, indicating that a classical autocrine-mechanism was responsible for their transformation. These IL-7-independent cells were tumorigenic, in contrast to the parental IL-7-dependent cells or those infected with a control vector. These results showed that IL-7 could participate in the malignant transformation of pre-B cells. However, neither of two Abelson murine leukemia virus (A-MuLV)-transformed pre-B-cell lines expressed detectable IL-7 mRNA, at a level of sensitivity corresponding to less than one molecule of mRNA per cell. Moreover, the proliferation of the A-MuLV transformants was unaffected by addition of the IL-7 antisera under conditions in which parallel experiments with IL-7 virus-infected cells resulted in greater than 70% growth inhibition. Thus, transformation of pre-B cells by A-MuLV was not associated with a demonstrable autocrine loop of IL-7 synthesis. These results show that IL-7 can participate in the malignant transformation of pre-B cells and suggest studies aimed at assessing the role of autocrine production of IL-7 in the generation of human leukemias and lymphomas.     

Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.

Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses.     

Modulation of ecotropic murine retroviruses by N-linked glycosylation of the cell surface receptor/amino acid transporter.

The cell surface receptor for ecotropic host-range (infection limited to mice or rats) murine leukemia viruses (MuLVs) is the widely expressed system y+ transporter for cationic amino acids (CAT-1). Like other retroviruses, ecotropic MuLV infection eliminates virus-binding sites from cell surfaces and results in complete interference to superinfection. Surprisingly, infection causes only partial (ca 40 to 60%) loss of mouse CAT-1 transporter activity. The NIH/Swiss mouse CAT-1 (mCAT-1) contains 622 amino acids with 14 hydrophobic potential membrane-spanning sequences, and it is known that the third extracellular loop from the amino terminus is required for virus binding. Although loop 3 is hypervariable in different species and mouse strains, consistent with its proposed role in virus-host coevolution, loop 3 sequences of both susceptible and resistant species contain consensus sites for N-linked glycosylation. Both of the consensus sites in loop 3 of mCAT-1 are known to be glycosylated and to contain oligosaccharides with diverse sizes (J. W. Kim and J. M. Cunningham, J. Biol. Chem. 268:16316-16320, 1993). We confirmed by several lines of evidence that N-linked glycosylation occludes a potentially functional virus-binding site in the CAT-1 protein of hamsters, thus contributing to resistance of that species. To study the role of receptor glycosylation in animals susceptible to infection, we eliminated loop 3 glycosylation sites by mutagenesis of an mCAT-1 cDNA clone, and we expressed wild-type and mutant receptors in mink fibroblasts and Xenopus oocytes. These receptors had indistinguishable transport properties, as determined by kinetic and voltage-jump electrophysiological studies of arginine uptake in oocytes and by analyses Of L-[3H]arginine uptake in mink cells. Bindings of ecotropic envelope glycoprotein gp7O to the accessible receptor sites on surfaces of mink cells expressing wild-type or mutant mCAT-1 were not significantly different in kinetics or in equilibrium affinities (i.e., K(D) approximately 3.7 X 10(-10) to 7.5 X 10(-10) M). However, when values were normalized to the same levels of mCAT-1 transporter expression, cells with wild-type glycosylated mCAT-1 had only approximately 50% as many sites for gp70 binding as cells with unglycosylated mCAT-1. Although infection with ecotropic MuLV had no effect on activity of the mink CAT-1 transporter that does not bind virus, it caused partial down-modulation of wild-type mCAT-1 and complete down-modulation of unglycosylated mutant mCAT-1. These results suggest that N-linked glycosylation causes wild-type mCAT-1 heterogeneity and that a significant proportion is inaccessible to virus. In part because only the interactive fraction of mCAT-1 can be down-modulated, infected murine cells conserve an amino acid transport capability that supports their viability.     

Neurologic disease induced by polytropic murine retroviruses: neurovirulence determined by efficiency of spread to microglial cells.

Several murine leukemia viruses (MuLV) induce neurologic disease in susceptible mice. To identify features of central nervous system (CNS) infection that correlate with neurovirulence, we compared two neurovirulent MuLV, Fr98 and Fr98/SE, with a nonneurovirulent MuLV, Fr54. All three viruses utilize the polytropic receptor and are coisogenic, each containing a different envelope gene within a common genetic background. Both Fr98 and Fr98/SE induce a clinical neurologic disease characterized by hyperexcitability and ataxia yet differ in incubation period, 16 to 30 and 30 to 60 days, respectively. Fr54 infects the CNS but fails to induce clinical signs of neurologic disease. In this study, we compared the histopathology, regional virus distribution, and cell tropism in the brain, as well as the relative CNS viral burdens. All three viruses induced similar histopathologic effects, characterized by intense reactive astrogliosis and microglial activation associated with minimal vacuolar degeneration. The infected target cells for each virus consisted primarily of endothelial and microglial cells, with rare oligodendrocytes. Infection localized predominantly in white matter tracts of the cerebellum, internal capsule, and corpus callosum. The only feature that correlated with relative neurovirulence was viral burden as measured by both viral CA protein expression in cerebellar homogenates and quantification of infected cells. Interestingly, Fr54 (nonneurovirulent) and Fr98/SE (slow disease) had similar viral burdens at 3 weeks postinoculation, suggesting that they entered the brain with comparable efficiencies. However, spread of Fr98/SE within the brain thereafter exceeded that of Fr54, reaching levels of viral burden comparable to that seen for Fr98 (rapid disease) at 3 weeks. These results suggest that the determinants of neurovirulence in the envelope gene may influence the efficiency of virus spread within the brain and that a critical number of infected cells may be required for induction of clinical neurologic disease.     

Horizontal transmission of Wolbachia in a Drosophila community

Abstract.  1.  Wolbachia bacteria are reproductive parasites of arthropods and infect an estimated 20% of all insect species worldwide. In order to understand patterns of Wolbachia infection, it is necessary to determine how infections are gained or lost. Wolbachia transmission is mainly vertical, but horizontal transmission between different host species can result in new infections, although its ecological context is poorly understood. Horizontal transmission is often inferred from molecular phylogenies, but could be confounded by recombination between different Wolbachia strains.
2. This study addressed these issues by using three genes: wsp , ftsZ , and groE , to study Wolbachia infections in fruit- and fungus-feeding Drosophila communities in Berkshire, U.K.
3. Identical sequences were found for all three genes in Drosophila ambigua and Drosophila tristis. This suggests horizontal transmission of Wolbachia between these two previously unstudied Drosophila species, which may be the result of the two host species sharing the same food substrates or parasites.
4.  Wolbachia infections might be lost from species due to curing by naturally occurring antibiotics and the presence of these is likely to vary between larval food substrates.
5. It was investigated whether Wolbachia incidence was lower in fungus-feeding than in fruit-feeding Drosophila species, but no significant difference based on food substrate was found.     

Horizontal transmission of begomoviruses between Bemisia tabaci biotypes

We have previously shown that the monopartite Tomato yellow leaf curl virus (TYLCV), a begomovirus (family Geminiviridae, genus Begomovirus) infecting tomato plants can be transmitted in a gender-dependent manner among its insect vector the whitefly Bemisia tabaci type B (Gennaduis) (Aleyrodidae: Hemiptera) during mating. Viruliferous females were able to transmit the virus to non-viruliferous males and vice versa, in the absence of any other virus source. The recipient insects were able to infect tomato plants. In this communication, we present evidence that two bipartite begomoviruses infecting cucurbits, Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV) can be transmitted in a gender-dependent manner among whiteflies. In addition we show that TYLCV can be transmitted during mating among individuals from the same biotype (from B-males to B-females and vice versa; and from Q-males to Q-females and vice versa). However, viruliferous males of the B biotype are unable to transmit the virus to females of the Q biotype (and vice versa); similarly, viruliferous males of the Q biotype are unable to transmit the virus to females of the B biotype (and vice versa). These findings support the hypothesis that a pre-zygotic mating barrier between the Q and B biotypes is the cause for the absence of gene flow between the two biotypes, and that virus transmission can be used as a marker for inter-biotype mating. To be transmitted during mating, the virus needs to be present in the haemolymph of the donor insect. Abutilon mosaic virus (AbMV), a bipartite begomovirus that can be ingested but not transmitted by B. tabaci, is absent in the whitefly haemolymph, and cannot be transmitted during mating. Mating was a precondition for horizontal virus transfer from male to female, or female to male. Virus was not transmitted when viruliferous B. tabaci were caged with the non-vector non-viruliferous whitefly Trialeurodes vaporariorum (Westwood) (Aleyrodidae: Hemiptera) and vice versa.     

The transmission of translational seat vibration to the head--II. Horizontal seat vibration

The second part of this study of the six axes of head motion caused by translational seat vibration is concerned with the effect of fore-and-aft (x-axis) and lateral (y-axis) seat vibration. Seat-to-head transmissibilities have been determined at frequencies up to 16 Hz for each of the three translational and three rotational axes of the head during exposure to random vibration of the seat. Repeatability measures within a single subject and studies of the variability across a group of twelve subjects have been conducted with two seating conditions: a rigid seat with a backrest, and the same seat with no backrest. Fore-and-aft seat motion mainly resulted in head motion within the mid-sagittal plane (x-z plane). Without the backrest, transmissibilities for the fore-and-aft, vertical and pitch axes of the head were greatest at about 2 Hz. The backrest greatly increased head vibration at frequencies above 4 Hz and caused a second peak in the transmissibility curves at about 6 to 8 Hz. Lateral seat motion mainly caused lateral head motion with a maximum transmissibility at about 2 Hz. The backrest had little effect on the transmission of lateral vibration to the head. For both axes of excitation inter-subject variability was much greater than intra-subject variability.     

Infectious entry of murine retroviruses into mouse cells: evidence of a postadsorption step inhibited by acidic pH.

The entry into cells by many enveloped RNA viruses is accomplished by endocytosis and subsequent penetration of the endosomal membrane by an acidic pH-dependent fusion event. In the current study, we examined early events in the infectious entry of mouse retroviruses, using as a framework the observation that infection of a mouse tail skin cell line by the ecotropic virus Friend murine leukemia virus was inhibited at mildly acidic pH (pH 6). This inhibition operated on a postadsorption step, since binding of virus was unaffected at this pH. The rate of penetration of preadsorbed virus, which displayed first-order kinetics, was markedly affected by changes in the pH of the medium. The half-time for disappearance of infectious cell surface virus at 37 degrees C was approximately 10 min at pH 7.6. At pH 6.0, however, greater than 98% of the adsorbed infectivity remained at the cell surface after 45 min. This cell surface virus, though not infecting the cell at pH 6.0, retained its capacity to enter and infect the cell when the pH of the medium was raised. Acidic pH had little effect on the rate of fluid uptake by the cells, as measured by internalization of [3H]sucrose, indicating that global inhibition of endocytosis had not occurred. In contrast, cell fusion induced by Friend murine leukemia virus was optimal at pH 7.6 but markedly inhibited at a pH of less than 6.4. This inhibitory effect of acidic pH on membrane fusion is unique among the enveloped viruses which have been studied and would preclude entry of Friend murine leukemia virus from within acidified endocytic vesicles. Entry of other members of the ecotropic, mink cell focus-forming, and xenotropic host range groups displayed similar pH sensitivity. However, one xenotropic virus was relatively resistant to the effect of acidic pH, suggesting that differences might exist in the requirements for entry of different retroviruses.