Patterns of Integration and Expression of Retroviral, Non-Replicative Vectors in Avian Embryos: Embryo Developmental Stage and Virus Subgroup Envelope Modulate Tissue-Tropism

We previously demonstrated that Avian Leukemia Viruses (ALV) carrying the v-myc gene specifically induce two types of tumors, cardiomyocytic tumors when the virus is injected before embryonic day 3 (E3), skin tumors when the virus is injected at E3 or E5.

Aiming to elucidate the mechanisms which determine this time-dependent change in target, we infected chick and quail embryos at E3 and E5 with replication-deficient, lacZ gene-carrying, ALV-based viruses produced by a packaging cell line. Three constructs driven by 3 different Long Terminal Repeats (LTRs) were tested and yielded similar results. When the constructs were inoculated at E3 and the lacZ gene product revealed 5 days later, around 70% of the embryos carried lacZ+ clones in the heart, around 50% had positive clones in the skin anywhere on the body, while a few embryos displayed clones in internal organs (liver, stomach, lungs). Immunocytological identification of the heart cell type(s) expressing the virus revealed that the only cells infected were cardiomyocytes. When the constructs were inoculated at E5, no lacZ+ clones appeared in the heart but all were located in the cephalic skin. In order to examine the relationship between viral integration and expression, DNA of different organs or tissues from lacZ stained embryos was analyzed by PCR. A tight correlation between integration and expression in the heart and in the skin was revealed in most cases. In contrast, a significant PCR signal was often detected in the liver or the stomach despite weak or absent expression as revealed by lacZ+ clones.

We then investigated the influence of envelope glycoprotein subgroups on the tropism of these constructs. The lacZ vector driven by RAV-2 LTRs was packaged as subgroups A, B or E viral particles. The A subgroup, used in the part of the study described above, infects both chick and quail while the B and E subgroups are specific for chick or quail respectively. These B and E subgroups induced lacZ+ clones in the heart (after E3 injection) while no clones or only a few were detected in the skin either after E3 or E5 injection. The following conclusions can be drawn: 1) cardiomyocytes are at E3 the major target for integration and expression of ALV-derived viruses in vivo; 2) targets change rapidly with embryonic age; and 3) tissue-specific infections depend on the envelope subgroup, thus presumably on the presence of the cognate receptor. This study clearly indicates that E3 inoculation of ALV-based retroviral vectors is a simple and powerful method to transfer gene sequences into cardiomyocytes and epidermal cells.     

Alteration of surfactant protein A expression in renal cell carcinoma

Surfactant protein-A (SP-A) belongs to a family of collagen-containing C-type lectins called collectins. SP-A is expressed by renal tubule epithelial cells. We investigated the distribution of SP-A in renal cell carcinomas (RCC) using immunohistochemical techniques and western blotting. We used 35 formalin fixed, paraffin embedded (FFPE) RCC tissue samples. We compared results with clinico-pathological parameters of RCC including age, sex, Fuhrman grade, tumor volume, tumor node metastasis (TNM) and clinical stage. SP-A was localized in the glomerulus and renal tubule epithelium in nontumor tissue and strong SP-A immunoreactivity was observed in tumor tissue. SP-A was expressed in the RCC tumor cells (64%) and nontumor cells (34%) in males and RCC tumor cells (90%) and nontumor cells (30%) in females. There was a significant correlation between SP-A immunoreactivity in tumor cells and gender, age, tumor diameter, Fuhrman grade and tumor diameter. Western blot analysis supported the immunohistochemical findings. We present evidence for involvement of SP-A in RCC and suggest that increased SP-A expression in RCC is associated with favorable prognosis.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.     

The T-stem determines the cytosolic or mitochondrial localization of trypanosomal tRNAsMet

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Organellar translation therefore depends on import of cytosolic, nucleus-encoded tRNAs. Except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The initiator tRNA(Met) is closely related to the imported elongator tRNA(Met). Thus, the distinct localization of the two tRNAs(Met) must be specified by the 26 nucleotides, which differ between the two molecules. Using transgenic T. brucei cell lines and subsequent cell fractionation, we show that the T-stem is both required and sufficient to specify the localization of the tRNAs(Met). Furthermore, it was shown that the tRNA(Met) T-stem localization determinants are also functional in the context of two other tRNAs. In vivo analysis of the modified nucleotides found in the initiator tRNA(Met) indicates that the T-stem localization determinants do not require modified nucleotides. In contrast, import of native tRNAs(Met) into isolated mitochondria suggests that nucleotide modifications might be involved in regulating the extent of import of elongator tRNA(Met).     

Relationship between SU subdomains that regulate the receptor-mediated transition from the native (fusion-inhibited) to the fusion-active conformation of the murine leukemia virus glycoprotein

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain.     

An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor

A new human endogenous retrovirus (HERV) family, termed HERV-W, was recently described (J.-L. Blond, F. Besème, L. Duret, O. Bouton, F. Bedin, H. Perron, B. Mandrand, and F. Mallet, J. Virol. 73:1175-1185, 1999). HERV-W mRNAs were found to be specifically expressed in placenta cells, and an env cDNA containing a complete open reading frame was recovered. In cell-cell fusion assays, we demonstrate here that the product of the HERV-W env gene is a highly fusogenic membrane glycoprotein. Transfection of an HERV-W Env expression vector in a panel of cell lines derived from different species resulted in formation of syncytia in primate and pig cells upon interaction with the type D mammalian retrovirus receptor. Moreover, envelope glycoproteins encoded by HERV-W were specifically detected in placenta cells, suggesting that they may play a physiological role during pregnancy and placenta formation.