Concentration of retroviruses by low-speed centrifugation

Concentration of retroviruses from volumes of up to 6 liters of medium by low-speed centrifugation is described. In contrast to pelleting, no damage or aggregation of particles was observed. Surface glycoproteins were also fully preserved. This method enables simple handling of relatively large volumes of medium. Highly purified mouse mammary tumor virus (MMTV) was obtained and its transframe protein p30 in SDS-PAGE was recognized as a double band.     

Quantal acetylcholine release induced by mediatophore transfection.

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.     

Mechanisms for Env glycoprotein acquisition by retroviruses

A mandatory step in the formation of an infectious retroviral particle is the acquisition of its envelope glycoprotein (Env). This step invariably occurs by Env positioning itself in the host membrane at the location of viral budding and being incorporated along with the host membrane into the viral particle. In some ways, this step of the viral life cycle would appear to be imprecise. There is no specific sequence in Env or in the retroviral structural protein, Gag, that is inherently required for the production of an infectious Env-containing particle. Additionally, Env-defective proviruses can efficiently produce infectious particles with any of a number of foreign retroviral Env glycoproteins or even glycoproteins from unrelated viral families, a process termed pseudotyping. However, mounting evidence suggests that Env incorporation is neither passive nor random. Rather, several redundant mechanisms appear to contribute to the carefully controlled process of Env acquisition, many of which are apparently used by a wide variety of enveloped viruses. This review presents and discusses the evidence for these different mechanisms contributing to incorporation.     

Induction of the early stages of Friend erythroleukemia with helper-free Friend spleen focus-forming virus.

The polycythemia-inducing strain of Friend virus (FV-P) causes a multistage erythroleukemia in susceptible mice. FV-P is a complex of two viruses, a replication-competent virus [Friend murine leukemia virus (F-MuLV)] and a replication-defective spleen focus-forming virus (SFFVp). We have addressed directly the role of SFFVp in the induction of the early stages of Friend disease by constructing stocks of SFFVp free of detectable F-MuLV, using a recently described retroviral helper-cell line. These preparations are capable of inducing erythroid bursts (vBFU-E) whose inducibility, kinetics, and responsiveness to erythropoietin suggest that they are very similar, if not identical, to the vBFU-E induced by FV-P. Single injections of helper-free SFFVp had no apparent effects in vivo, although the addition of exogenous helper virus to the inoculum resulted in the induction of classic Friend disease. Increasing the effective titer by giving mice five daily virus injections resulted in the induction of splenomegaly and a large increase in the number of erythroid colony-forming units that were independent of erythropoietin. When the injections were discontinued, the spleens regressed and all the mice survived. When the injections were continued, all the mice died within 25 days of the first injection. These results demonstrate that SFFVp alone can alter the growth characteristics of erythroid progenitors and is directly responsible for the induction of vBFU-E in vitro and the erythroid hyperplasia in vivo. They also demonstrate that the initial polyclonal stage of Friend disease is reversible and can be reproduced by using preparations of SFFVp free of detectable F-MuLV.     

Therapeutic choices for patients with hemophilia and high-titer inhibitors.

Effective treatment of bleeding episodes in hemophilia with high titer inhibitors (HTI) remains a challenge, despite the fact that the therapeutic armamentarium has expanded considerably over the past few years. Treatment safety has improved with the availability of porcine factor VIII (FVIII) and bypassing products such as recombinant factor VIIa (rFVIIa), and plasma‐derived activated Prothrombin Complex Concentrates (aPCCs) that are virally inactivated. The major drawbacks of rFVIIa and aPCCs are their unpredictable hemostatic effect, lack of laboratory assays to monitor efficacy and dosing frequency, and the risk of thrombosis. The proceedings of a one‐day workshop of physicians who specialized in treating patients with hemophilia held in Vienna on May 13, 2000 have been summarized. In making a decision regarding the choice of product, physicians often consider the type of bleeding episode (life or limb threatening), age of the patient, volume of the reconstituted product, previous exposure to plasma derived products, cost, efficacy, and safety. For plasma naïve patients, to achieve rapid hemostasis a majority of the panelists used porcine FVIII (for patients who lack porcine inhibitory antibodies) or rFVIIa. For patients previously treated with plasma derived factors, in addition to the above concentrates, aPCCs were recommended. Although no data exists regarding safety and efficacy, switching products was routinely practiced either because of availability or cost. Furthermore, the panelists were uncertain about the efficacy of bypassing agents in the prevention of joint disease in inhibitor patients. The workshop participants felt that future research offers the best solution to resolve some of the dilemmas faced by clinicians and may help individualise treatment in a hemophilia patient with a high titer inhibitor. Am. J. Hematol. 67:240–246, 2001. © 2001 Wiley‐Liss, Inc.     

Sperm-associated retroviruses in the mouse epididymis.

Sperm adsorbed with retrovirus particles were recovered from the epididymis of apparently normal male mice. Epididymal semen from all four mouse strains examined was positive for retrovirus (10(5) to 10(8) particles per microgram of protein) indicating that epididymal fluids and sperm may be important vehicles for murine retrovirus spread. Immunoblot analyses revealed that the banding patterns of electrophoretically separated epididymal viral proteins from the four strains of males were more similar to each other than to either xenotropic New Zealand Black virus or ecotropic Rauscher leukemia virus proteins. The results indicate that retrovirus particles, possibly a unique strain, are commonly expressed at relatively high titers in the reproductive tract of male mice and are sperm-associated.     

Long-term reinfection of the human genome by endogenous retroviruses

Endogenous retrovirus (ERV) families are derived from their exogenous counterparts by means of a process of germ-line infection and proliferation within the host genome. Several families in the human and mouse genomes now consist of many hundreds of elements and, although several candidates have been proposed, the mechanism behind this proliferation has remained uncertain. To investigate this mechanism, we reconstructed the ratio of nonsynonymous to synonymous changes and the acquisition of stop codons during the evolution of the human ERV family HERV-K(HML2). We show that all genes, including the env gene, which is necessary only for movement between cells, have been under continuous purifying selection. This finding strongly suggests that the proliferation of this family has been almost entirely due to germ-line reinfection, rather than retrotransposition in cis or complementation in trans, and that an infectious pool of endogenous retroviruses has persisted within the primate lineage throughout the past 30 million years. Because many elements within this pool would have been unfixed, it is possible that the HERV-K(HML2) family still contains infectious elements at present, despite their apparent absence in the human genome sequence. Analysis of the env gene of eight other HERV families indicated that reinfection is likely to be the most common mechanism by which endogenous retroviruses proliferate in their hosts.     

Cationic liposome-mediated RNA transfection.

We have developed an efficient and reproducible method for RNA transfection, using a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), incorporated into a liposome (lipofectin). Transfection of 10 ng to 5 micrograms of Photinus pyralis luciferase mRNA synthesized in vitro into NIH 3T3 mouse cells yields a linear response of luciferase activity. The procedure can be used to efficiently transfect RNA into human, rat, mouse, Xenopus, and Drosophila cells. Using the RNA/lipofectin transfection procedure, we have analyzed the role of capping and beta-globin 5' and 3' untranslated sequences on the translation efficiency of luciferase RNA synthesized in vitro. Following transfection of NIH 3T3 cells, capped mRNAs with beta-globin untranslated sequences produced at least 1000-fold more luciferase protein than mRNAs lacking these elements.     

Immunosuppressive actions of retroviruses

The immunosuppressive properties of retroviruses were first demonstrated by Old et al. We later showed that Gross Passage A retrovirus superinfection in mice resulted in decreased antibody production and diminished allograft rejection. We have studied in some detail the immunosuppression which occurs subsequent to infection with feline leukemia virus (FeLV) as characterized by profoundly depressed T and B lymphocyte responses and decreased production of gamma-interferon. Injection of staphylococcal protein A (SPA) corrected these deficient immune responses, cleared circulating FeLV from blood and produced a regression of FeLV-induced lymphomas and leukemias. The immunosuppressive properties of FeLV and certain other retroviruses have been linked to the transmembrane viral envelope peptide, p15E. Cianciolo et al synthesized a 17-amino acid viral component which shares sequence homology with a highly conserved region of p15E. In vitro analyses have shown that this synthetic retroviral peptide suppresses T and B cell functions, inhibits the generation of cytotoxic lymphocyte (CTL) responses and dramatically alters the morphology and distribution of monocytes. The latter finding, along with reports that cells of the monocyte/macrophage lineage play a critical role in the initiation of human immunodeficiency infection, suggests that monocytes and macrophages may play a crucial role in retroviral infection and some of the associated immunodeficiencies associated with retroviral infection.     

Highly efficient induction of type C retroviruses by a human tumor in athymic mice.

We have found that 1 of 20 human tumors transplanted and passaged in nude mice was associated with a massive induction of endogenous murine leukemia virus (MuLV). Separation and growth of these viruses on various substrates indicated that both ecotropic and xenotropic MuLV were present in the induced mixture. Tryptic peptide fingerprints of the p30 and gp70 structural elements of the viruses indicated that all of the known endogenous muLVs of BALB/c mice were present in the mixture. In addition, a new xenotropic MuLV was identified. The human tumor that induced the viruses was an oat cell carcinoma. The oat cell carcinoma possibly produced a specific hormone or factor that acts as a potent inducer of endogenous type C retroviruses.     

Immunosuppression by retroviruses in tumors and immune deficiency diseases

Most retroviruses are immunosuppressive in vitro and in vivo. They are able to enhance virus-induced tumor development and/or to induce acquired immune deficiency syndromes (AIDS) which are characterized by malignant tumors and opportunistic infections. Experimental evidence for the immunosuppressive properties of several type D viruses derived from human cell lines and other retroviruses is presented.     

Mechanisms of cell killing/cytopathic effects by nonhuman retroviruses

Retroviruses have been studied primarily for their role in cancer. However, with the AIDS epidemic, attention has shifted to the other effects of retroviral infection. In particular, since human immunodeficiency virus results in killing of CD4-positive T cells, there is interest in the mechanism of cell killing by retroviruses. Numerous other retroviruses are also cytopathic. Studies of these other retroviruses are reviewed in this article. In particular, the hypothesis that cell killing is the result of superinfection or other increased viral envelope protein-cell receptor interaction is suggested.     

Relocation of a protease-like gene segment between two retroviruses.

An anomalous sequence in certain lentiviruses was found to be related to a region in a completely different part of the simian retrovirus type I (SRV-I) and its close relative, the hamster intracisternal A particle (IAP-H18). The segment is not present in the human immunodeficiency virus (HIV), which is also a lentivirus, nor is it found in any one of a dozen other retroviruses whose sequences have been reported. These observations imply that a horizontal transfer of newly acquired genetic information has taken place between an SRV-I-type virus and one of the lentivirus type, and that this event occurred more recently than did the divergence of members of this latter group and HIV. Comparison of the viral nucleic acid sequences that encode these segments revealed the presence of imperfect direct nucleotide repeats resembling the retroviral endonuclease cleavage sites at the 5' and 3' ends of these regions.     

Production of dysplastic platelets by peripheral blood megakaryoblasts in transient myeloproliferative disorder in Down syndrome

Monitoring the course of platelet function in HELLP (haemolysis, elevated liver-enzymes and low platelets) syndrome is important for clinical decision-making. We present a primigravid woman developing HELLP syndrome at 29 weeks and 6 days. Platelet function was monitored by multiple electrode aggregometry (MEA), platelet function analyzer (PFA-100®), platelet count and mean platelet volume (MPV) over an 11-day period. MPV and PFA-100® seem better predictors for platelet function than platelet levels.