Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction.
(33/1905)
The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed. (+info)
Analysis of receptor usage by ecotropic murine retroviruses, using green fluorescent protein-tagged cationic amino acid transporters.
(34/1905)
Entry of ecotropic murine leukemia virus (MuLV) into host cells is initiated by interaction between the receptor-binding domain of the viral SU protein and the third extracellular domain (TED) of the receptor, cationic amino acid transporter 1 (CAT1). To study the molecular basis for the retrovirus-receptor interaction, mouse CAT1 (mCAT1) was expressed in human 293 cells as a fusion protein with jellyfish green fluorescent protein (GFP). Easily detected by fluorescence microscopy and immunoblot analysis with anti-GFP antibodies, the mCAT1-GFP fusion protein was expressed in an N-glycosylated form on the cell surface and in the Golgi apparatus, retaining the ecotropic receptor function. The system was applied to compare Friend MuLV (F-MuLV) and its neuropathogenic variant, PVC-211 MuLV, which exhibits a unique cellular tropism and host range, for the ability to use various CAT family members as a receptor. The results indicated that F-MuLV and PVC-211 MuLV could infect the cells expressing wild-type mCAT1 at comparable efficiencies and that rat CAT3, but not mCAT2, conferred a low but detectable level of susceptibility to F-MuLV and PVC-211 MuLV. The data also suggested that CAT proteins might be expressed in an oligomeric form. Further application of the system developed in this study may provide useful insights into the entry mechanism of ecotropic MuLV. (+info)
Efficacy of antiretroviral agents against murine replication-competent retrovirus infection in human cells.
(35/1905)
Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone. (+info)
Development of murine leukemia virus-based self-activating vectors that efficiently delete the selectable drug resistance gene during reverse transcription.
(36/1905)
Expression of the selectable drug resistance gene in retroviral vectors used for gene therapy can lead to a decreased expression of the gene of interest and may induce a host immune response, resulting in a decreased efficiency of gene therapy. In this study, we demonstrate that high-frequency deletion of direct repeats, an inherent property of reverse transcriptases, can be used to efficiently excise the drug resistance gene during reverse transcription. One retroviral vector containing a direct repeat deleted the neomycin resistance expression cassette during a single replication cycle at >99% efficiency. (+info)
Effect of substrate residues on the P2' preference of retroviral proteinases.
(37/1905)
The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts. (+info)
High levels of IL-4 and IL-10 mRNA and low levels of IL-2, IL-9 and IFN-gamma mRNA in MuLV-induced lymphomas.
(38/1905)
The expression of cytokines may influence the development of lymphoma in retrovirally infected animals in at least two ways: (1) cytokines in the tumor environment may stimulate the proliferation of tumor cells and/or (2) cytokines in the tumor environment may diminish the cell-mediated antitumor immune response. To evaluate these possibilities, a semiquantitative RT-PCR approach was utilized to permit a broad screening of cytokine mRNAs in a large number of tissue samples. Examination of MuLV-induced end-stage lymphomas revealed the absence of mRNA for cytokines known to stimulate the proliferation of T cells (i.e., IL-2, IL-9), the absence of mRNA for cytokines known to enhance cell-mediated antitumor immune responses (i.e., IL-2, IFNgamma), and the presence of mRNA for cytokines known to diminish such responses (i.e., IL-4, IL-10). Similar patterns of cytokine mRNA expression were detected in tumor-derived cell lines. Spleen and thymus from animals collected longitudinally during infection and from age-matched uninfected mice also demonstrated a similar pattern, except that IFNgamma mRNA was readily detectable. These findings do not support the hypothesis that the developing tumor depends on cytokines to provide proliferative signals. The findings suggest that cytokines in the immediate environment of the lymphoma support tumor development by acting to diminish an effective antitumor immune response. (+info)
Use of recombinant viruses to assess the pattern of early human immunodeficiency virus breakthrough infection in the presence of stavudine.
(39/1905)
A variety of cell lines were infected with replication-defective recombinant retroviruses in the presence of stavudine (d4T). Cells which were infected despite the presence of d4T were isolated and subjected to infection with other retroviruses [replication-competent human immunodeficiency virus (HIV), replication-defective HIV or replication-defective recombinant murine retroviruses]. Each of the host cell types tested had a small subset of cells that were infected with HIV or murine retroviruses in the presence of d4T. Some of these infected cells could be infected repeatedly at high efficiency in the presence of d4T. This phenotype of 'persistent refractoriness' to the antiviral effects of d4T could be overcome by the addition of 5-fluoro-2-deoxyuridine (floxuridine) to d4T. The d4T-floxuridine combination also had potent antiretroviral effects in primary blood mononuclear cells. (+info)
The nucleocapsid domain is responsible for the ability of spleen necrosis virus (SNV) Gag polyprotein to package both SNV and murine leukemia virus RNA.
(40/1905)
Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements. (+info)