Time-resolved fluorescence investigation of the human immunodeficiency virus type 1 nucleocapsid protein: influence of the binding of nucleic acids.
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Depending on the HIV-1 isolate, MN or BH10, the nucleocapsid protein, NCp7, corresponds to a 55- or 71-amino acid length product, respectively. The MN NCp7 contains a single Trp residue at position 37 in the distal zinc finger motif, and the BH10 NCp7 contains an additional Trp, at position 61 in the C-terminal chain. The time-resolved intensity decay parameters of the zinc-saturated BH10 NCp7 were determined and compared to those of single-Trp-containing derivatives. The fluorescence decay of BH10 NCp7 could be clearly represented as a linear combination (with respect to both lifetimes and fractional intensities) of the individual emitting Trp residues. This suggested the absence of interactions between the two Trp residues, a feature that was confirmed by molecular modeling and fluorescence energy transfer studies. In the presence of tRNAPhe, taken as a RNA model, the same conclusions hold true despite the large fluorescence decrease induced by the binding of tRNAPhe. Indeed, the fluorescence of Trp37 appears almost fully quenched, in keeping with a stacking of this residue with the bases of tRNAPhe. Despite the multiple binding sites in tRNAPhe, the large prevalence of ultrashort lifetimes, associated with the stacking of Trp37, suggests that this stacking constitutes a major feature in the binding process of NCp7 to nucleic acids. In contrast, Trp61 only stacked to a small extent with tRNAPhe. The behavior of this residue in the tRNAPhe-NCp7 complexes appeared to be rather heterogeneous, suggesting that it does not constitute a major determinant in the binding process. Finally, our data suggested that the binding of NCp7 proteins from the two HIV-1 strains to nonspecific nucleic acid sequences was largely similar. (+info)
Tagging the human immunodeficiency virus gag protein with green fluorescent protein. Minimal evidence for colocalisation with actin.
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The assembly and budding of human immunodeficiency virus type 1, encoded solely in the Gag protein precursor Pr55Gag, occur at the plasma membrane of infected cells. However, little is known about the routing of the Gag molecule from its site of synthesis in the cytoplasm to the site of budding, with past studies suggesting that the cytoskeleton, particularly actin, may be involved in the translocation. We have constructed a T7 promoter-driven gag gene fusion with green fluorescent protein (GFP) that expresses Gag-GFP in both cells and supernatant. The distribution of Gag-GFP was the same as Gag only, suggesting that cellular routing was not affected by fusion to GFP, and using colabelling techniques, Gag-GFP was shown to have no particular colocalisation with actin. After detergent extraction of expressing cells, Gag and Gag-GFP remained cell associated, whereas GFP only was wholly released. These data suggest that Gag may associate with other cytoskeletal components or, perhaps more likely, that a partial assembly to a large-molecular-weight intermediate occurs before localisation at the plasma membrane. (+info)
Two types of HTLV-1 particles are released from MT-2 cells.
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The MT-2 cell line transformed by human T-cell leukemia virus type 1 (HTLV-1) contains one complete provirus and seven defective proviruses. Four defective genomes have an identical structure (LTR-MA-deltaCA-pX-LTR) with an open reading frame that spans from MA to pX, giving rise to a 3.4-kb (24S) RNA transcript encoding a chimeric Gag-pX protein, p28. MT-2 cells release two distinct types of virions. The major "classic" type of particle has a buoyant density of 1.155-1.16 g/cm3 and contains the standard HTLV-I structural proteins and reverse transcriptase (RT). In addition, about 5% of particles are "light," approximately 1.12 g/cm3, and contain p28, RT activity, and the 3.4-kb RNA transcript. RT-PCR and in vitro translation indicate that some of the classic HTLV-1 particles package 3.4-kb RNA as well as full-length 8.5-kb RNA. In addition to matrix features, the p28 protein has a motif resembling a zinc finger at the C-terminal, pX0 region, which may play a role in the assembly of the defective light virions. (+info)
Opposing effects of human immunodeficiency virus type 1 matrix mutations support a myristyl switch model of gag membrane targeting.
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Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55(gag) to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55(gag) with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane. (+info)
Vif and the p55(Gag) polyprotein of human immunodeficiency virus type 1 are present in colocalizing membrane-free cytoplasmic complexes.
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The Vif protein of human immunodeficiency virus type 1 (HIV-1) is a potent regulator of viral infectivity. Current data posit that Vif functions late in replication to modulate assembly, budding, and/or maturation. Consistent with this model, earlier indirect immunofluorescence analyses of HIV-1-infected cells demonstrated that Vif and Gag colocalize to a substantial degree (J. H. M. Simon, R. A. M. Fouchier, T. E. Southerling, C. B. Guerra, C. K. Grant, and M. H. Malim, J. Virol. 71:5259-5267, 1997). Here, we describe a series of subcellular fractionation studies which indicate that Vif and the p55(Gag) polyprotein are present in membrane-free cytoplasmic complexes that copurify in sucrose density gradients and are stable in nonionic detergents. Both Vif and Gag are targeted to these complexes independent of each other, and their association with them appears to be mediated by protein-protein interactions. We propose that these complexes may represent viral assembly intermediates and that Vif is appropriately localized to influence the final stages of the viral life cycle and, therefore, the infectivity of progeny virions. (+info)
Detection and induction of equine infectious anemia virus-specific cytotoxic T-lymphocyte responses by use of recombinant retroviral vectors.
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Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses. (+info)
Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin.
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The nucleocapsid (NC) domain of the retrovirus Gag protein plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of human immunodeficiency virus type 1 (HIV-1) Gag was found to drastically reduce virus particle production in CD4(+) T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type HIV-1 Gag, unmyristylated Gag, and p6(Gag) domain-truncated Gag molecules copurified with the host cell cytoskeleton, most of the mutant Gag molecules lacking both the NC and p6(Gag) domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against actin coimmunoprecipitated full-length Gag and p6(Gag) domain-truncated Gag molecules from cell lysates but failed to precipitate the truncated mutant Gag molecules lacking NC plus p6(Gag). Purified recombinant NCp7, but not CAp24, was able to bind F-actin in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular actin-binding protein (the villin headpiece), bound F-actin in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind F-actin directly and that interaction between HIV-1 Gag and the actin cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle. (+info)
Human immunodeficiency virus types 1 and 2 differ in the predominant mechanism used for selection of genomic RNA for encapsidation.
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Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors. (+info)