Association of Nef with the human immunodeficiency virus type 1 core.
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Highly conserved among primate lentiviruses, the human immunodeficiency virus type 1 (HIV-1) Nef protein enhances viral infectivity by an unknown mechanism. Nef-defective virions are blocked at a stage of the HIV-1 life cycle between entry and reverse transcription, possibly virus uncoating. Nef is present in purified HIV-1 particles; however, it has not been determined whether Nef is specifically recruited into HIV-1 particles or whether virion-associated Nef plays a functional role in HIV-1 replication. To address the specificity and potential functionality of virion-associated Nef, we determined the subviral localization of Nef. HIV-1 cores were isolated by detergent treatment of concentrated virions followed by equilibrium density gradient sedimentation. Relative to HIV-1 virions, HIV-1 cores contained equivalent amounts of reverse transcriptase and integrase, decreased amounts of the viral matrix protein, and trace quantities of the viral transmembrane glycoprotein gp41. Examination of the particles by electron microscopy revealed cone-shaped structures characteristic of lentiviral cores. Similar quantities of proteolytically processed Nef protein were detected in gradient fractions of HIV-1 cores and intact virions. In addition, detergent-resistant subviral complexes isolated from immature HIV-1 particles contained similar quantities of Nef as untreated virions. These results demonstrate that Nef stably associates with the HIV-1 core and suggest that virion-associated Nef plays a functional role in accelerating HIV-1 replication. (+info)
The induction of virus-specific CTL as a function of increasing epitope expression: responses rise steadily until excessively high levels of epitope are attained.
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The role of epitope expression levels in CD8+ T cell priming has been controversial. Yet this parameter is of great importance in the design of rational approaches to optimize CTL responses to a variety of pathogens. In this paper we examine the influence of epitope production on CD8+ T cell priming by exploiting a system that allows a 200-fold range of cell surface epitope expression in vitro with a fixed dose of vaccinia virus. Our results demonstrate that, with the exception of a notable decline at the highest level of epitope, the magnitude of the responding CTL population generated in vivo following equivalent viral infections is essentially proportional to epitope density. (+info)
Structural features of a chimeric peptide inducing cytotoxic T lymphocyte responses in saline.
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Little information is available correlating the structural properties of peptides with their immunogenicity in terms of responses via cytotoxic T lymphocytes (CTLs). The TT-NP6 chimeric peptide, consisting of two copies of a promiscuous T-helper epitope (T: residues 288-302 from the fusion protein of the measles virus) linked to the NP6 T-cytotoxic epitope (NP6: residues 52-60 from the nucleoprotein of measles virus) was able to induce virus-specific CTL responses in the absence of any adjuvant and hydrophobic component. The present work was undertaken to gain insight into structural features of the TT-NP6 peptide that may be important in optimizing the CTL immunogenicity of the peptide. Circular dichroism data, obtained in a buffer of physiological ionic strength and pH, strongly suggest a self-associated state for the peptide, which was confirmed by a sedimentation velocity experiment. However, helix association is accompanied by loss of overall helical content. Thermal-dependence studies show that the unfolding of self-associated alpha-helices is significantly more pronounced than the unfolding of isolated alpha-helices. Circular dichroism data, together with tryptic limited proteolysis, suggest the presence of a charged amino acid within the hydrophobic core. This study should provide a basis for engineering more effective immunogenic peptides against the measles virus by increasing the stability of the TT-NP6 peptide. (+info)
TH cells primed during influenza virus infection provide help for qualitatively distinct antibody responses to subsequent immunization.
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The quality of the primary Ab-forming cell (AFC) response in cervical lymph nodes and mediastinal lymph nodes of mice to intranasal influenza virus was strongly influenced by viral replicative capacity. IgA secretors were prominent in the early AFC response to infectious virus in mediastinal lymph nodes, while IgG expression was more frequent among isotypically switched AFC in cervical lymph nodes of the same mice; this pattern was reversed in the response to inactivated virus. Influenza viruses A/Puerto Rico/8/34 (A/PR8) and A/X-31 share six of eight genome segments, differing only in hemagglutinin (H1 in A/PR8, H3 in A/X-31) and neuraminidase (N1 in A/PR8, N2 in A/X-31) genes. These viruses therefore elicit extensively cross-reactive TH populations, though their glycoproteins are serologically unrelated. Mice recovered from an A/X-31 infection thus mount a primary B cell response against A/PR8 glycoproteins, when challenged with the latter virus, though this response can call upon memory TH cells. To assess the impact of memory TH populations on a primary Ab response, we compared the AFC response to inactivated A/PR8 in naive mice and mice that had cleared an A/X-31 infection. A/X-31 immune mice mounted a more vigorous AFC response against A/PR8 H1 and N1 glycoproteins than naive animals, when immunized intranasally with inactivated A/PR8. However the distribution of isotypes among H1/N1-specific AFC in lymph nodes of A/X-31-primed mice resembled that of naive mice. Evidently, in this functional context, memory TH cells retained the ability to help Ab responses different in quality from that generated during their primary reaction. (+info)
Activation of p53 tumor suppressor by hepatitis C virus core protein.
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In addition to being a structural protein that packages the viral genomic RNA, hepatitis C virus (HCV) core protein possesses regulatory functions. In this report, we demonstrate that the HCV core protein could enhance the gene transactivation activity of the tumor suppressor p53, regardless of whether p53 was derived from an exogenous or an endogenous gene. The activation of p53 by the HCV core protein was supported by the observation that the HCV core protein could enhance the expression of p21(waf1/Cip1), a downstream effector gene of p53, in a p53-dependent manner. Further studies indicated that the HCV core protein could also suppress hepatocellular growth via p53. The HCV core protein and p53 could bind to each other in vitro, which was evidenced by the coimmunoprecipitation, the GST pull-down, and the Far-Western blot assays. The deletion-mapping analysis indicated that the carboxy-terminal sequence of p53 located between amino acids 366 and 380 was required for the core protein binding. These results raised the possibility that the HCV core protein might activate p53 through direct physical interaction. The persistent perturbation of p53 activity by the HCV core protein during chronic infection may have important consequences in HCV pathogenesis. (+info)
Selective expansion of cross-reactive CD8(+) memory T cells by viral variants.
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The role of memory T cells during the immune response against random antigenic variants has not been resolved. Here, we show by simultaneous staining with two tetrameric major histocompatibility complex (MHC)-peptide molecules, that the polyclonal CD8(+) T cell response against a series of natural variants of the influenza A nucleoprotein epitope is completely dominated by infrequent cross-reactive T cells that expand from an original memory population. Based on both biochemical and functional criteria, these cross-reactive cytotoxic T cells productively recognize both the parental and the mutant epitope in vitro and in vivo. These results provide direct evidence that the repertoire of antigen-specific T cells used during an infection critically depends on prior antigen encounters, and indicate that polyclonal memory T cell populations can provide protection against a range of antigenic variants. (+info)
Characterization of the Hantaan nucleocapsid protein-ribonucleic acid interaction.
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The nucleocapsid (N) protein functions in hantavirus replication through its interactions with the viral genomic and antigenomic RNAs. To address the biological functions of the N protein, it was critical to first define this binding interaction. The dissociation constant, K(d), for the interaction of the Hantaan virus (HTNV) N protein and its genomic S segment (vRNA) was measured under several solution conditions. Overall, increasing the NaCl and Mg(2+) in these binding reactions had little impact on the K(d). However, the HTNV N protein showed an enhanced specificity for HTNV vRNA as compared with the S segment open reading frame RNA or a nonviral RNA with increasing ionic strength and the presence of Mg(2+). In contrast, the assembly of Sin Nombre virus N protein-HTNV vRNA complexes was inhibited by the presence of Mg(2+) or an increase in the ionic strength. The K(d) values for HTNV and Sin Nombre virus N proteins were nearly identical for the S segment open reading frame RNA, showing weak affinity over several binding reaction conditions. Our data suggest a model in which specific recognition of the HTNV vRNA by the HTNV N protein resides in the noncoding regions of the HTNV vRNA. (+info)
Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation.
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To clarify the binding properties of hepatitis C virus (HCV) core protein and its viral RNA for the encapsidation, morphogenesis, and replication of HCV, the specific interaction of HCV core protein with its genomic RNA synthesized in vitro was examined in an in vivo system. The positive-sense RNA from the 5' end to nucleotide (nt) 2327, which covers the 5' untranslated region (5'UTR) and a part of the coding region of HCV structural proteins, interacted with HCV core protein, while no interaction was observed in the same region of negative-sense RNA and in other regions of viral and antiviral sense RNAs. The internal ribosome entry site (IRES) exists around the 5'UTR of HCV; therefore, the interaction of the core protein with this region of HCV RNA suggests that there is some effect on its cap-independent translation. Cells expressing HCV core protein were transfected with reporter RNAs consisting of nt 1 to 709 of HCV RNA (the 5'UTR of HCV and about two-thirds of the core protein coding regions) followed by a firefly luciferase gene (HCV07Luc RNA). The translation of HCV07Luc RNA was suppressed in cells expressing the core protein, whereas no significant suppression was observed in the case of a reporter RNA possessing the IRES of encephalomyocarditis virus followed by a firefly luciferase. This suppression by the core protein occurred in a dose-dependent manner. The expression of the E1 envelope protein of HCV or beta-galactosidase did not suppress the translation of both HCV and EMCV reporter RNAs. We then examined the regions that are important for suppression of translation by the core protein and found that the region from nt 1 to 344 was enough to exert this suppression. These results suggest that the HCV core protein interacts with viral genomic RNA at a specific region to form nucleocapsids and regulates the expression of HCV by interacting with the 5'UTR. (+info)