Oral AttenuatedSalmonella EntericaSerovarTyphimuriumVaccine Expressing Codon-Optimized HIV Type 1GagEnhanced Intestinal Immunity in Mice

Tsunetsugu-Yokota, Yasuko; Ishige, Masayuki; Murakami, Masahiro

doi:10.1089/aid.2006.0098

Cited by 21 publications

(15 citation statements)

References 30 publications

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“…Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. The membrane was incubated with mouse anti-HIV-1 p17MA (Advanced Biotechnologies), mouse antip24CA (40), rabbit anti-p7NC (41), sheep anti-p6 (Aalto, Ireland), rabbit anti-PR (NIH AIDS Research and Reference Reagent Program), mouse anti-RT (ICN Pharmaceuticals), mouse anti-FLAG (Sigma), and rabbit anti-HA (Sigma) antibodies and subsequently with horseradish peroxidase-conjugated secondary antibodies (ICN Pharmaceuticals). Anti-caveolin antibody was purchased from Abcam.…”

Section: Methodsmentioning

confidence: 99%

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

Sudo

Haraguchi

Hirai

et al. 2013

J Virol

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d Efavirenz (EFV), a nonnucleoside reverse transcriptase (RT) inhibitor, also inhibits HIV-1 particle release through enhanced Gag/Gag-Pol processing by protease (PR). To better understand the mechanisms of the EFV-mediated enhancement of Gag processing, we examined the intracellular localization of Gag/Gag-Pol processing products and their precursors. Confocal microscopy revealed that in the presence of EFV, the N-terminal p17 matrix (p17MA) fragment was uniformly distributed at the plasma membrane (PM) but the central p24 capsid (p24CA) and the Pol-encoded RT antigens were diffusely distributed in the cytoplasm, and all of the above were observed in puncta at the PM in the absence of EFV. EFV did not impair PM targeting of Gag/ Gag-Pol precursors. Membrane flotation analysis confirmed these findings. Such uniform distribution of p17MA at the PM was not seen by overexpression of Gag-Pol and was suppressed when EFV-resistant HIV-1 was used. Forster's fluorescence resonance energy transfer assay revealed that Gag-Pol precursor dimerization occurred mainly at the PM and that EFV induced a significant increase of the Gag-Pol dimerization at the PM. Gag-Pol dimerization was not enhanced when HIV-1 contained the EFV resistance mutation in RT. Bacterial two-hybrid assay showed that EFV enhanced the dimerization of PR-RT fragments and restored the dimerization impaired by the dimerization-defective mutation in the RT tryptophan repeat motif but not that impaired by the mutation at the PR dimer interface. Collectively, our data indicate that EFV enhances Gag-Pol precursor dimerization, likely after PM targeting but before complete particle assembly, resulting in uniform distribution of p17MA to and dissociation of p24CA and RT from the PM.

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Section: Methodsmentioning

confidence: 99%

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

Sudo

Haraguchi

Hirai

et al. 2013

J Virol

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“…The membrane was incubated with anti-HIV-1 p24 mouse antibody [48] and subsequently with horseradish peroxidase-conjugated secondary antibodies (Cappel).…”

Section: Methodsmentioning

confidence: 99%

A Large Extension to HIV-1 Gag, Like Pol, Has Negative Impacts on Virion Assembly

et al. 2012

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The GagPol protein of HIV-1 harbors viral enzymes, such as protease (PR), reverse transcriptase, and integrase, that are all crucial for virion infectivity. Previous studies have suggested that expression of GagPol alone does not produce viral particles and that the budding defect is caused by the presence of the Pol region. However, it has remained unknown why GagPol fails to produce viral particles. We show here that HIV-1 GagPol is incapable of membrane binding and subsequent particle assembly. Our confocal data indicated that, despite full N-myristoylation, GagPol protein failed to target plasma membrane with diffuse distribution in the cytoplasm. Membrane flotation analysis confirmed these findings. Progressive C-terminal truncation of GagPol to give GagPR allowed for plasma membrane targeting but still not for particle production. Conversely, the C-terminal addition of a noncognate protein, such as ß-galactosidase or 4 tandem GFP, to Gag impaired the membrane affinity, indicating that the Pol region, a large extension to Gag, inhibits membrane binding in the context of GagPol. The addition of the 10 N-terminal amino acids of Fyn kinase [Fyn(10)], a tight membrane-binding signal, conferred plasma membrane targeting on GagPol, but the Fyn(10)GagPol did not produce viral particles. The defect in particle budding was not rescued by the introduction of the PTAP motif, which is responsible for a late stage of viral particle budding. Rather, electron microscopy suggested that the budding defect of GagPR occurred at an early stage of particle morphogenesis. Our data, which were consistent with previous observations, demonstrate the defects of GagPol in membrane binding and particle assembly.

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“…Oral vaccination of mice with the recombinant Salmonella vector induced MV-specific serum antibodies and CD4 + T cell response [94]. In another study, it was also shown that an attenuated Salmonella vaccine expressing codon optimized HIV-1 Gag was efficient in inducing Gag-specific mucosal IgA and CD8 + T cell responses in intestinal lymphoid tissues of orally vaccinated mice [95]. Therefore, in developing recombinant Salmonella vaccines for HIV-1, it is critical to optimize the viral genes for expression by the vector as this improves the nature, quality, and magnitude of the immune responses elicited.…”

Section: Salmonella Vaccine Vectors Expressing Hiv-1 Gagmentioning

confidence: 99%

Recombinant Salmonella enterica Serovar Typhimurium as a Vaccine Vector for HIV-1 Gag

Chin'ombe

2013

Viruses

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The HIV/AIDS epidemic remains a global health problem, especially in Sub-Saharan Africa. An effective HIV-1 vaccine is therefore badly required to mitigate this ever-expanding problem. Since HIV-1 infects its host through the mucosal surface, a vaccine for the virus needs to trigger mucosal as well as systemic immune responses. Oral, attenuated recombinant Salmonella vaccines offer this potential of delivering HIV-1 antigens to both the mucosal and systemic compartments of the immune system. So far, a number of pre-clinical studies have been performed, in which HIV-1 Gag, a highly conserved viral antigen possessing both T- and B-cell epitopes, was successfully delivered by recombinant Salmonella vaccines and, in most cases, induced HIV-specific immune responses. In this review, the potential use of Salmonella enterica serovar Typhimurium as a live vaccine vector for HIV-1 Gag is explored.

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Oral AttenuatedSalmonella EntericaSerovarTyphimuriumVaccine Expressing Codon-Optimized HIV Type 1GagEnhanced Intestinal Immunity in Mice

Cited by 21 publications

References 30 publications

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

Efavirenz Enhances HIV-1 Gag Processing at the Plasma Membrane through Gag-Pol Dimerization

A Large Extension to HIV-1 Gag, Like Pol, Has Negative Impacts on Virion Assembly

Recombinant Salmonella enterica Serovar Typhimurium as a Vaccine Vector for HIV-1 Gag

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