Abstract
BackgroundHost proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. Although the identification efficiency of host proteins is improved by mass spectrometry, how those host proteins affect HIV-1 replication has not yet been fully clarified.ResultsIn this study, we show that virion-associated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) does not allosterically inactivate HIV-1 reverse transcriptase (RT) but decreases the efficiency of reverse transcription reactions by decreasing the packaging efficiency of lysyl-tRNA synthetase (LysRS) and tRNALys3 into HIV-1 virions. Two-dimensional (2D) gel electrophoresis demonstrated that some isozymes of GAPDH with different isoelectric points were expressed in HIV-1-producing CEM/LAV-1 cells, and a proportion of GAPDH was selectively incorporated into the virions. Suppression of GAPDH expression by RNA interference in CEM/LAV-1 cells resulted in decreased GAPDH packaging inside the virions, and the GAPDH-packaging-defective virus maintained at least control levels of viral production but increased the infectivity. Quantitative analysis of reverse transcription products indicated that the levels of early cDNA products of the GAPDH-packaging-defective virus were higher than those of the control virus owing to the higher packaging efficiencies of LysRS and tRNALys3 into the virions rather than the GAPDH-dependent negative allosteric modulation for RT. Furthermore, immunoprecipitation assay using an anti-GAPDH antibody showed that GAPDH directly interacted with Pr55gag and p160gag-pol and the overexpression of LysRS in HIV-1-producing cells resulted in a decrease in the efficiency of GAPDH packaging in HIV particles. In contrast, the viruses produced from cells expressing a high level of GAPDH showed decreased infectivity in TZM-bl cells and reverse transcription efficiency in TZM-bl cells and peripheral blood mononuclear cells (PBMCs).ConclusionsThese findings indicate that GAPDH negatively regulates HIV-1 infection and provide insights into a novel function of GAPDH in the HIV-1 life cycle and a new host defense mechanism against HIV-1 infection.
Highlights
Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection
Multiple glyceraldehyde 3-phosphate dehydrogenase (GAPDH) isozymes were incorporated into virions To examine the host proteins found in virions produced by an HIV-infected cell line, CEM/LAV-1 cells, a highly purified HIV-1LAV-1 preparation was analyzed by 2D gel electrophoresis, followed by MALDI-TOF Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MS)/MS (Figure 1A)
To confirm whether GAPDH is incorporated into virions, contaminating particles such as microvesicles and exosomes were removed according to previously described methods [19]
Summary
Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. GAPDH phosphorylates the hepatitis B virus core protein [15] and binds with the cis-acting RNAs of several viruses, such as the 50 UTR of the hepatitis A virus [16], the 30 UTR sequences of the human parainfluenza virus [17], and the hepatitis C virus [18]. These findings indicated that GAPDH might play a role in regulating
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