Biomedical subjects

Hsu-Chen Chiu

Publications and source records attributed to Hsu-Chen Chiu.

6 recordsLinked to original sources

PubMed2006

Effects of human immunodeficiency virus type 1 transframe protein p6* mutations on viral protease-mediated Gag processing.

The proteolytic processing of human immunodeficiency virus (HIV) particles mediated by the viral pol-encoded protease (PR) is essential for viral infectivity. The pol coding sequence partially overlaps with the gag coding sequence and is translated as a Gag-Pol polyprotein precursor. Within Gag-Pol, the C-terminal p6(gag) domain is replaced by a transframe peptide referred to as p6*, which separates the Gag nucleocapsid domain from PR. Several previous in vitro studies have ascribed a PR-suppression regulatory function to p6*. Here, it was demonstrated that an HIV-1 Gag-Pol lacking p6* is efficiently incorporated into virions when coexpressed with HIV-1 Gag precursor. However, the released virions are not processed appropriately and show a greatly reduced viral infectivity. This suggests that the p6* is indispensable during the process of PR-mediated virus particle maturation.

Amino Acid Sequence↗

PubMed2004

The virus-associated human immunodeficiency virus type 1 Gag-Pol carrying an active protease domain in the matrix region is severely defective both in autoprocessing and in trans processing of gag particles.

We have previously demonstrated that a human immunodeficiency virus (HIV) chimeric Gag protein containing a partial replacement of the matrix domain by the viral protease domain (PR) could undergo autoprocessing with no virus particle production [J. Virol. 74 (2000) 3418]. To further analyze the effects of repositioned PR on virus particle production and Gag-Pol incorporation, we introduced the chimeric PR construct into a PR-negative Gag-Pol expression plasmid and coexpressed the resultant construct with a Pr55(gag) expression plasmid (pGAG) in 293T cells. Analysis indicated that the chimeric PR was similar to native PR in that both could prevent virus particle production in cotransfections with an equivalent amount of pGAG plasmid DNA, suggesting an efficient trans processing of Pr55(gag) by the chimeric PR. In cotransfections with the pGAG at a DNA ratio of 1:10 to 1:20, which resembles the normal intracellular expression ratio of Gag-Pol to Gag, Gag-Pol carrying the PR in the Gag coding region could undergo autoprocessing in cells and was incorporated into virus particles at a level about 20-40% of that of wild-type Gag-Pol. However, the incorporated chimeric Gag-Pol was unable to autocleave and unable to process the Gag particles properly, as mature particle-associated reverse transcriptase (RT) and p24(gag) proteins were barely detected. Our data strongly suggest that positioning an active HIV PR in the matrix region significantly affects the PR-mediated virus particle maturation.

Catalytic Domain↗

PubMed2004

Effects of mutations in an HIV-1 gag gene containing a 107-codon tandem repeat in the matrix region on assembly and processing of the protein product.

It has been demonstrated previously that a human immunodeficiency virus (HIV) type 1 Gag mutant (MA2) with a tandem repeat of 107-matrix codons in the matrix domain could direct virus particle assembly and budding [Wang et al. (2000c): J Med Virol 61:423-432]. Since the regions involved functionally in HIV Gag assembly and transport have been mapped to the matrix domain, it was interesting to test the effects of the duplicated matrix-coding sequence on Gag assembly, transport, and virus processing of some assembly-defective HIV matrix mutants. In this study, a number of HIV matrix mutations were introduced into either the proximal or distal copy of the duplicated matrix-coding sequence. Assembly, release, processing, and subcellular localization of the Gag mutants were analyzed by transient expression in 293T cells. The result indicates that the budding defect of HIV matrix mutants could be moderately or significantly reversed when the additional 107-matrix codons were present; however, these matrix double mutations affected significantly the virus particle processing. Mislocalized matrix mutants could also be redistributed to a certain degree in the presence of the duplicated matrix copy. Although the subcellular distribution patterns of the matrix mutants did not correlate completely with the budding efficiency, the data suggest that the budding defect caused by the matrix mutations could be masked to some extent by the duplicated matrix coding sequence.

Cell Culture Techniques↗

PubMed2002

Effects of gag mutations on human immunodeficiency virus type 1 particle assembly, processing, and cyclophilin A incorporation.

A series of human immunodeficiency virus (HIV) mutants was constructed either by deletion or by linker insertion at various regions in the gag coding sequences. The ability of each mutant to assemble virus particles and to process them proteolytically, as well as incorporate cyclophilin A, was analyzed by Western immunoblot. This investigation indicated that most of the gag mutants were assembled and released at a level comparable to that of wild-type virus. In an assay involving a single cycle of infection, mutants containing significant levels of cyclophilin A showed less in trans interference effects on wild-type infectivity than did cyclophilin A-deficient mutants. Mutations in the N-terminal two-thirds of capsid protein severely disrupted cyclophilin A incorporation, but they affected virus processing only slightly to moderately. Virions released from cyclosporine-treated cells were processed, as well as virions made by the mock-treated cells. Also, protease inhibitor treatment had no detectable effect on the cyclophilin A incorporation. These results indicate that cyclophilin A incorporation is not required for virus particle processing and that virus processing does not affect cyclophilin A incorporation.

Amino Acid Sequence↗

PubMed2002

Coding sequences upstream of the human immunodeficiency virus type 1 reverse transcriptase domain in Gag-Pol are not essential for incorporation of the Pr160(gag-pol) into virus particles.

Incorporation of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virions is thought to be mediated by the N-terminal Gag domain via interaction with the Gag precursor. However, one recent study has demonstrated that the murine leukemia virus Pol can be incorporated into virions independently of Gag-Pol expression, implying a possible interaction between the Pol and Gag precursor. To test whether the HIV-1 Pol can be incorporated into virions on removal of the N-terminal Gag domain and to define sequences required for the incorporation of Gag-Pol into virions in more detail, a series of HIV Gag-Pol expression plasmids with various extensive deletions in the region upstream of the reverse transcriptase (RT) domain was constructed, and viral incorporation of the Gag-Pol deletion mutants was examined by cotransfecting 293T cells with a plasmid expressing Pr55(gag). Analysis indicated that deletion of the N-terminal two-thirds of the gag coding region did not significantly affect the incorporation of Gag-Pol into virions. In contrast, Gag-Pol proteins with deletions covering the capsid (CA) major homology regions and the adjacent C-terminal CA regions were impaired with respect to assembly into virions. However, Gag-Pol with sequences deleted upstream of the protease, or of the RT domain but retaining 15 N-terminal gag codons, could still be rescued into virions at a level about 20% of the wild-type level. When assayed in a nonmyristylated Gag-Pol context, all of the Gag-Pol deletion mutants were incorporated into virions at a level comparable to their myristylated counterparts, suggesting that the incorporation of the Gag-Pol deletion mutants into virions is independent of the N-terminal myristylation signal.

Capsid↗

PubMedDate not supplied

The human immunodeficiency virus type 1 carboxyl-terminal third of capsid sequence in Gag-Pol is essential but not sufficient for efficient incorporation of Pr160(gag-pol) into virus particles.

To elucidate the role of the C-terminal portion of Gag in the incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-Pol into virus particles, a series of HIV-1 Gag-Pol mutants with deletions in the C-terminal gag sequence was constructed and viral incorporation of the Gag-Pol deletion mutants was analyzed using co-transfecting 293T cells with a Pr55(gag) expression plasmid. The biological function of the incorporated HIV-1 pol gene product was tested using an infectivity assay of the released virus particles which were pseudotyped with the murine leukemia virus Env. Analysis indicated that Gag-Pol deletion mutants, with a removal of the matrix (MA) and/or nucleocapsid (NC) or of the N-terminal two thirds of the gag coding sequence, could be incorporated efficiently into virus particles and produce significant amounts of infectious virions when assayed in a single-cycle infection assay. In contrast, mutations involving a deletion of the major homology region and the adjacent C-terminal capsid sequence significantly affected Gag-Pol incorporation. However, incorporation into virus particles of a Gag-Pol deletion mutant retaining both the major homology region and the adjacent C-terminal capsid intact was still severely impaired. This suggests that the capsid major homology region and the adjacent C-terminal capsid sequence in Gag-Pol are necessary but not sufficient for the incorporation of HIV-1 Pr160(gag-pol) into virus particles.

Base Sequence↗