Growth characteristics of T-cell tropic HIV-1 vpu gene mutants in human peripheral blood mononuclear cells

Growth characteristics of T-cell tropic HIV-1 vpu gene mutants in human peripheral blood mononuclear cells

Shinya Iida, Tomoharu Fukumori, Yoko Oshima, A. Hajime Koyama, and Akio Adachi

Department of Virology, The University of Tokushima School of Medicine, Tokushima, Japan

Abstract:A mutant designated NL-E65, which lacks the expression of entire vpu gene, was constructed from T-cell tropic wild-type (wt) human immunodeficiency virus type1(HIV-1) clone and monitored for its replication property in human cells, along with a mutant NL-Ss which expresses a C-terminal truncated Vpu. The mutant NL-Ss could grow in two cell lines and in all peripheral blood mononuclear cell (PBMC) preparations to some extent, with kinetics similar to those of wt virus. Likewise, the mutant NL-E65exhibited a replication property typical to the vpu mutant in the two cell lines and in all PBMC cultures, growing at a low level. Along with the results previously reported, these data indicate that HIV-1 Vpu is dispensable for virus replication in any of the types of cells so far tested. J. Med. Invest. 46:43-47, 1999

Keywords:HIV-1, Vpu, accessory gene

INTRODUCTION
Human immunodeficiency virus type1 (HIV-1)carries several auxiliary genes not found in the other simple retroviruses (1). Of the six auxiliary genes, four accessory genes designated nef, vif, vpr, and vpu are dispensable for viral replication at least in certain types of cells (2-5). However, the products of these accessory genes certainly modulate virus replication in tissue culture systems, and more importantly, virus replication and pathogenesis in vivo. Of the four accessory proteins, Vpu is always non-essential for virus replication in any kinds of cells tested (6-11), although it has been demonstrated to enhance virion release from cells of various types (8,12). Even in natural target cells such as periph-eral blood mononuclear cells (PBMCs), vpu mutant viruses grow quite well (9, 11). It is possible, however, that the results described above may not be general, and may be dependent on the vpu mutant clones (10) and cells used in the experiments. It has been reported that vpu mutants grow very poorly in macrophage cultures (7, 9).
In this study, a vpu mutant, which lacks the expres-sion of the entire vpu gene, was newly constructed and monitored for its replication in various types of cells in comparison with another mutant expressing a truncated form of Vpu (13) and a wild-type (wt) clone. We show here that this new vpu mutant does replicate in two lymphocytic cell lines and in all PBMC cultures of six individuals, thus demonstrat-ing the dispensability of the HIV-1 Vpu for virus replication in lymphocytic cells.

MATERIALS AND METHODS
Cells, transfection, and infection
A human cervical carcinoma cell line, HeLa (ATCC CCL2), was maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum. CD4-positive human lymphocytic cell lines A3.01 (14) and CEMx174 (15) were main-tained in RPMI1640 medium supplemented with 10% heat-inactivated fetal calf serum. Human PBMCs were prepared and cultured as previously described (16). For transfection, uncleaved plasmid DNA was introduced into HeLa cells by the calcium-phosphate coprecipitation method (17). Cells were infected with cell-free virus samples prepared from transfections as previously described (14, 18).

RT assay
Virion-associated reverse transcriptase (RT) activity was measured as described previously (19).

Western blotting
Western immunoblotting was performed as previously described (19). MULTI GEL system (Daiichi Pure Chemicals) was used for polyacrylamide gel electrophoresis. Specific viral Vpu protein was detected by the ECL system (Amersham) using an antibody, a generous gift from Dr. Klaus Strebel, raised against amino acids 33-81 of the HIV-1 Vpu (C-terminus) (20). A serum of an individual infected with HIV-1 was also used for detection of viral structural proteins.

DNA constructs
Molecular clones of HIV-1 designated pNL432and pNL-Ss have been previously described (8, 13, 17, 21). A vpu mutant clone pNL-E65, which lacks the initiation codon (AGG instead of ATG), was constructed by the LA PCR in vitro Mutagenesis Kit (Takara). The structure of this mutant was confirmed by dideoxy sequencing.

RESULTS
Western blot analysis of vpu mutants
Our previous genetic studies (8, 11, 13, 21) involved the use of a mutant, NL-Ss, which carries a linker insertion resulting in a translational frameshift after the first 32 amino acids of Vpu like a mutant desig-nated Vpu35 (10, 22) and therefore has the potential to express the entire N-terminal transmembrane (TM) domain (22). We constructed a second HIV-1 vpu mutant , NL-E65, which lacks the initiation codon and is therefore unable to express any Vpu-specific sequence. The parental clone of the two vpu mutants was T-cell tropic pNL432 (17). We first confirmed the inability of our new vpu mutant NL-E65 to express the Vpu protein. HeLa cells were transfected with various clones, and cell lysates were made at 2days post-transfection. The expression of Vpu was moni-tored by Western blotting analysis. As shown in Fig.1A, the two vpu mutants were able to produce viral structural proteins normally upon transfection. In contrast, as shown in Fig.1B, the mutants did not generate Vpu detectable by the anti-Vpu antiserum, as was expected.

Growth property of vpu mutant viruses in established cell lines
When inoculated into CD4-positive cell lines, HIV-1 vpu mutant viruses display unique and char-acteristic growth curves (6, 8, 12, 22). Namely, the growth kinetics of the vpu mutant virus are similar to those of wt virus but the expression of virus pro-duction in culture medium is much lower than that of wt virus.
We initially examined the growth property of a new HIV-1vpu mutant virus derived from transfection of plasmid pNL-E65 in two CD4-positive cell lines. HeLa cells were transfected with various clones, and cell-free virus samples were prepared at 2days post-transfection. As shown in Fig.2, in A3.01 and CEMx174 cells, both vpu mutants, NL-Ss (8, 13) and NL-E65, showed the growth charac-teristics typical to the vpu mutant.

Growth property vpu mutant viruses in PBMCs
To determine whether Vpu is critical for the replication of HIV-1 in natural target cells, we monitored the growth potential of the NL-E65 vpu mutant virus in stimulated and unstimulated PBMCs. Our previous study using a vpu mutant of macrophage tropic clone has shown that Vpu is quite important for virus replication in macrophages (7). However, only a mild effect of vpu muta-tion on virus replication was observed in PBMCs, (7, 11).
Cell-free virus samples were prepared as described above, and inoculated into PBMCs prepared from six healthy indi-viduals. We were especially interested in infection of unstimulated PBMCs with NL-E65. It has been recently reported that the accessory protein Nef is very important for virus replication in the unstimulated cells (18). As shown in Fig.3, in stimulated PBMCs, both vpu mutants grew considerably well relative to wt virus in the all six independent preparations as reported previously (11). This was the case in most unstimulated PBMC preparations, as shown in Fig.4. Although the peak level of virus produc-tion varied, the two vpu mutants grew relatively well. In one unstimulated PBMC preparation, the two vpu mutants, espe-cially NL-E65, grew very poorly relative to wt virus (Fig.4B).

DISCUSSION
The major conclusion of this study is that the HIV-1 Vpu is dispensable for virus replication in any types of CD4-positive cells. Although a critical requirement of Vpu for HIV-1growth in macrophages has been reported previously (7, 9), HIV-1 lacking the entire vpu gene could replicate to some extent in all the cell types tested so far. Whether the Vpu is essential for HIV-1 replication in cells other than those examined to date needs to be determined. It is also possible that Vpu plays an impor-tant functional role for in vivo replication and pathogenesis like Nef and Vpr (2-5).
It has been reported that the HIV-1Vpu has two structural domains which are important for two distinct biological activities (10). The TM and cytoplasmic regions represent active domains for the regulation of virus release from cells and the CD4 degradation in cells, respectively. The results of our experiments described here suggest that the TM region of the Vpu does not contribute much to the growth property of the vpu mutant. Further genetic study is required to determine the functional domains of HIV-1 Vpu.

ACKNOWLEDGMENTS
This work was supported in part by a grant-in-aid for AIDS research from the Ministry of Education, Science and Culture of Japan. We thank Dr. Klaus Strebel for the anti-HIV-1 Vpu antiserum.

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Received for publication October 19, 1998;accepted October 28, 1998.

Address correspondence and reprint requests to Akio Adachi, Ph.D., Department of Virology, The University Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7080.