JOURNAL OF VIROLOGY, May 2000, p. 4891–4893 0022-538X/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 10
Cell-Dependent Requirement of Human Immunodeficiency Virus Type 1 gp41 Cytoplasmic Tail for Env Incorporation into Virions HIROFUMI AKARI, TOMOHARU FUKUMORI,
AND
AKIO ADACHI*
Department of Virology, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan Received 8 November 1999/Accepted 16 February 2000
Growth kinetics in lymphocytic H9 and M8166 cells of two mutants of human immunodeficiency virus type 1 (HIV-1) with deleted gp41 cytoplasmic tails were examined. While the mutant viruses designated CTdel-44 and CTdel-144 were able to grow in M8166 cells, they were unable to grow in H9 cells. Transfection and single-round infectivity assays demonstrated that they are defective in the early phase of viral replication in H9 cells. Analysis of the mutant virions revealed drastically reduced incorporation of Env gp120 (compared with the incorporation of wild-type virions) in H9 cells but normal incorporation in M8166 cells. These results indicate that the HIV-1 cytoplasmic tail of gp41 determines virus infectivity in a cell-dependent manner by affecting incorporation of Env into virions and suggest the involvement of a host cell factor(s) in the Env incorporation.
CT mutants CTdel-44 (previously referred as pNL-Hp) (2) and CTdel-144 (a generous gift of Eric Freed, National Institutes of Health [NIH]) (7), an env-negative mutant pNL-Kp (2), and a vif-negative mutant pNL-Nd (2, 16). CTdel-44 and CTdel144 lack 44 and 144 C-terminal amino acids of Env gp41, respectively. The vif mutant virus NL-Nd, which displays a celldependent growth phenotype (16), was used as a control. Lymphocytic H9 and M8166 cells are nonpermissive and permissive for the NL-Nd virus, respectively. H9 and M8166 cells were infected with each virus and were monitored for virus production in the culture supernatants by 32P-based reverse transcriptase (RT) assay (19). As shown in Fig. 1, H9 cells were not productively infected by NL-Nd, CTdel-44, or CTdel-144 virus. In contrast, in M8166 cells, these viruses grew to various extents. While wt and NL-Nd viruses grew in a similar manner, CTdel-44 and CTdel-144 viruses showed delayed growth kinetics. CTdel-144 grew especially poorly. These results indicated that HIV-1 gp41 CT is essential for virus replication in H9 cells but not in M8166 cells. To determine which step of the viral replication cycle in H9 cells is affected by the truncated gp41, pNL432, and pNL-Nd, CT mutants were directly transfected into H9 cells by electroporation (3). Levels of virus production in the culture supernatants at 24 h postelectroporation were determined by Gag p24 enzyme-linked immunosorbent assay (ELISA) (Cellular Products, Buffalo, N.Y.). No major difference in the p24 level was observed (Table 1), and it appeared that the late replication phase of the CT mutants in H9 cells as monitored by virus production (Gag p24) is normal. The single-round infectivity of the viruses produced in H9 and M8166 cells was then examined by multinuclear activation of galactosidase inhibitor (MAGI) assay (14). As shown in Fig. 2, CTdel-44, CTdel-144, and NL-Nd viruses produced in H9 cells demonstrated reduced infectivity, while those obtained from M8166 cells exhibited a level of infectivity comparable with that of wt virus. From these results, it was concluded that the two CT mutant viruses produced in H9 cells are defective in the early stage of viral replication cycle and suggested that the virions of the CT mutants released from H9 cells are abnormal. To examine virion proteins of the CT mutants, viruses were harvested from electroporated H9 and M8166 cells and were
The Env glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of surface subunit gp120 and transmembrane subunit gp41, which are proteolytic products of the gp160 precursor encoded by the env gene. In HIV-1 infection, the gp120-gp41 complex interacts with CD4 and a batch of chemokine receptors, and a fusion peptide and an N-terminal ectodomain of gp41 dissociated from gp120 promote fusion between viral and target cell membranes (6). The gp41 comprises two other functional domains, i.e., the transmembrane subunit and cytoplasmic domains. The gp41 cytoplasmic tail (CT) regions of HIV-1 and the other primate immunodeficiency viruses are unusually long compared with those of the other retroviruses (11). The CT regions of HIV-1 and HIV-2 are generally around 150 amino acid residues in length; in contrast, those of avian and murine leukemia viruses are typically 20 to 30 residues long. The CT region of HIV-1 contains a number of functional domains: a tyrosine-based motif in the N-terminus as a signal for the sorting and trafficking of Env (4); two amphipathic motifs, which form an alphahelix and bind calmodulin (15, 17) and which are responsible for induction of apoptosis in cells (12); and a C-terminal region important for viral infectivity and Env incorporation into virions (5, 7, 8, 10, 15, 18, 20). During our mutational analysis of the HIV-1 genome, we have noticed that some mutants, including those with altered CTs, exhibit cell-dependent growth properties (2, 13, 16; our unpublished results). In this report, we have biologically and biochemically characterized the CT mutants and demonstrated that the gp41 CT is a critical determinant for producing cell-dependent virion infectivity. To obtain input viruses to determine growth potentials of CT mutants in lymphocytic cells, HeLa cells were transfected with proviral DNA clones by the calcium phosphate coprecipitation method, and cell-free solutions of viruses were prepared 48 h posttransfection as previously described (1, 16). Proviral clones used here were wild-type (wt) pNL432 (1), its
* Corresponding author. Mailing address: Department of Virology, The University of Tokushima School of Medicine, 3 Kuramoto, Tokushima 770-8503, Japan. Phone: 81-88-633-7078. Fax: 81-88-6337080. E-mail:
[email protected]. 4891
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FIG. 2. Infectivity of wt and mutant viruses. Single-round infectivity of viruses derived from H9 and M8166 cells electroporated with pNL432 (WT), pNL-Nd (vif mutant [Nd]), pNL-Kp (env mutant [Kp]), CTdel-44 (44), or CTdel144 (144) was monitored by MAGI assay. Infectivity was determined by counting blue foci of X-Gal-treated MAGI cells 2 days after inoculation of viruses. Average and standard deviation of three independent experiments are shown.
FIG. 1. Growth kinetics of wt and mutant viruses in H9 and M8166 cells. Viruses were prepared from HeLa cells transfected with pNL432 (WT), pNL-Nd (vif mutant [Nd]), CTdel-44, or CTdel-144. Cells (106) were infected with 5 ⫻ 105 RT units of viruses. RT production in the culture supernatants was monitored at intervals.
from Cosmobio (Tokyo, Japan). As shown in Fig. 3, the level of gp120 was much lower in CTdel-44 and CTdel-144 virions from H9 cells than in wt and NL-Nd virions, whereas the level was comparable in all M8166-derived virions. Figure 3 also shows no obvious differences in the amounts of Gag and RT or in the levels of their processing among the virions derived from H9 and M8166 cells. Protein profiles of wt and mutant virions prepared from transfected HeLa cells were similar to those of
concentrated by ultracentrifugation through 20% sucrose cushion at 20,000 ⫻ g for 2 h at 4°C (3). The prepared virion lysates were then analyzed by Western blotting assay (3). The lysates containing an equal amount of the viral antigen as judged by p24 ELISA were subjected to electrophoresis through sodium dodecyl sulfate gradient (4 to 20%) polyacrylamide gels. The separated proteins were blotted onto nitrocellulose membranes, were treated with antibodies to HIV-1 proteins, and were visualized by using an ECL system (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). The antisera to Env gp160 and RT were provided by M. Page through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH. The anti-p24/p55 Gag monoclonal antibody was obtained TABLE 1. Virus production in H9 cells transfected with various clonesa Clone
pNL432 pNL-Nd pNL-CTdel-44 pNL-CTdel-144 pNL-Kp Mock
Result of assay no. (ng/ml): 1
2
8.1 7.7 7.6 4.6 5.8 0.0
5.2 3.8 3.1 5.0 3.0 0.0
a Cells were electroporated with 10 g of each proviral clone, and after 24 h, culture supernatants were harvested and quantitated by p24 ELISA. For details of the clones, see the text.
FIG. 3. Analysis of virion proteins by Western blotting. The lysates of virions prepared from H9 (A to C) and M8166 (D to F) cells electroporated with pNL432 (WT), pNL-Nd (vif mutant [Nd]), CTdel-44, CTdel-144, or pUC19 (control) were analyzed by Western blotting with a sheep anti-Env gp160 antiserum (A and D), a mouse anti-p24 monoclonal antibody (B and E), and a sheep anti-RT antiserum (C and F).
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from the Ministry of Health and Welfare of Japan and by a grant-in-aid for biomedical research from the Uehara Memorial Foundation.
ADDENDUM While the manuscript was being reviewed, Murakami and Freed published similar results (15a).
FIG. 4. Analysis of viral proteins expressed in cells by Western blotting. The lysates of H9 (A and B) and M8166 (C and D) cells electroporated with pNL432 (WT), pNL-Nd (vif mutant [Nd]), CTdel-44, CTdel-144, or pUC19 (control) were analyzed by Western blotting with a sheep anti-Env gp160 antiserum (A and C) and a mouse anti-p24 monoclonal antibody (B and D).
virions from transfected M8166 cells (data not shown). To further address this issue, the expression of Env within cells electroporated with various clones was similarly monitored. As shown in Fig. 4, the expression level of Env was comparable among clone-transfected H9 and M8166 cells. In addition, syncytia formation was similarly observed in the H9 cells transfected with the clones. This observation suggested that the precursor of the mutant Env was processed into biologically active mature proteins and that these proteins were expressed on the surface of the cells as previously reported (9). These data indicated that CTdel-44 and CTdel-144 produced in H9 cells but not in M8166 cells are defective for Env incorporation into virions. The results presented here strongly suggest that HIV-1 gp41 CT plays a critical role during virus assembly and entry in the virus replication cycle in a cell-dependent way. The key finding is the producer-cell-dependent role of the gp41 CT in Env incorporation into virions (Fig. 3). In the absence of gp41 CT, HIV-1 virions produced in certain cell types virtually lack Env gp120 and are defective for initiating the next round of the virus replication cycle. In normal virus assembly, functional association of an unknown cell factor(s) with some domain in the C-terminal 44 amino acid residues of gp41 is important. Although H9 and M8166 cells are nonpermissive and permissive, respectively, for both of the vif and CT mutants, we have recently demonstrated that the function of Vif is unrelated to Env (3). Further study is needed to ascertain the molecular basis for the cell-dependent functional role of HIV-1 gp41 CT, and the identification of the cell factor(s) responsible for Env incorporation is critically required. We thank Eric O. Freed for providing a plasmid clone. The antisera to Env gp160 and RT (from M. Page) were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. We also thank Kazuko Yoshida for editorial assistance. This work was supported by grants-in-aid for AIDS research from the Ministry of Education, Science, Sports, and Culture of Japan and
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