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J Virol, July 1998, p. 6257-6259, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Virion Entry by Dominant-Negative Hck

Kenzo Tokunaga,^1, Etsuko Kiyokawa,¹ Mie Nakaya,² Naomi Otsuka,² Asato Kojima,¹ Takeshi Kurata,¹ and Michiyuki Matsuda^1,2,*

Department of Pathology, National Institute of Infectious Diseases, Toyama, Shinjuku-ku, Tokyo 162-8640,¹ and Department of Pathology, Research Institute, International Medical Center of Japan, Toyama, Shinjuku-ku, Tokyo 162-8655,² Japan

Received 22 December 1997/Accepted 27 February 1998

	ABSTRACT

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To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa-CD4-LTR--gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.

	TEXT

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Src family non-receptor-type tyrosine kinases are involved in the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in many ways (for a review, see reference 6). Accordingly, it has been reported that Src family tyrosine kinases are activated upon infection with HIV-1 (10, 13). However, little is known about the mechanism by which Src family tyrosine kinases regulate HIV-1 infection.

The dominant-negative mutant is one of the most potent tools for deciphering the signal transduction cascade. A Src mutant that is deficient in its catalytic activity has been shown to inhibit the Src-dependent signaling cascade (3). In the present study, we found a decrease in the infectivity of HIV-1 due to the expression of a dominant-negative Hck protein.

Inhibition of HIV-1 infectivity by the expression of a dominant-negative Hck protein. An expression vector for the dominant-negative Hck mutant pCAGGS-HckN, which consists solely of the amino-terminal regulatory domain (amino acids 1 to 230), was constructed by use of PCR (Fig. 1A). Amino acid substitution of Hck was also performed by PCR-mediated mutagenesis. Arg151, which is essential for the function of SH2, was substituted with Ser in HckN-R151S. Similarly, Trp93, which is essential for the function of SH3, was substituted with Phe in HckN-W93F.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Inhibition of HIV-1 infectivity by dominant-negative Hck. (A) Structures of the wild-type Hck and of the mutant protein used in this study. (B) HIV-1 proviral DNA, pNL-432, and expression plasmids of HckN, HckN-W93F, HckN-R151S, and CrkII were transfected into 293T cells. Virus stocks harvested at 36 h posttransfection were used to infect MAGI cells. Forty-eight hours later, infected cells were identified by staining with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside X-Gal). Each bar represents the average of two determinations. (C) 293T cells used to produce virus stock analyzed by immunoblotting with monoclonal antibody against HckN or CrkII.

HIV-1 proviral DNA (pNL-432) and expression plasmids were transfected into 293T cells by the calcium phosphate method (1). Virus stocks were harvested at 36 h posttransfection and filtered through a 0.45-µm-pore-size filter. We used virus stocks containing equal amounts of p24^gag to infect HeLa-CD4-LTR--gal (MAGI) cells as described elsewhere (8). Expression of HckN and HckN-R151S, the SH2 mutant, significantly decreased the infectivity of HIV-1 (Fig. 1B). The SH3 mutant of HckN, HckN-W93F, did not affect the infectivity of HIV-1. Thus, the decrease in HIV-1 infectivity caused by the dominant-negative Hck depends solely on its SH3 domain. We also tested SrcN, which was constructed similarly to HckN from mouse c-src cDNA. HIV-1 virions harvested from SrcN-expressing cells showed reduced infectivity in MAGI cells (18% ± 7% of that of the wild type). CrkII adaptor protein, which consists mostly of the SH2 and SH3 domains, was used as a control for HckN (9). We could not see any decrease in the infectivity of HIV-1 caused by coexpression of CrkII, suggesting that the inhibition of HIV-1 infectivity is specific to the Src family of tyrosine kinases.

Expression of HckN and CrkII was examined by immunoblotting. 293T cells, which were used to produce virus stock, were lysed in lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 1 µg of aprotinin per ml), cleared by centrifugation, separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred to a polyvinylidene difluoride (PVDF) membrane. HckN and CrkII were detected by use of monoclonal antibody against HckN or CrkII (Transduction Lab, Lexington, Ky.). We confirmed that the wild-type HckN and the mutant HckN were expressed equally (Fig. 1C).

Because Hck is not expressed in 293T cells to a detectable level (data not shown), it is likely that HckN interfered with other Src family tyrosine kinases expressed in 293T cells. Src and Yes, which are present rather ubiquitously (7), may regulate HIV-1 infectivity in 293T cells.

Additive effect of HckN on Nef-deficient HIV-1. We examined the effect of HckN on Nef-deficient HIV-1 because Nef is known to activate Src family kinases (Fig. 2). We assumed that HckN did not affect the infectivity of Nef-deficient HIV-1 when Src family tyrosine kinases functioned downstream of Nef. Nef-deficient HIV-1 was less infectious than the wild type, as reported by many groups (for a review, see reference 15). Against our expectation, we found that HckN further decreased the infectivity of Nef-deficient HIV-1. We confirmed that expression of HckN did not decrease the quantity of Nef in the virion (Fig. 2B). Thus, HckN appears to inhibit HIV-1 infectivity independently of Nef.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Inhibition of the infectivity Nef-deficient HIV-1 by HckN. (A) Proviral HIV-1 DNAs of the wild type (WT; pNL-432) and of the Nef mutant (Nef; pNL-432-Xh) transfected with or without the HckN expression vector into 293T cells. We used the virus stocks to infect MAGI cells. (B) Virions collected from the virus stock by ultracentrifugation on a 20% sucrose cushion, separated on an SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-Nef monoclonal antibody.

Restoration of the infectivity by VSV pseudotyping. It has been reported that pseudotyping by vesicular stomatitis virus (VSV) G protein (VSV-G) can restore the infectivity of Nef-deficient HIV-1 (2). We transfected 293T cells with pNL-432 and pV-G, a VSV-G expression vector (11), with or without the HckN expression vector. We found that VSV pseudotyping restored the infectivity of HIV-1 produced in HckN-expressing cells (Fig. 3). However, Nef expression did not affect the infectivity of HIV-1 produced in HckN-expressing cells. This result argues that HckN may inhibit the entry or uncoating of HIV-1. We also observed that overexpression of HIV-1 env did not restore the infectivity of HIV-1 from HckN-expressing cells (data not shown).

View larger version (31K): [in this window] [in a new window]   FIG. 3.   Restoration of infectivity by VSV pseudotyping. (A) Proviral DNAs of the wild type (WT) and of the Nef mutant (Nef) transfected into 293T cells with vector alone (closed bars), Nef expression vector (open bars), or VSV-G expression vector (shaded bars). Infectivity of the virion was examined as described in the legend to Fig. 1. Each bar represents the average of two determinations. (B) 293T cells used for virus production lysed in lysis buffer, separated on an SDS-polyacrylamide gel, and transferred to a PVDF membrane. VSV-G was detected by anti-G protein monoclonal antibody, followed by an enhanced chemiluminescence detection system (ECL).

Decreased efficiency of HIV-1 entry by the expression of dominant-negative Hck. We then compared the efficiencies of virus entry among wild-type HIV-1, Nef-deficient HIV-1, and HIV-1 produced in HckN-expressing cells. Proviral HIV-1 DNAs of the wild type and of the Nef mutant were transfected with or without the HckN expression vector into 293T cells. CEMx174 (12) or M8166 (14) cells were incubated at 37°C for 1 h with virus stocks containing 10 ng of p24^gag. The cells were trypsinized to remove viruses attached to the cell surfaces nonspecifically and were lysed in 200 µl of phosphate-buffered saline containing 1% Nonidet P-40. The quantity of p24^gag antigen in the lysates was determined by an anti-p24^gag enzyme-linked immunosorbent assay. The entry of the HIV-1 produced in HckN-expressing cells was inhibited significantly compared to that of the wild type, whereas that of the Nef-deficient HIV-1 was not (Fig. 4). The level of the decrease in virus entry was comparable to that of the infectivity shown in Fig. 1, indicating that impaired virus entry could account for the overall decrease in the infectivity of HIV-1 produced in the HckN-expressing cells.

View larger version (14K): [in this window] [in a new window]   FIG. 4.   Inhibition of HIV-1 entry by HckN. 293T cells were transfected with wild-type pNL-432 alone (WT), pNL-432 plus pCAGGS-HckN (WT+HckN), pNL-432-Xh (Nef), or pNL-432 without the env region [Env()]. The viruses produced were used for infection of M8166 (top) and CEMx174 (bottom) cells. The quantities of viruses that entered into the cells were measured by an anti-p24gag enzyme-linked immunosorbent assay.

Further study of how HckN inhibits HIV-1 infectivity is awaited. Because SrcN inhibited HIV-1 infectivity as effectively as did HckN, a target molecules(s) common to the Src family kinases appears to be inhibited by HckN. The SH3 domain of Src family kinases also binds intramolecularly to an SH3-binding motif between the catalytic and the regulatory domains. Thus, HckN may bind to the Src family kinases and may inhibit their enzymatic activity. In either case, our data indicate that signaling from Src family tyrosine kinases is required for the full infectivity of the produced HIV-1 virion.

Previous reports have implicated Src family tyrosine kinases in syncytium formation by HIV-1. Herbimycin A, which is a Src-specific tyrosine kinase inhibitor, prevented HIV-1-infected cells from having a cytopathic effect, including syncytium formation (5). More directly, expression of Lck in Lck-deficient T-cell lines enhances syncytium formation by HIV-1 (4). In conjunction with these preceding reports, our observation that dominant-negative Hck decreases HIV-1 entry suggests that Src family tyrosine kinases are involved in the regulation of membrane fusion by HIV-1. We observed that the overexpression of HIV-1 env did not restore the infectivity of HIV-1 from HckN-expressing cells. Therefore, quantitative and qualitative study of gp120 and gp41 in the virion should be examined further by using the HIVs produced from control and HckN-expressing cells.

	ACKNOWLEDGMENTS

This work was supported in part by grants from the Ministry of Education, Science, and Culture, from the Ministry of Health and Welfare, and from the Japanese Health Sciences Foundation, Tokyo, Japan. K.T. and E.K. are awardees of Research Resident fellowships from the Japanese Health Sciences Foundation and Japanese Foundation for AIDS Prevention, respectively.

We thank S. Hattori for cDNA of human Hck, J. Miyazaki for pCAGGS, A. Adachi for pNL-432, M. Okada for mouse N-Src cDNA, K. Ikuta for the anti-Nef monoclonal antibody, S. Nagata for the anti-VSV antibody, J. Rose for VSV-G cDNA, and W. W. Hall for helpful discussions. MAGI cells were obtained from M. Emerman through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655. Japan. Phone: 81-3-3202-7181. Fax: 81-3-3202-7364. E-mail address: mmatsuda @nih .go .jp.

Present address. Unite de Biologie des Retrovirus, Departement de Virologie, Institut Pasteur, Paris, France.

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J Virol, July 1998, p. 6257-6259, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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