Department of Virology, School of Medicine, The University of Tokushima, 3 Kuramoto, Tokushima 770-8503, Japan1
Author for correspondence: Hirofumi Akari. Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460, USA. Fax +1 301 402 0226.
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The function of Nef protein in the early stage of the HIV-1 replication cycle has some similarities with that of Vif; Nef-dependent enhancement of HIV-1 replication is cell type-dependent (Miller et al., 1994 ; Spina et al., 1994
; Tokunaga et al., 1998a
, b
) and defective mutations in the gene result in restricted proviral DNA synthesis in infected cells (Aiken & Trono, 1995
; Schwartz et al., 1995
). Recent reports (Aiken, 1997
; Luo et al., 1998
) have shown that pseudotyping HIV-1 by the envelope glycoprotein of vesicular stomatitis virus (VSV-G) targets HIV- 1 entry to an endocytic pathway and suppresses the requirement of Nef for infectivity. These results suggest that Nef acts not only on post- entry step(s) such as uncoating and reverse-transcription but also on binding-entry step(s), which can be bypassed through an endocytic pathway. Actually, we have recently reported the cell-dependent requirement of Nef for efficient entry of HIV-1 (Tokunaga et al. , 1998b
).
In this study, we examined whether the endocytic pathway induced by VSV-G pseudotyping suppresses the requirement of Vif for virus infectivity as well as Nef. We also evaluated the effects of Vif on the efficiency of virus binding and entry, virion incorporation of Env, and processing of Gag proteins.
First, to determine the effect of VSV-G pseudotyping on the ability of Vif to enhance infectivity of HIV-1 produced in non-permissive cells, HIV-1 particles defective in either Env (Env) or both Vif and Env (
Vif
Env) pseudotyped by VSV-G were made. As a control, the envelope protein of amphotropic murine leukaemia virus (A- MLV-Env), which uses the membrane fusion pathway for virus entry like HIV-1, was used for pseudotyping. Infectious molecular clones of HIV-1 pNL-432 and its Vif-, Env- and Nef-defective mutants, pNL-Nd, pNL-Kp and pNL-M1T, respectively, have been described previously (Adachi et al., 1986
, 1991
) or elsewhere (H. Akari, S. Arold, T. Fukumori, T. Okazaki & A. Adachi, unpublished results). To make a Vif/Env or a Env/Nef double-defective mutant pNL- NdKp or pNL-KpM1T, respectively, a frameshift mutation was introduced into the KpnI site of the env gene in pNL-Nd and pNL- M1T as previously described (Adachi et al., 1991
). A VSV-G expression construct pCMV-G (Yee et al., 1994
) and an A-MLV-Env expression plasmid SA-A-MLV-env (Page et al., 1990
) were kindly provided by A. Miyanohara (University of California, San Diego) and D. Littman (New York University Medical Center, Skirball Institute, New York), respectively. To obtain wild-type (WT),
Vif
Env or
Nef virus from non-permissive cells, 10 µg of plasmid pNL-432, pNL-Nd, pNL-Kp or pNL-M1T was transfected to 4x106 H9 cells (Popovic et al., 1984
) by electroporation at 0·2 kV on a Gene Pulser (Bio-Rad). For preparation of pseudotyped viruses, 10 µg of pNL-Kp, pNL-NdKp or pNL-KpM1T and pCMV-G or SA-A-MLV-env were co-transfected into H9 or HeLa cells. The culture supernatants were harvested 24 h after electroporation, filtered through a 0·45 µm filter, and aliquoted at -80 °C. Quantification of the amounts of virus was conducted by a p24 antigen ELISA kit (Cellular Products). At 24 h post-electroporation, no clear differences in the amount of p24 antigen in the culture supernatants were observed among any combination of plasmids (data not shown). The infectivity of the viruses was measured by MAGI assay as previously described (Kimpton & Emerman, 1992
). As shown in Fig. 1
(A),
Vif showed 20-fold lower infectivity than WT virus as previously described (Fouchier et al. , 1996
). Interestingly, pseudotyping HIV-1 by not only A-MLV-Env but also VSV-G did not restore the lower infectivity of the Vif-defective mutant (Fig. 1 B
). On the other hand,
Nef exhibited 10-fold lower infectivity than WT virus, and pseudotyping HIV-1 by VSV-G suppressed the requirement of Nef as previously reported (Aiken, 1997
; Luo et al., 1998
) (Fig. 1 C
). These results indicated that the infectivity enhancement by Vif is independent of the entry pathway and suggested that Vif function is unrelated to envelope glycoprotein coating on the virion.
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The present results in this study indicated that Vif does not influence the entry step(s) of HIV-1 in the replication cycle (Figs 1 and 2
). Together with the consensus finding that
Vif is defective for proviral DNA synthesis in the infected cells (Chowdhury et al., 1996
; Courcoul et al., 1995
; Goncalves et al., 1996
; Reddy et al., 1995
; Schwedler et al., 1993
; Simon & Malim, 1996
; Sova & Volsky, 1993
), it can be concluded that the
Vif mutation reduces the infectivity due to the defect(s) in a step(s) from uncoating to reverse transcription.
Our data revealed that no envelope glycoprotein among HIV-1, VSV and A-MLV is associated with the function of Vif in non-permissive cells. It has been shown that HIV-1 matrix (MA) protein directly associates with the cytoplasmic tail of Env gp41 and that a mutation in MA results in the loss of Env incorporation into the virion (Cosson, 1996 ; Freed & Martin, 1995
). Furthermore, truncation of the C-terminal 144 amino acids of gp41 results in sufficient infectivity without association of gp41 with MA (Reil et al., 1998
). It is possible that the Vif defect affects the function of MA, resulting in inefficient incorporation of Env into virions. To test this possibility, we examined whether a truncation of the cytoplasmic tail of Env gp41 rescues the Vif defect. The truncation did not compensate for the defective phenotype of
Vif (H. Akari & A. Adachi, unpublished results). This result also supports our conclusion that the function of Vif is unrelated to Env.
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References |
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Received 1 June 1999;
accepted 26 July 1999.