Journal of General Virology (1998), 79, 2447–2453. Printed in Great Britain ................................................................................................................................................................................................................................................................................... Enhancement of human immunodeﬁciency virus type 1 infectivity by Nef is producer cell-dependent Kenzo Tokunaga, 1 Asato Kojima, 1 Takeshi Kurata, 1 Kazuyoshi Ikuta, 2 Hirofumi Akari, 3 A. Hajime Koyama, 3 Meiko Kawamura, 3 Ritsuko Inubushi, 3 Reika Shimano 3 and Akio Adachi 3 1 Department of Pathology, National Institute of Infectious Diseases, Tokyo 162, Japan 2 Institute of Immunological Science, Hokkaido University, Sapporo 060, Japan 3 Department of Virology, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan The growth kinetics of wild-type and nef mutant viruses of human immunodeﬁciency virus type 1 were comparatively analysed in several human CD4 M cell lines. Delayed replication of nef mutant virus was observed in all cell lines examined. To determine the stage in the virus replication cycle that is affected by Nef, a single-round replication assay was performed. Initially, the expression of marker genes in transfected cells was examined in order to study the role of Nef in the late phase of infection. The results obtained indicated that Nef is dispensable during the transcription to virion pro- Introduction One of the accessory genes of human immunodeﬁciency virus type 1 (HIV-1), designated nef, encodes a 25 or 27 kDa protein. In early studies, it was suggested that the gene product, Nef, acted as a negative factor in virus replication (Terwilliger et al., 1986) by repressing transcription from the long terminal repeat (LTR) promoter (Ahmad & Venkatesan, 1988 ; Niederman et al., 1989). However, these ﬁndings were not conﬁrmed in a subsequent report (Hammes et al., 1989). Several studies have reported that Nef could moderately facilitate the virus replication rate (de Ronde et al., 1992; Kim et al., 1989 ; Terwilliger et al., 1991 ; Zazopoulos & Haseltine, 1992, 1993). However, a drastic eﬀect of Nef as a positive factor has been observed in experimental infections in vivo, which demonstrated that Nef of simian immunodeﬁciency virus SIVmac is necessary for maintaining high virus loads and for disease progression of AIDS in rhesus monkeys (Kestler et al., 1991). This result was supported by Jamieson et al. (1994) using HIV-1-infected severe combined immunodeﬁcient mice Author for correspondence : Akio Adachi. Fax 81 886 33 7080. e-mail adachibasic.med.tokushima-u.ac.jp duction stage. Next, the effect of Nef on the early phase was investigated with a single-round infec- tion. It was demonstrated that Nef is required in the early phase of the virus replication cycle, from virion adsorption to integration. Finally, the infectivity of virus stocks prepared from four cell lines was determined. The relative infectivity of the nef mutant from the four cell lines differed. Taken together, we conclude that Nef acts via modulation of viral particles to enhance virus infectivity in a cell- dependent manner. which had been transplanted with foetal human thymus and liver tissues. Subsequently, this positive eﬀect was clearly revealed in in vitro infection experiments using quiescent primary T-lympho- cytes (Miller et al., 1994; Spina et al., 1994) and was further observed in CD4 + cell lines infected with low virus inputs (Chowers et al., 1994). In the latter observation, it was also suggested that Nef enhances viral particle infectivity (Chowers et al., 1994). Miller et al. (1995) have recently shown that the increased infectivity by Nef is independent of virus entry, and is manifested at the stage after entry but prior to or coincident with viral gene expression. Schwartz et al. (1995) have indicated that Nef acts at an early stage of the virus replication cycle, but not when the virus binds to the receptor and before the completion of reverse transcription. Moreover, Aiken & Trono (1995) have suggested that Nef functions at the stage of particle formation, leading to the eﬃcient completion of proviral DNA synthesis, not to enhanced virus internalization. This study was conﬁrmed by Chowers et al. (1995). In the present study, we ﬁrstly conﬁrmed the delayed replication kinetics of nef mutant virus in several cell lines. To determine the replication stage inﬂuenced by Nef, we used the single-round replication assay (Helseth et al., 1989 ; Sakai et al., 1995). The results obtained showed that Nef is dispensable in 0001-5657  1998 SGM CEEH