- Email: [email protected]
Simian immunodeficiency viruses containing mutations in the long terminal repeat NF-κB or Sp1 binding sites replicate efficiently in T cells and PHA-stimulated PBMCs
Virus Research 49 (1997) 205 – 213
Short communication
Simian immunodeficiency viruses containing mutations in the long terminal repeat NF-kB or Sp1 binding sites replicate efficiently in T cells and PHA-stimulated PBMCs Jianbo Zhang a, Francis Novembre b, Arnold B. Rabson a,* a
New Jersey Center for Ad6anced Biotechnology and Medicine (CABM) and the Cancer Institute of New Jersey, Department of Molecular Genetics and Microbiology, Uni6ersity of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, CABM room 139, 679 Hoes Lane, Piscataway, NJ 08854, USA b Yerkes Primate Center, Emory Uni6ersity, Atlanta, GA 30322, USA Received 26 September 1996; received in revised form 28 January 1997; accepted 5 February 1997
Abstract The long terminal repeats (LTRs) of primate lentiviruses contain conserved binding sites for the NF-kB and Sp1 cellular transcription factors. In order to study the role that these sites play in simian immunodeficiency virus (SIV) replication, we have introduced mutations that disrupt either the NF-kB or Sp1 binding sites in the LTR of an infectious molecular clone of SIVmac239. An additional mutation also disrupted the SF3 transcription factor binding site that overlaps the NF-kB site. Viruses containing point mutations or deletions of the NF-kB, SF3, or Sp1 binding sites retained the ability to replicate efficiently in the CEMx174 and MT4 cell lines, as well as in PHA-stimulated primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Efficient replication of SIVs mutated in either NF-kB or Sp1 binding sites suggests that the SIV LTR promoter contains multiple functionally redundant elements capable of supporting sufficient transcription to allow productive viral replication. © 1997 Elsevier Science B.V. Keywords: Simian immunodeficiency virus; Long terminal repeat; NF-kB; Sp1
1. Introduction
* Corresponding author. Fax: +1 908 2354850; e-mail: [email protected]
The SIVs are primate lentiviruses that exhibit numerous genetic and phenotypic similarities to the human immunodeficiency viruses (HIVs). Like
0168-1702/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 8 - 1 7 0 2 ( 9 7 ) 0 1 4 6 2 - 7
206
J. Zhang et al. / Virus Research 49 (1997) 205–213
the HIVs, SIVs infect both CD4 + T cells and cells of the monocyte/macrophage series, and are capable of inducing severe AIDS-like immunodeficiency diseases in susceptible monkey strains (Letvin and King, 1990; Hirsch and Johnson, 1994; Simon et al., 1994). SIV-induced AIDS represents an excellent animal model for HIV disease and the availability of disease-inducing molecular clones of SIV has allowed the identification of SIV genes, such as nef, that play important roles in pathogenesis (Kestler et al., 1991). The replication of primate lentiviruses is dependent on transcription of viral RNAs from integrated proviral DNA. Transcription of viral RNAs is determined both by cellular transcriptional factors interacting with viral LTR DNA sequences and by the activity of the virally-encoded Tat transactivator which binds to the TAR element at the 5% end of nascent viral RNAs (reviewed in Cullen, 1992; Gaynor, 1992; and Antoni et al., 1994). The LTRs of both the HIVs and the SIVs contain a partially conserved arrangement of DNA sequences proximal to the transcription start site including binding sites for the NF-kB and Sp1 cellular transcription factors (Clements and Payne, 1994; Jeang and Gatignol, 1994). The roles of these sites in the regulation of LTR transcription and viral replication for HIV-1 have been studied in detail by mutagenesis in several laboratories (Leonard et al., 1989; McClure et al., 1990; Parrott et al., 1991; Ross et al., 1991; Kim et al., 1993; Alcami et al., 1995). While these sites do not appear to be absolutely required for replication in most cells studied, they do have differential effects on HIV replication in different cell types. The prototypical SIV LTR contains a single NF-kB site adjacent to three or four Sp1 binding sites. An analysis of the functional activities and protein-DNA interactions in the LTRs derived from two SIV isolates, SIVmac239 and SIVmac251, demonstrated differences in protein binding to these two LTRs in sequences overlapping the 3% most Sp1 site (Anderson and Clements, 1991). Studies of the LTR of SIVmac142 identified a distinct protein binding site, referred to as SF3, overlapping the 3% end of the NF-kB site (Winandy et al., 1992). Mutations in the NF-kB site of SIVmac239 demonstrated that similar to
HIV, these sites were not required for SIV replication in T cells (Bellas et al., 1993), however, when the LTR NF-kB mutations were introduced into a macrophage-tropic SIV, the NF-kB sites were shown to be required for efficient infection of monocytes. Evidence for a possible contribution of NF-kB sites to SIV replication and disease pathogenesis has come from an analysis of the molecular determinants of the acute pathogenicity associated with infection with SIVsmmPBj14, which contains a duplication of the NF-kB site (Dewhurst et al., 1990). Although the duplication of this site was not sufficient to induce the acutely lethal syndrome associated with SIVsmmPBj14 infection (Novembre et al., 1993), viruses with the duplicated NFkB site do replicate more rapidly than isogenic viruses containing a single LTR NF-kB site (Dollard et al., 1994), suggesting that enhanced replication may contribute to SIVsmmPBj14 pathogenesis. Recently, Ilyinskii and Desrosiers (1996) have studied the replication of SIVs containing mutations in the Sp1 and NF-kB binding sites and have shown that SIV is capable of replicating without these elements. We have studied the effects of these LTR elements on SIV replication. In this study, we have systematically mutated the NF-kB or Sp1 binding sites of the LTRs of SIVmac239 and have assessed their replication properties in CD4+ T cell lines and PHAstimulated rhesus macaque PBMCs.
2. Materials and methods In order to study the effects of the SIV LTR NF-kB and Sp1 binding sites, mutations were introduced into these sites in the U3 region of an SIV LTR. Mutated LTRs were reconstructed into full length proviruses by concatemerization of segments of the SIV provirus as illustrated in Fig. 1. In the initial step, a BamHI fragment containing the LTR from the 5% molecular clone of SIVmac239, p239SpSp5% (Kestler et al., 1990) was subdoned into the pLitmus 38 vector. Mutations in the NF-kB and Sp1 binding sites were introduced by oligonucleotide-directed, site-specific mutagenesis. The mutated LTR was reinserted back into p239SpSp5% in place of the wild-type LTR.
J. Zhang et al. / Virus Research 49 (1997) 205–213
207
Fig. 1. Construction of SIV proviral DNAS containing LTR mutations. The structure of the full-length SIVmac239 provirus with 2 LTRs is shown on line A. The structure of the 5%, molecular clone of SIVmac239, p239SpSp5% (Kestler et al., 1990) is shown on line B. A BamHI fragment containing 256 bp of 5% cellular flanking sequence, the 5% LTR and 1332 bp of adjacent gag sequences was purified from p239SpSp5% (Kestler et al., 1990) and subdoned into the pLitmus 38 vector (New England Biolabs) to create pLitmus 5% LTR (shown in line C). pLitmus 5%LTR was subjected to oligonucleotide-directed, site-specific mutagenesis using the Chameleon mutagenesis system (Stratagene). Introduction of the desired mutation was confirmed by DNA sequence analysis and the BamHI fragment containing the mutated LTR was reinserted back into p239SpSp5% in place of the wild-type LTR. The presence of the mutated LTR was reconfirmed by DNA sequence analysis in p239SpSp5% prior to further analysis. In order to assay for infectivity of the SIVs containing the LTR mutations, two fragments of the SIV genome were purified and used for subsequent ligation and transfection. An Nde1–Sph1 fragment of p239SpSp5% from the Nde1 site in the middle of U3 at position 288 to the Sph1 site at 6445 bp contained mutated U3 and wild-type R and US LTR sequences as well as 5% proviral gag and pol, 6if, 6px and 6pr sequences. This fragment was ligated to an Sph1-Nde1 fragment derived from p239SpE3%nef-open (shown in line D) that contained sequences from the Sph1 site at 6445 to the Nde1 site in the U3 region of the 3% LTR (9749 bp) including tat, re6, en6 and nef sequences as well as U3 sequences upstream of the site of the introduced mutations. The ligation mixture, containing concatemerized Nde1–Sph1 and Sph1–Nde1 fragments, would be expected to include reconstituted full length proviruses with two LTRs, both of which contain the introduced mutations (line E).
In order to assay for infectivity of the SIVs containing the LTR mutations, two fragments of the SIV genome were purified and used for subsequent ligation and transfection (Fig. 1). An Nde1–Sph1 fragment of p2395p5p5% that contained the mutated U3 LTR sequence, as well as R and U5 LTR sequences and 5% proviral sequences was ligated to an Sph1 – Nde1 fragment
derived from p239SpE3%nef-open (Kestler et al., 1991), (kind gift of Dr R. Desrosiers), that contains sequences comprising the 3% half of the provirus to the Nde1 site in the U3 region of the 3% LTR upstream of the NF-kB and Sp1 sites. The ligation mixture, containing concatemerized Nde1–Sph1 and Sph1-Nde1 fragments, would be expected to include reconstituted full length
208
J. Zhang et al. / Virus Research 49 (1997) 205–213
Fig. 2. Mutations introduced into the SIVmac239 LTR. The positions of the LTR U3, R and US regions and of the NF-kB and Sp1 binding sites and TAR region are shown at top. The nucleotide sequences of the NF-kB and Sp1 sites (from −103 to −54 bp with respect to the start site of transcription) are shown for the wild-type and mutant LTRs. Deletions are represented by dashes and the point mutations are underlined.
proviruses with two LTRs, both of which contain the introduced mutations (Fig. 1). This ligation mix was transfected into the SW480 colon carcinoma cells as described (Leonard et al., 1989). Two days following transfection, the SW480 cells were co-cultivated with CD4 +, CEMx174 cells permissive for SIV replication and supernatants were monitored for the production of reverse transcriptase (RT) as previously described (Leonard et al., 1989). Virus produced within 2 – 4 days of cocultivation was harvested as viral stock for use in subsequent infections. Because our use of uncloned, ligated DNA fragments in the construction of the mutant viruses raised the remote, but real possibility that recovered viruses may contain contaminating wild-type U3 LTR sequences, we confirmed that the recovered viruses contained the introduced mutations. LTR DNA sequences from the U3 and R regions were amplified by PCR from DNA prepared following a second round of infection of CEMx174 cells with the original virus stocks, and were subjected to DNA sequencing.
The presence of the originally introduced, unaltered LTR mutations was confirmed by DNA sequence analysis of all of the mutant viruses.
3. Results A series of mutations in the SIV LTR NF-kB and Sp1 binding sites were prepared for these studies. The DNA sequence of the SIV LTR in the region of the NF-kB and Sp1 sites is shown in Fig. 2 as are the nucleotide sequences of the mutations used for these studies. Mutations of the NF-kB site included a complete deletion of the NF-kB and overlapping SF3 sites (dlNFkB), and point mutations in bases important for NF-kB binding (NFkBM1) or in nucleotides that define the SF3 binding site (NFkBM2) (Winandy et al., 1992). Mutations of the Sp1 binding sites include a deletion of all three Sp1 sites (dlSp1), as well as point mutations of the 5% most Sp1 site (Sp1 M1), the 5% and middle Sp1 sites (Sp1 M2) and all three
J. Zhang et al. / Virus Research 49 (1997) 205–213
Sp1 sites (Sp1 M3). Mutations in the NF-kB site and the Sp1 sites were designed based upon sequence changes previously demonstrated to disrupt protein binding to these sites (Duh et al., 1989; Jones et al., 1986) and the mutation affecting the SF3 site is predicted to disrupt SF3 binding based upon the results of previously published methylation interference assays (Winandy et al., 1992). Transfection of each of the concatemerized SIV DNAs with the different LTR mutations resulted in recovery of infectious SIV following cocultivation with the CEMx174 cells (data not shown). These results demonstrated that neither the NF-kB sites nor the Sp1 sites were required for replication of SIV in CEMx174 cells and allowed the generation of viral stocks for assays of replication of the mutant viruses in different CD4+ lymphoid cells. The replication of the SIVs containing LTR mutations was compared to that of wild-type SIV in CD4+ T cell lines permissive for SIV infection, as well as in PHA-stimulated primary rhesus peripheral blood mononuclear cells (PBMCs) (Fig. 3). Virus stocks from the wild-type and mutant viruses were normalized for reverse transcriptase activity and equivalent amounts of virus (10 000 cpm) were used in all infections. As the virus stocks were recovered from co-cultivations with CEMx174 cells, we initially assessed replication kinetics of the different mutant viruses in this cell line (Fig. 3A). Cells were infected with equivalent amounts of wild-type and mutant viruses and SIV production was monitored by assay for viral reverse transcriptase production. All 4 Sp1 mutants and 3 NF-kB mutant viruses replicated efficiently in CEMx174 cells, exhibiting no significant difference in kinetics from wild type virus. All mutant viruses induced equivalent levels of SIV-associated cytopathicity as monitored by syncytia formation and cell death, and similar results of virus production and cytotoxicity were seen in triplicate infections (data not shown). We were also interested in the effects of the LTR mutations on SIV replication in other CD4+ T cell lines. In preliminary experiments, we found that SIVmac239 replicated efficiently in the HTLV-1+ , MT4 cell line, but not in Jurkat cells or Sup T cells, two other cell lines highly
209
permissive for HIV replication. We therefore also assayed replication of the LTR mutant viruses in MT4 cells (Fig. 3B). Again, all LTR mutants replicated efficiently without demonstrating significant differences from wild-type virus. SIV infection by either wild-type or mutant viruses resulted in essentially complete killing of the MT4 cells (data not shown). Similar results were seen in duplicate infections. It was important to determine if any of the LTR mutations affected the efficiency of replication of SIV in PHA-stimulated primary rhesus macaque peripheral blood mononuclear cells (Fig. 3C). Previous studies had suggested that mutation of the LTR NF-kB sites did not affect SIV replication in these cells. As expected, neither the NF-kB deletions or point mutations altered the kinetics of SIV replication in these cells. Similarly, even the complete deletion or introduction of point mutations into all three Sp1 binding sites still resulted in efficient production of SIV in the PBMCs. A repeat infection in PHA-stimulated PBMCs derived from a second rhesus macaque also demonstrated replication of all the LTR mutant SIVs (data not shown).
4. Discussion The results of these studies demonstrate that mutations of either the LTR NF-kB or Sp1 binding sites do not prevent efficient replication of SIV in permissive T cell lines or PHA-stimulated primary rhesus PBMCs. In fact, relatively little effect on viral replication could be detected even with complete deletion of either of these sites. This work confirms the previous observations that the NF-kB and Sp1 binding sites are not required for SIV replication in PBMCs (Bellas et al., 1993; Ilyinskii and Desrosiers, 1996) and extends the analysis to include the role of the overlapping SF3 binding site as well as the effects of these mutations in a human CD4+ T cell line that is highly susceptible to HIV replication (MT4 cells). Mutations that interrupt the SF3 binding site overlapping the NF-kB site also did not impair viral replication, suggesting that this binding site, although important for high basal transcription in
210
J. Zhang et al. / Virus Research 49 (1997) 205–213
Fig. 3. Replication of wild-type and LTR mutant SIVs in CD4+ T cell lines and PHA-stimulated rhesus macaque PBMCs. T-cell cultures in 24-well dishes (4×105 cells in 1 ml. per well) were inoculated with equivalent amounts of wild-type and mutant virus stock as determined by reverse transcriptase (RT) activity (10 000 cpm per well). Production of progeny virus was monitored by production of viral RT in the culture supernatants. (A) Infection of CEMx174 cells; (B) infection of MT4 cells and (C) infection of PHA-stimulated rhesus macaque PBMCs. PBMCs were stimulated with phytohemagglutinin (PHA-P, Sigma) for 72 h prior to infection. PBMCs were maintained in RPMI 1640 supplemented with 20% fetal bovine serum (GIBCO-BRL) and 10% recombinant human interleukin 2 (Boehringer Mannheim).
J. Zhang et al. / Virus Research 49 (1997) 205–213
transfection assays (Winandy et al., 1992), also was not required for viral production. Thus, mutations or deletions of the NF-kB sites, the SF3 site and the Sp1 sites failed to markedly impair SIV replication in the cell types studied, demonstrating that these sites were also not essential for sufficient viral transcription to allow productive replication. These observations in the SIV system are similar to those observed for HIV, in which deletion or point mutations of either the NFkB or Sp1 binding sites failed to inhibit HIV replication in either MT4 cells or human PBMCs (Leonard et al., 1989; Parrott et al., 1991; Ross et al., 1991). Interestingly, there have been reports of an absolute requirement for the HIV NF-kB sites for replication of HIV in PHA-stimulated purified human CD4+ cells (Alcami et al., 1995). As our experiments did not utilize purified simian CD4 + T cells, we cannot determine the effects of the SIV LTR NF-kB sites in these cells. The most likely explanation for the ability of SIV to replicate in the absence of either the Sp1 binding sites or the NF-kB sites is the exterisive redundancy of positive acting transcriptional regulatory elements in this promoter. This redundancy may be due to the effects of several different LTR elements. One possibility is that the NF-kB and Sp1 sites may functionally substitute for each other. The cell types studied may contain both activated, nuclear NF-kB and Sp1, either of which would complement the loss of binding to the other site. Similar functional complementation appears to be responsible for the ability of Sp1deleted HIV proviruses to replicate in a variety of human cells (Parrott et al., 1991). In fact, both MT 4 cells and PHA-stimulated T cells have been reported to have induced nuclear NF-kB (Parrott et al., 1991) which could be sufficient to allow transcription and replication of the SIV LTR Sp1 mutants. Although we have not directly determined the NF-kB status of CEMx174 cells, it would not be surprising if they also contained constitutive nuclear. NF-kB activation, as this cell line was derived as a hybrid of CEM cells which lack constitutive nuclear NF-kB (Parrott et al., 1991), with a transformed B cell line (Salter et al., 1985) which is likely to contain nuclear NF-kB,
211
as activation of this factor is a hallmark of B cell maturation (Miyamoto and Verma, 1995). Clearly, in view of our results, the replication potential of an SIV deleted in the NFkB, Sp1 and SF3 binding sites would be important to assess the possible functional redundancy of these elements. In fact such a mutation has been recently reported by Ilyinskii and Desrosiers (Ilyinskii and Desrosiers, 1996), who observed that an SIV deleted in the region spanning both the NF-kB, SF3, and Sp1 sites still can replicate. This observation strongly suggests that other LTR sites participate in supporting viral transcription in place of these sites. Such candidate regulatory sequences include those identified in the LTR mapping between − 162 and − 114, shown to contain both constitutive and inducible enhancer activity (Renjifo et al., 1990) that is responsive to SIV Tat (Ilyinskii and Desrosiers, 1996). This region contains binding sites for proteins designated SF1, SF2, as well as an additional binding site for SF3 (Winandy et al., 1992). Any of these upstream regulatory elements may be sufficient to support SIV replication. Even only relatively low levels of transcription would likely be sufficient to promote viral replication, as the production of low levels of Tat protein and TAR-containing RNAs would allow rapid amplification of SIV transcription through the potent transactivating effects of Tat. An important question raised by these results relates to the possible significance of the LTR NF-kB and Sp1 sites in SIV replication in vivo, particularly in view of their evolutionary conservation in essentially all primate lentiviruses. The presence of these mutations in disease-inducing molecular clones of SIV should allow direct analysis of their contributions to the induction of simian AIDS. The fact that these mutant viruses all replicate efficiently in activated rhesus PBMCs might suggest that they will not play an important role in induction of AIDS. On the other hand, a nef-SIV replicates well in activated PBMCs in culture and yet exhibits profound defects in disease induction (Kestler et al., 1991). An interesting hypothesis with regard to the role of the NF-kB site in the LTRs of AIDS-inducing primate lentiviruses relates to recent observations that immune stimulation may enhance viral repli-
212
J. Zhang et al. / Virus Research 49 (1997) 205–213
cation in vivo (Fultz et al., 1992; Schwiebert and Fultz, 1994; O’Brien et al., 1995; Staprans et al., 1995; Stanley et al., 1996). Immune activation results in NF-kB induction in T cells and monocytes (reviewed in (Miyamoto and Verma, 1995) and thus could be hypothesized to lead to enhanced viral replication in immune-stimulated animals. According to this model, deletion of the LTR NF-kB site might result in a loss of immune activation mediated enhancement of AIDS pathogenesis. The mutant viruses studied here may provide reagents to shed light on this important issue. Another interesting issue relates to the effects that LTR mutations have on the replication of SIV in cells of the monocyte/macrophage lineage. Although not specifically addressed in our studies due to our use of an SIVmac239 viral background which does not infect monocyte/ macrophages efficiently, previous studies have suggested that the NF-kB and Sp1 sites may be more important for SIV replication in monocytes (Bellas et al., 1993; Ilyinskii and Desrosiers, 1996). Thus, these LTR mutant SIVs may also be useful in dissecting the role of monocyte/macrophage infection in the pathogenesis of AIDS in vivo.
Acknowledgements We would like to thank Dr D. Regier and Dr R. Desrosiers for kindly supplying us with the p2395pE3%nef-open plasmid. The following reagents were obtained from the NIH, NIAID AIDS Research and Reference Reagent Program, Division of AIDS, NIAID: p239SPSP5% (contributed by R. Desrosiers), CEMx174 cells (contributed by P. Cresswell). This work was supported by a grant from NIAID, NIH (A130901), by a grant to CABM from the New Jersey Commission on Science and Technology, and by an NCRR, NIH grant to the Yerkes Primate Center (RR-00165).
References Alcami, J., Lain de Lera, T., Folgueira, L., Pedraza, M.-A., Jacque, J.M., Bachelerie, F., Noriega, A.R., Hay, R.T.,
Harrich, D., Gaynor, R.B., Virelizier, J.-L. and ArenzanaSeisdedos, F. (1995) Absolute dependence on kB responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO. J. 14, 1552 – 1560. Anderson, M.G. and Clements, J.E. (1991) Comparison of the transcriptional activity of the long terminal repeats of simian immunodeficiency viruses SIVmac251 and SIVmac239 in T-celI lines and macrophage cell lines. J. Virol. 65, 51 – 60. Antoni, B.A., Stein, S.B. and Rabson, A.B. (1994) Regulation of human immunodeficiency virus infection: implications for pathogenesis. In: K. Maramorosch, F.A. Murphy and A.J. Shatkin (Eds), Advances in Virus Research, Academic Press, New York, Vol. 43, pp. 53 – 145. Bellas, R.E., Hopkins, N. and Li, Y. (1993) The NF-kB binding site is necessary for efficient replication of simian immunodeficiency virus of macaques in primary macrophages but not in T cells in vitro. J. Virol. 67, 2908 – 2913. Clements, J.E. and Payne, S.L. (1994) Molecular basis of the pathobiology of lentiviruses. Virus Res. 32, 97 – 109. Cullen, B.R. (1992) Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56, 375 – 394. Dewhurst, S., Embretson, J.E. Anderson, D.C., Mullins, J.I. and Fultz, P.N. (1990) Sequence analysis and acute pathogenicity of molecularly cloned SIVsmm-pbj14. Nature (London) 345, 636 – 639. Dollard, S.C., Gummuluru, S., Tsang, S., Fultz, P.N. and Dewhurst, S. (1994) Enhanced responsiveness to nuclear factor kB contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14. J. Virol. 68, 7800 – 7809. Duh, E.J., Maury, W.J., Folks, T.M., Fauci, A.S. and Rabson, A.B. (1989) Tumor necrosis factor a activates human immunodeficiency virus-1 through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA 86, 5974 – 5978. Fultz, P.N., Gluckman, J.C., Muchmore, E. and Girard, M. (1992) Transient increases in numbers of infectious cells in an HIV-1 infected chimpanzee following immune stimulation. AIDS Res. Hum. Retraviruses 8, 313 – 317. Gaynor, R. (1992) Cellular transcriptional factors involved in the transcriptional regulation of HIV-1 gene expression. AIDS 6, 347 – 363. Hirsch, V.M. and Johnson, P.R. (1994) Pathogenic diversity of simian immunodeficiency viruses. Virus Res. 32, 183 – 203. Ilyinskii, P.O. and Desrosiers, R.C. (1996). Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kB and Sp1 binding elements. J. Virol. 70, 3118 – 3126. Jeang, K. and Gatignol, A. (1994). Comparison of regulatory features among primate lentiviruses. Curr. Top. Microbial. Immunol. 188, 123 – 144. Jones, K.A., Kadonaga, J.T., Luciw, P.A. and Tjian, R. (1986). Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. Science. 232, 755 – 759.
J. Zhang et al. / Virus Research 49 (1997) 205–213 Kestler, H., Kodama, T., Ringler, D., Marthas, M., Pedersen, N., Lackner, A., Regier, D., Sehgal, P., Daniel, M., King, N. and Desroslers, R. (1990) Induction of AIDS in Rhesus Monkeys by molecularly cloned simian immunodeficiency virus. Science 248, 1109–1112. Kestler III, H.W., Ringler, D.J., Mon, K., Panicali, D.L., Sehgal, P.K., Daniel, M.D. and Desrosiers, R.C. (1991) Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65, 651–662. Kim, J.Y., Gonzalez-Scarano, F., Zeichner, S.L. and Alwine, J.C. (1993) Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the − 201 to − 130 region of the long terminal repeat. J. Virol. 67, 1658 – 1662. Leonard, J., Parrott, C., Buckler-White, A.J., Turner, W., Ross, E.K., Martin, M.A. and Rabson, A.B. (1989) The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. J. Virol. 63, 4919–4924. Letvin, N.L. and King, N.W. (1990) Immunologic and pathologic manifestations of the infection of rhesus monkeys with simian immunodeficiency virus of macaques. J. Acquir. Immune Def. Syndr. 3, 1023–1040. McClure, H.M., Anderson, D.C., Ansari, A.A., Fultz, P.N., Klumpp, S.A. and Schinazi, R.F. (1990) Nonhuman primate models for evaluation of AIDS therapy. Ann. NY Acad. Sci. 616, 287 – 298. Miyamoto, S. and Verma, I.M. (1995) Rel/NF-kB/lkB story. Adv. Cancer Res. 66, 255–292. Novembre, F.J., Johnson, P.R., Lewis, M.G., Anderson, D.C., Klumpp, S., McClure, H.M. and Hirsch, V.M. (1993) Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant. J. Virol. 67, 2466–2474. O’Brien, W.A., Grovit-Ferbas, K., Namazi, A., Ovcak-Derzic, S., Wang, H.J., Park, J., Yeramlan, C., Mao, S.H. and Zack, J.A. (1995) Human immunodeficiency virus-type 1 replication can be increased in peripheral blood of seropositive patients after influenza vaccination. Blood 86, 1082– 1089.
.
213
Parrott, C., Seidner, T., Duh, E., Leonard, J., Theodore, T.S., Buckler-White, A., Martin, M.A. and Rabson, A.B. (1991) Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T Lymphocytes. J. Virol. 65, 1414 – 1419. Renjifo, B., Speck, N.A., Winandy, S., Hopkins, N. and Li, Y. (1990) cis-acting elements in the U3 region of a simian immunodeficiency virus. J. Virol. 64, 3130 – 3134. Ross, E.K., Buckler-White, A.J., Rabson, A.B., Englund, G. and Martin, M.A. (1991) Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. J. Virol. 65, 4350 – 4358. Salter, R.D., Howell, D.N. and Cresswell, P. (1985) Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21, 235 – 246. Schwiebert, R. and Fultz, P.N. (1994) Immune activation and viral burden in acute disease induced by simian immunodeficiency virus SIVsmmPBj14: correlation between in vitro and in vivo events. J. Virol. 68, 5538 – 5547. Simon, M.A., Brodie, S.J., Sasseville, V.G., Chalifoux, L.V., Desrosiers, R. and Ringler, D.J. (1994) Immunopathogenesis of SIVmac. Virus Res. 32, 227 – 251. Stanley, S.K., Ostrowski, M.A., Justement, J.S., Gantt, K., Heydayati, S., Mannix, M., Roche, K., Schwartzentruber, D.J., Fox, C.H. and Fauci, A.S. (1996) Effect of immunization with a common recall antigen on viral expression in patients infected with human immunodeficiency virus type 1. N. Engl. J. Med. 334, 1222 – 1230. Staprans, S.I., Hamilton, B.L., Follansbee, S.E., Elbelk, T., Barbosa, P., Grant, R.M. and Feinberg, M.B. (1995) Activation of virus replication after vaccination of HIV-1-infected individuals. J. Exp. Med. 182, 1727 – 1737. Winandy, S., Renjifo, B., Li, Y. and Hopkins, N. (1992) Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat. J. Virol. 66, 5216 – 5223.
Short communication
Simian immunodeficiency viruses containing mutations in the long terminal repeat NF-kB or Sp1 binding sites replicate efficiently in T cells and PHA-stimulated PBMCs Jianbo Zhang a, Francis Novembre b, Arnold B. Rabson a,* a
New Jersey Center for Ad6anced Biotechnology and Medicine (CABM) and the Cancer Institute of New Jersey, Department of Molecular Genetics and Microbiology, Uni6ersity of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, CABM room 139, 679 Hoes Lane, Piscataway, NJ 08854, USA b Yerkes Primate Center, Emory Uni6ersity, Atlanta, GA 30322, USA Received 26 September 1996; received in revised form 28 January 1997; accepted 5 February 1997
Abstract The long terminal repeats (LTRs) of primate lentiviruses contain conserved binding sites for the NF-kB and Sp1 cellular transcription factors. In order to study the role that these sites play in simian immunodeficiency virus (SIV) replication, we have introduced mutations that disrupt either the NF-kB or Sp1 binding sites in the LTR of an infectious molecular clone of SIVmac239. An additional mutation also disrupted the SF3 transcription factor binding site that overlaps the NF-kB site. Viruses containing point mutations or deletions of the NF-kB, SF3, or Sp1 binding sites retained the ability to replicate efficiently in the CEMx174 and MT4 cell lines, as well as in PHA-stimulated primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Efficient replication of SIVs mutated in either NF-kB or Sp1 binding sites suggests that the SIV LTR promoter contains multiple functionally redundant elements capable of supporting sufficient transcription to allow productive viral replication. © 1997 Elsevier Science B.V. Keywords: Simian immunodeficiency virus; Long terminal repeat; NF-kB; Sp1
1. Introduction
* Corresponding author. Fax: +1 908 2354850; e-mail: [email protected]
The SIVs are primate lentiviruses that exhibit numerous genetic and phenotypic similarities to the human immunodeficiency viruses (HIVs). Like
0168-1702/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 8 - 1 7 0 2 ( 9 7 ) 0 1 4 6 2 - 7
206
J. Zhang et al. / Virus Research 49 (1997) 205–213
the HIVs, SIVs infect both CD4 + T cells and cells of the monocyte/macrophage series, and are capable of inducing severe AIDS-like immunodeficiency diseases in susceptible monkey strains (Letvin and King, 1990; Hirsch and Johnson, 1994; Simon et al., 1994). SIV-induced AIDS represents an excellent animal model for HIV disease and the availability of disease-inducing molecular clones of SIV has allowed the identification of SIV genes, such as nef, that play important roles in pathogenesis (Kestler et al., 1991). The replication of primate lentiviruses is dependent on transcription of viral RNAs from integrated proviral DNA. Transcription of viral RNAs is determined both by cellular transcriptional factors interacting with viral LTR DNA sequences and by the activity of the virally-encoded Tat transactivator which binds to the TAR element at the 5% end of nascent viral RNAs (reviewed in Cullen, 1992; Gaynor, 1992; and Antoni et al., 1994). The LTRs of both the HIVs and the SIVs contain a partially conserved arrangement of DNA sequences proximal to the transcription start site including binding sites for the NF-kB and Sp1 cellular transcription factors (Clements and Payne, 1994; Jeang and Gatignol, 1994). The roles of these sites in the regulation of LTR transcription and viral replication for HIV-1 have been studied in detail by mutagenesis in several laboratories (Leonard et al., 1989; McClure et al., 1990; Parrott et al., 1991; Ross et al., 1991; Kim et al., 1993; Alcami et al., 1995). While these sites do not appear to be absolutely required for replication in most cells studied, they do have differential effects on HIV replication in different cell types. The prototypical SIV LTR contains a single NF-kB site adjacent to three or four Sp1 binding sites. An analysis of the functional activities and protein-DNA interactions in the LTRs derived from two SIV isolates, SIVmac239 and SIVmac251, demonstrated differences in protein binding to these two LTRs in sequences overlapping the 3% most Sp1 site (Anderson and Clements, 1991). Studies of the LTR of SIVmac142 identified a distinct protein binding site, referred to as SF3, overlapping the 3% end of the NF-kB site (Winandy et al., 1992). Mutations in the NF-kB site of SIVmac239 demonstrated that similar to
HIV, these sites were not required for SIV replication in T cells (Bellas et al., 1993), however, when the LTR NF-kB mutations were introduced into a macrophage-tropic SIV, the NF-kB sites were shown to be required for efficient infection of monocytes. Evidence for a possible contribution of NF-kB sites to SIV replication and disease pathogenesis has come from an analysis of the molecular determinants of the acute pathogenicity associated with infection with SIVsmmPBj14, which contains a duplication of the NF-kB site (Dewhurst et al., 1990). Although the duplication of this site was not sufficient to induce the acutely lethal syndrome associated with SIVsmmPBj14 infection (Novembre et al., 1993), viruses with the duplicated NFkB site do replicate more rapidly than isogenic viruses containing a single LTR NF-kB site (Dollard et al., 1994), suggesting that enhanced replication may contribute to SIVsmmPBj14 pathogenesis. Recently, Ilyinskii and Desrosiers (1996) have studied the replication of SIVs containing mutations in the Sp1 and NF-kB binding sites and have shown that SIV is capable of replicating without these elements. We have studied the effects of these LTR elements on SIV replication. In this study, we have systematically mutated the NF-kB or Sp1 binding sites of the LTRs of SIVmac239 and have assessed their replication properties in CD4+ T cell lines and PHAstimulated rhesus macaque PBMCs.
2. Materials and methods In order to study the effects of the SIV LTR NF-kB and Sp1 binding sites, mutations were introduced into these sites in the U3 region of an SIV LTR. Mutated LTRs were reconstructed into full length proviruses by concatemerization of segments of the SIV provirus as illustrated in Fig. 1. In the initial step, a BamHI fragment containing the LTR from the 5% molecular clone of SIVmac239, p239SpSp5% (Kestler et al., 1990) was subdoned into the pLitmus 38 vector. Mutations in the NF-kB and Sp1 binding sites were introduced by oligonucleotide-directed, site-specific mutagenesis. The mutated LTR was reinserted back into p239SpSp5% in place of the wild-type LTR.
J. Zhang et al. / Virus Research 49 (1997) 205–213
207
Fig. 1. Construction of SIV proviral DNAS containing LTR mutations. The structure of the full-length SIVmac239 provirus with 2 LTRs is shown on line A. The structure of the 5%, molecular clone of SIVmac239, p239SpSp5% (Kestler et al., 1990) is shown on line B. A BamHI fragment containing 256 bp of 5% cellular flanking sequence, the 5% LTR and 1332 bp of adjacent gag sequences was purified from p239SpSp5% (Kestler et al., 1990) and subdoned into the pLitmus 38 vector (New England Biolabs) to create pLitmus 5% LTR (shown in line C). pLitmus 5%LTR was subjected to oligonucleotide-directed, site-specific mutagenesis using the Chameleon mutagenesis system (Stratagene). Introduction of the desired mutation was confirmed by DNA sequence analysis and the BamHI fragment containing the mutated LTR was reinserted back into p239SpSp5% in place of the wild-type LTR. The presence of the mutated LTR was reconfirmed by DNA sequence analysis in p239SpSp5% prior to further analysis. In order to assay for infectivity of the SIVs containing the LTR mutations, two fragments of the SIV genome were purified and used for subsequent ligation and transfection. An Nde1–Sph1 fragment of p239SpSp5% from the Nde1 site in the middle of U3 at position 288 to the Sph1 site at 6445 bp contained mutated U3 and wild-type R and US LTR sequences as well as 5% proviral gag and pol, 6if, 6px and 6pr sequences. This fragment was ligated to an Sph1-Nde1 fragment derived from p239SpE3%nef-open (shown in line D) that contained sequences from the Sph1 site at 6445 to the Nde1 site in the U3 region of the 3% LTR (9749 bp) including tat, re6, en6 and nef sequences as well as U3 sequences upstream of the site of the introduced mutations. The ligation mixture, containing concatemerized Nde1–Sph1 and Sph1–Nde1 fragments, would be expected to include reconstituted full length proviruses with two LTRs, both of which contain the introduced mutations (line E).
In order to assay for infectivity of the SIVs containing the LTR mutations, two fragments of the SIV genome were purified and used for subsequent ligation and transfection (Fig. 1). An Nde1–Sph1 fragment of p2395p5p5% that contained the mutated U3 LTR sequence, as well as R and U5 LTR sequences and 5% proviral sequences was ligated to an Sph1 – Nde1 fragment
derived from p239SpE3%nef-open (Kestler et al., 1991), (kind gift of Dr R. Desrosiers), that contains sequences comprising the 3% half of the provirus to the Nde1 site in the U3 region of the 3% LTR upstream of the NF-kB and Sp1 sites. The ligation mixture, containing concatemerized Nde1–Sph1 and Sph1-Nde1 fragments, would be expected to include reconstituted full length
208
J. Zhang et al. / Virus Research 49 (1997) 205–213
Fig. 2. Mutations introduced into the SIVmac239 LTR. The positions of the LTR U3, R and US regions and of the NF-kB and Sp1 binding sites and TAR region are shown at top. The nucleotide sequences of the NF-kB and Sp1 sites (from −103 to −54 bp with respect to the start site of transcription) are shown for the wild-type and mutant LTRs. Deletions are represented by dashes and the point mutations are underlined.
proviruses with two LTRs, both of which contain the introduced mutations (Fig. 1). This ligation mix was transfected into the SW480 colon carcinoma cells as described (Leonard et al., 1989). Two days following transfection, the SW480 cells were co-cultivated with CD4 +, CEMx174 cells permissive for SIV replication and supernatants were monitored for the production of reverse transcriptase (RT) as previously described (Leonard et al., 1989). Virus produced within 2 – 4 days of cocultivation was harvested as viral stock for use in subsequent infections. Because our use of uncloned, ligated DNA fragments in the construction of the mutant viruses raised the remote, but real possibility that recovered viruses may contain contaminating wild-type U3 LTR sequences, we confirmed that the recovered viruses contained the introduced mutations. LTR DNA sequences from the U3 and R regions were amplified by PCR from DNA prepared following a second round of infection of CEMx174 cells with the original virus stocks, and were subjected to DNA sequencing.
The presence of the originally introduced, unaltered LTR mutations was confirmed by DNA sequence analysis of all of the mutant viruses.
3. Results A series of mutations in the SIV LTR NF-kB and Sp1 binding sites were prepared for these studies. The DNA sequence of the SIV LTR in the region of the NF-kB and Sp1 sites is shown in Fig. 2 as are the nucleotide sequences of the mutations used for these studies. Mutations of the NF-kB site included a complete deletion of the NF-kB and overlapping SF3 sites (dlNFkB), and point mutations in bases important for NF-kB binding (NFkBM1) or in nucleotides that define the SF3 binding site (NFkBM2) (Winandy et al., 1992). Mutations of the Sp1 binding sites include a deletion of all three Sp1 sites (dlSp1), as well as point mutations of the 5% most Sp1 site (Sp1 M1), the 5% and middle Sp1 sites (Sp1 M2) and all three
J. Zhang et al. / Virus Research 49 (1997) 205–213
Sp1 sites (Sp1 M3). Mutations in the NF-kB site and the Sp1 sites were designed based upon sequence changes previously demonstrated to disrupt protein binding to these sites (Duh et al., 1989; Jones et al., 1986) and the mutation affecting the SF3 site is predicted to disrupt SF3 binding based upon the results of previously published methylation interference assays (Winandy et al., 1992). Transfection of each of the concatemerized SIV DNAs with the different LTR mutations resulted in recovery of infectious SIV following cocultivation with the CEMx174 cells (data not shown). These results demonstrated that neither the NF-kB sites nor the Sp1 sites were required for replication of SIV in CEMx174 cells and allowed the generation of viral stocks for assays of replication of the mutant viruses in different CD4+ lymphoid cells. The replication of the SIVs containing LTR mutations was compared to that of wild-type SIV in CD4+ T cell lines permissive for SIV infection, as well as in PHA-stimulated primary rhesus peripheral blood mononuclear cells (PBMCs) (Fig. 3). Virus stocks from the wild-type and mutant viruses were normalized for reverse transcriptase activity and equivalent amounts of virus (10 000 cpm) were used in all infections. As the virus stocks were recovered from co-cultivations with CEMx174 cells, we initially assessed replication kinetics of the different mutant viruses in this cell line (Fig. 3A). Cells were infected with equivalent amounts of wild-type and mutant viruses and SIV production was monitored by assay for viral reverse transcriptase production. All 4 Sp1 mutants and 3 NF-kB mutant viruses replicated efficiently in CEMx174 cells, exhibiting no significant difference in kinetics from wild type virus. All mutant viruses induced equivalent levels of SIV-associated cytopathicity as monitored by syncytia formation and cell death, and similar results of virus production and cytotoxicity were seen in triplicate infections (data not shown). We were also interested in the effects of the LTR mutations on SIV replication in other CD4+ T cell lines. In preliminary experiments, we found that SIVmac239 replicated efficiently in the HTLV-1+ , MT4 cell line, but not in Jurkat cells or Sup T cells, two other cell lines highly
209
permissive for HIV replication. We therefore also assayed replication of the LTR mutant viruses in MT4 cells (Fig. 3B). Again, all LTR mutants replicated efficiently without demonstrating significant differences from wild-type virus. SIV infection by either wild-type or mutant viruses resulted in essentially complete killing of the MT4 cells (data not shown). Similar results were seen in duplicate infections. It was important to determine if any of the LTR mutations affected the efficiency of replication of SIV in PHA-stimulated primary rhesus macaque peripheral blood mononuclear cells (Fig. 3C). Previous studies had suggested that mutation of the LTR NF-kB sites did not affect SIV replication in these cells. As expected, neither the NF-kB deletions or point mutations altered the kinetics of SIV replication in these cells. Similarly, even the complete deletion or introduction of point mutations into all three Sp1 binding sites still resulted in efficient production of SIV in the PBMCs. A repeat infection in PHA-stimulated PBMCs derived from a second rhesus macaque also demonstrated replication of all the LTR mutant SIVs (data not shown).
4. Discussion The results of these studies demonstrate that mutations of either the LTR NF-kB or Sp1 binding sites do not prevent efficient replication of SIV in permissive T cell lines or PHA-stimulated primary rhesus PBMCs. In fact, relatively little effect on viral replication could be detected even with complete deletion of either of these sites. This work confirms the previous observations that the NF-kB and Sp1 binding sites are not required for SIV replication in PBMCs (Bellas et al., 1993; Ilyinskii and Desrosiers, 1996) and extends the analysis to include the role of the overlapping SF3 binding site as well as the effects of these mutations in a human CD4+ T cell line that is highly susceptible to HIV replication (MT4 cells). Mutations that interrupt the SF3 binding site overlapping the NF-kB site also did not impair viral replication, suggesting that this binding site, although important for high basal transcription in
210
J. Zhang et al. / Virus Research 49 (1997) 205–213
Fig. 3. Replication of wild-type and LTR mutant SIVs in CD4+ T cell lines and PHA-stimulated rhesus macaque PBMCs. T-cell cultures in 24-well dishes (4×105 cells in 1 ml. per well) were inoculated with equivalent amounts of wild-type and mutant virus stock as determined by reverse transcriptase (RT) activity (10 000 cpm per well). Production of progeny virus was monitored by production of viral RT in the culture supernatants. (A) Infection of CEMx174 cells; (B) infection of MT4 cells and (C) infection of PHA-stimulated rhesus macaque PBMCs. PBMCs were stimulated with phytohemagglutinin (PHA-P, Sigma) for 72 h prior to infection. PBMCs were maintained in RPMI 1640 supplemented with 20% fetal bovine serum (GIBCO-BRL) and 10% recombinant human interleukin 2 (Boehringer Mannheim).
J. Zhang et al. / Virus Research 49 (1997) 205–213
transfection assays (Winandy et al., 1992), also was not required for viral production. Thus, mutations or deletions of the NF-kB sites, the SF3 site and the Sp1 sites failed to markedly impair SIV replication in the cell types studied, demonstrating that these sites were also not essential for sufficient viral transcription to allow productive replication. These observations in the SIV system are similar to those observed for HIV, in which deletion or point mutations of either the NFkB or Sp1 binding sites failed to inhibit HIV replication in either MT4 cells or human PBMCs (Leonard et al., 1989; Parrott et al., 1991; Ross et al., 1991). Interestingly, there have been reports of an absolute requirement for the HIV NF-kB sites for replication of HIV in PHA-stimulated purified human CD4+ cells (Alcami et al., 1995). As our experiments did not utilize purified simian CD4 + T cells, we cannot determine the effects of the SIV LTR NF-kB sites in these cells. The most likely explanation for the ability of SIV to replicate in the absence of either the Sp1 binding sites or the NF-kB sites is the exterisive redundancy of positive acting transcriptional regulatory elements in this promoter. This redundancy may be due to the effects of several different LTR elements. One possibility is that the NF-kB and Sp1 sites may functionally substitute for each other. The cell types studied may contain both activated, nuclear NF-kB and Sp1, either of which would complement the loss of binding to the other site. Similar functional complementation appears to be responsible for the ability of Sp1deleted HIV proviruses to replicate in a variety of human cells (Parrott et al., 1991). In fact, both MT 4 cells and PHA-stimulated T cells have been reported to have induced nuclear NF-kB (Parrott et al., 1991) which could be sufficient to allow transcription and replication of the SIV LTR Sp1 mutants. Although we have not directly determined the NF-kB status of CEMx174 cells, it would not be surprising if they also contained constitutive nuclear. NF-kB activation, as this cell line was derived as a hybrid of CEM cells which lack constitutive nuclear NF-kB (Parrott et al., 1991), with a transformed B cell line (Salter et al., 1985) which is likely to contain nuclear NF-kB,
211
as activation of this factor is a hallmark of B cell maturation (Miyamoto and Verma, 1995). Clearly, in view of our results, the replication potential of an SIV deleted in the NFkB, Sp1 and SF3 binding sites would be important to assess the possible functional redundancy of these elements. In fact such a mutation has been recently reported by Ilyinskii and Desrosiers (Ilyinskii and Desrosiers, 1996), who observed that an SIV deleted in the region spanning both the NF-kB, SF3, and Sp1 sites still can replicate. This observation strongly suggests that other LTR sites participate in supporting viral transcription in place of these sites. Such candidate regulatory sequences include those identified in the LTR mapping between − 162 and − 114, shown to contain both constitutive and inducible enhancer activity (Renjifo et al., 1990) that is responsive to SIV Tat (Ilyinskii and Desrosiers, 1996). This region contains binding sites for proteins designated SF1, SF2, as well as an additional binding site for SF3 (Winandy et al., 1992). Any of these upstream regulatory elements may be sufficient to support SIV replication. Even only relatively low levels of transcription would likely be sufficient to promote viral replication, as the production of low levels of Tat protein and TAR-containing RNAs would allow rapid amplification of SIV transcription through the potent transactivating effects of Tat. An important question raised by these results relates to the possible significance of the LTR NF-kB and Sp1 sites in SIV replication in vivo, particularly in view of their evolutionary conservation in essentially all primate lentiviruses. The presence of these mutations in disease-inducing molecular clones of SIV should allow direct analysis of their contributions to the induction of simian AIDS. The fact that these mutant viruses all replicate efficiently in activated rhesus PBMCs might suggest that they will not play an important role in induction of AIDS. On the other hand, a nef-SIV replicates well in activated PBMCs in culture and yet exhibits profound defects in disease induction (Kestler et al., 1991). An interesting hypothesis with regard to the role of the NF-kB site in the LTRs of AIDS-inducing primate lentiviruses relates to recent observations that immune stimulation may enhance viral repli-
212
J. Zhang et al. / Virus Research 49 (1997) 205–213
cation in vivo (Fultz et al., 1992; Schwiebert and Fultz, 1994; O’Brien et al., 1995; Staprans et al., 1995; Stanley et al., 1996). Immune activation results in NF-kB induction in T cells and monocytes (reviewed in (Miyamoto and Verma, 1995) and thus could be hypothesized to lead to enhanced viral replication in immune-stimulated animals. According to this model, deletion of the LTR NF-kB site might result in a loss of immune activation mediated enhancement of AIDS pathogenesis. The mutant viruses studied here may provide reagents to shed light on this important issue. Another interesting issue relates to the effects that LTR mutations have on the replication of SIV in cells of the monocyte/macrophage lineage. Although not specifically addressed in our studies due to our use of an SIVmac239 viral background which does not infect monocyte/ macrophages efficiently, previous studies have suggested that the NF-kB and Sp1 sites may be more important for SIV replication in monocytes (Bellas et al., 1993; Ilyinskii and Desrosiers, 1996). Thus, these LTR mutant SIVs may also be useful in dissecting the role of monocyte/macrophage infection in the pathogenesis of AIDS in vivo.
Acknowledgements We would like to thank Dr D. Regier and Dr R. Desrosiers for kindly supplying us with the p2395pE3%nef-open plasmid. The following reagents were obtained from the NIH, NIAID AIDS Research and Reference Reagent Program, Division of AIDS, NIAID: p239SPSP5% (contributed by R. Desrosiers), CEMx174 cells (contributed by P. Cresswell). This work was supported by a grant from NIAID, NIH (A130901), by a grant to CABM from the New Jersey Commission on Science and Technology, and by an NCRR, NIH grant to the Yerkes Primate Center (RR-00165).
References Alcami, J., Lain de Lera, T., Folgueira, L., Pedraza, M.-A., Jacque, J.M., Bachelerie, F., Noriega, A.R., Hay, R.T.,
Harrich, D., Gaynor, R.B., Virelizier, J.-L. and ArenzanaSeisdedos, F. (1995) Absolute dependence on kB responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO. J. 14, 1552 – 1560. Anderson, M.G. and Clements, J.E. (1991) Comparison of the transcriptional activity of the long terminal repeats of simian immunodeficiency viruses SIVmac251 and SIVmac239 in T-celI lines and macrophage cell lines. J. Virol. 65, 51 – 60. Antoni, B.A., Stein, S.B. and Rabson, A.B. (1994) Regulation of human immunodeficiency virus infection: implications for pathogenesis. In: K. Maramorosch, F.A. Murphy and A.J. Shatkin (Eds), Advances in Virus Research, Academic Press, New York, Vol. 43, pp. 53 – 145. Bellas, R.E., Hopkins, N. and Li, Y. (1993) The NF-kB binding site is necessary for efficient replication of simian immunodeficiency virus of macaques in primary macrophages but not in T cells in vitro. J. Virol. 67, 2908 – 2913. Clements, J.E. and Payne, S.L. (1994) Molecular basis of the pathobiology of lentiviruses. Virus Res. 32, 97 – 109. Cullen, B.R. (1992) Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56, 375 – 394. Dewhurst, S., Embretson, J.E. Anderson, D.C., Mullins, J.I. and Fultz, P.N. (1990) Sequence analysis and acute pathogenicity of molecularly cloned SIVsmm-pbj14. Nature (London) 345, 636 – 639. Dollard, S.C., Gummuluru, S., Tsang, S., Fultz, P.N. and Dewhurst, S. (1994) Enhanced responsiveness to nuclear factor kB contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14. J. Virol. 68, 7800 – 7809. Duh, E.J., Maury, W.J., Folks, T.M., Fauci, A.S. and Rabson, A.B. (1989) Tumor necrosis factor a activates human immunodeficiency virus-1 through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA 86, 5974 – 5978. Fultz, P.N., Gluckman, J.C., Muchmore, E. and Girard, M. (1992) Transient increases in numbers of infectious cells in an HIV-1 infected chimpanzee following immune stimulation. AIDS Res. Hum. Retraviruses 8, 313 – 317. Gaynor, R. (1992) Cellular transcriptional factors involved in the transcriptional regulation of HIV-1 gene expression. AIDS 6, 347 – 363. Hirsch, V.M. and Johnson, P.R. (1994) Pathogenic diversity of simian immunodeficiency viruses. Virus Res. 32, 183 – 203. Ilyinskii, P.O. and Desrosiers, R.C. (1996). Efficient transcription and replication of simian immunodeficiency virus in the absence of NF-kB and Sp1 binding elements. J. Virol. 70, 3118 – 3126. Jeang, K. and Gatignol, A. (1994). Comparison of regulatory features among primate lentiviruses. Curr. Top. Microbial. Immunol. 188, 123 – 144. Jones, K.A., Kadonaga, J.T., Luciw, P.A. and Tjian, R. (1986). Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. Science. 232, 755 – 759.
J. Zhang et al. / Virus Research 49 (1997) 205–213 Kestler, H., Kodama, T., Ringler, D., Marthas, M., Pedersen, N., Lackner, A., Regier, D., Sehgal, P., Daniel, M., King, N. and Desroslers, R. (1990) Induction of AIDS in Rhesus Monkeys by molecularly cloned simian immunodeficiency virus. Science 248, 1109–1112. Kestler III, H.W., Ringler, D.J., Mon, K., Panicali, D.L., Sehgal, P.K., Daniel, M.D. and Desrosiers, R.C. (1991) Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell 65, 651–662. Kim, J.Y., Gonzalez-Scarano, F., Zeichner, S.L. and Alwine, J.C. (1993) Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the − 201 to − 130 region of the long terminal repeat. J. Virol. 67, 1658 – 1662. Leonard, J., Parrott, C., Buckler-White, A.J., Turner, W., Ross, E.K., Martin, M.A. and Rabson, A.B. (1989) The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. J. Virol. 63, 4919–4924. Letvin, N.L. and King, N.W. (1990) Immunologic and pathologic manifestations of the infection of rhesus monkeys with simian immunodeficiency virus of macaques. J. Acquir. Immune Def. Syndr. 3, 1023–1040. McClure, H.M., Anderson, D.C., Ansari, A.A., Fultz, P.N., Klumpp, S.A. and Schinazi, R.F. (1990) Nonhuman primate models for evaluation of AIDS therapy. Ann. NY Acad. Sci. 616, 287 – 298. Miyamoto, S. and Verma, I.M. (1995) Rel/NF-kB/lkB story. Adv. Cancer Res. 66, 255–292. Novembre, F.J., Johnson, P.R., Lewis, M.G., Anderson, D.C., Klumpp, S., McClure, H.M. and Hirsch, V.M. (1993) Multiple viral determinants contribute to pathogenicity of the acutely lethal simian immunodeficiency virus SIVsmmPBj variant. J. Virol. 67, 2466–2474. O’Brien, W.A., Grovit-Ferbas, K., Namazi, A., Ovcak-Derzic, S., Wang, H.J., Park, J., Yeramlan, C., Mao, S.H. and Zack, J.A. (1995) Human immunodeficiency virus-type 1 replication can be increased in peripheral blood of seropositive patients after influenza vaccination. Blood 86, 1082– 1089.
.
213
Parrott, C., Seidner, T., Duh, E., Leonard, J., Theodore, T.S., Buckler-White, A., Martin, M.A. and Rabson, A.B. (1991) Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T Lymphocytes. J. Virol. 65, 1414 – 1419. Renjifo, B., Speck, N.A., Winandy, S., Hopkins, N. and Li, Y. (1990) cis-acting elements in the U3 region of a simian immunodeficiency virus. J. Virol. 64, 3130 – 3134. Ross, E.K., Buckler-White, A.J., Rabson, A.B., Englund, G. and Martin, M.A. (1991) Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types. J. Virol. 65, 4350 – 4358. Salter, R.D., Howell, D.N. and Cresswell, P. (1985) Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids. Immunogenetics 21, 235 – 246. Schwiebert, R. and Fultz, P.N. (1994) Immune activation and viral burden in acute disease induced by simian immunodeficiency virus SIVsmmPBj14: correlation between in vitro and in vivo events. J. Virol. 68, 5538 – 5547. Simon, M.A., Brodie, S.J., Sasseville, V.G., Chalifoux, L.V., Desrosiers, R. and Ringler, D.J. (1994) Immunopathogenesis of SIVmac. Virus Res. 32, 227 – 251. Stanley, S.K., Ostrowski, M.A., Justement, J.S., Gantt, K., Heydayati, S., Mannix, M., Roche, K., Schwartzentruber, D.J., Fox, C.H. and Fauci, A.S. (1996) Effect of immunization with a common recall antigen on viral expression in patients infected with human immunodeficiency virus type 1. N. Engl. J. Med. 334, 1222 – 1230. Staprans, S.I., Hamilton, B.L., Follansbee, S.E., Elbelk, T., Barbosa, P., Grant, R.M. and Feinberg, M.B. (1995) Activation of virus replication after vaccination of HIV-1-infected individuals. J. Exp. Med. 182, 1727 – 1737. Winandy, S., Renjifo, B., Li, Y. and Hopkins, N. (1992) Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat. J. Virol. 66, 5216 – 5223.