Chimeric Human Immunodeficiency Virus Type 1 - Journal of Virology

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Jul 1, 2003 - 35. Miller, M. D., M. T. Warmerdam, S. S. Ferrell, R. Benitez, and W. C.. Greene. 1997. Intravirion generation of the C-terminal core domain of.
JOURNAL OF VIROLOGY, Feb. 2004, p. 1843–1850 0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.4.1843–1850.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 78, No. 4

Chimeric Human Immunodeficiency Virus Type 1 (HIV-1) Virions Containing HIV-2 or Simian Immunodeficiency Virus Nef Are Resistant to Cyclosporine Treatment Mahfuz Khan, Lingling Jin, Ming Bo Huang, Lesa Miles, Vincent C. Bond, and Michael D. Powell* Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310 Received 1 July 2003/Accepted 20 October 2003

The viral protein Nef and the cellular factor cyclophilin A are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions. In contrast, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate cyclophilin A into virions or need it for full infectivity. Since Nef and cyclophilin A appear to act in similar ways on postentry events, we determined whether chimeric HIV-1 virions that contained either HIV-2 or SIV Nef would have a direct effect on cyclophilin A dependence. Our results show that chimeric HIV-1 virions containing either HIV-2 or SIV Nef are resistant to treatment by cyclosporine and enhance the infectivity of virions with mutations in the cyclophilin A binding loop of Gag. Amino acids at the C terminus of HIV-2 and SIV are necessary for inducing cyclosporine resistance. However, transferring these amino acids to the C terminus of HIV-1 Nef is insufficient to induce cyclosporine resistance in HIV-1. These results suggest that HIV-2 and SIV Nef are able to compensate for the need for cyclophilin A for full infectivity and that amino acids present at the C termini of these proteins are important for this function. isomerase activity of the protein appears to be dispensable for the enhancement of infectivity (39). Therefore, if these two proteins influence the disassembly process, they must do so by some means other than directly affecting core stability. It has also been shown that there is some mechanistic independence in the actions of the two proteins on enhancement of infectivity (3). In addition, Nef has been found to be associated with the ribonucleoprotein complex following disassembly (15). All of these observations leave doubts as to the exact mechanism of action of Nef and CyPA in disassembly and how or whether they are independent in their action. In contrast to HIV-1, HIV-2 and simian immunodeficiency virus (SIV) do not incorporate CyPA into viral particles or appear to need it for full infectivity (7, 16, 44). This appears to be due to differences in the sequence of the Gag polyprotein which result in disruption of its interactions with CyPA and prevent the incorporation of CyPA into particles (16, 31). This renders the viruses insensitive to treatment by the immunosuppressive drug cyclosporine (CsA) (7). It has been shown that chimeric HIV-1 virions that contain a small portion of SIV Gag can replicate in a CyPA-independent fashion (18). Although differences in the Gag polyprotein help explain why CyPA is not incorporated into HIV-2 and SIV virions, the reason that these viruses do not require CyPA for full infectivity remains unclear. In addition to differences in the CA proteins of HIV-2 and SIV there are also differences in the Nef proteins (38). The Nef proteins from HIV-2 (7312A) and SIVmac239 contain an additional 20 and 27 amino acids, respectively. Both the SIV and HIV-1 Nef proteins can enhance infectivity in similar ways and appear to be functionally interchangeable (40). When amino acid alignments between HIV-1 Nef and SIV or HIV-2 Nef were compared, it was found that some of the additional amino

The human protein cyclophilin A (CyPA) and the viral protein Nef are both required for full infectivity of human immunodeficiency virus type 1 (HIV-1) virions (10, 12, 16, 20, 24, 34, 35, 42, 44). CyPA-depleted virions and virions with nef deleted exhibit similar 5- to 20-fold reductions in infectivity compared to wild-type (WT) virions (7, 9, 12, 16, 36). Both proteins are present in the intact virion (13, 16, 37). Nef has been shown to be associated with the viral core (29); however, CyPA is not present in immunoblots of purified HIV-1 cores (48). Each protein must be present at the time of particle assembly to enhance virion infectivity (4, 16, 36). These features suggest that both proteins need to be present in the infecting viral particle to exert their effect. Pseudotyping virions with nef deleted or CyPA-depleted virions with the envelope glycoprotein from vesicular stomatitis virus targets entry to the endosomal compartment and restores infectivity (2). Likewise, treatment of CyPA-depleted virions and virions with nef deleted to induce natural endogenous reverse transcription induces premature disassembly of the viral core (14, 50–52) and restores infectivity to WT levels (25, 26). These features suggest that both of these proteins are dispensable when the viral core is allowed to disassemble through alternate means. Because of the similarity of their actions, it appears that both proteins are involved in some common aspect of early entry, such as core disassembly. However, neither protein appears to directly affect the stability of the viral core (15, 47), and, although CyPA is a cis-trans prolyl isomerase (19, 21) and can induce changes in the structure of the mature core (1, 8), the

* Corresponding author. Mailing address: Morehouse School of Medicine, Dept. of Microbiology, Biochemistry, Immunology, 720 Westview Dr. SW, Atlanta, GA 30310. Phone: (404) 752-1582. Fax: (404) 752-1179. E-mail: [email protected]. 1843

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acids present in HIV-2 and SIV are located at the C terminus. The C-terminal regions of both HIV-2 and SIV have been implicated in increased pathogenicity (30, 43). In addition, when the additional C-terminal amino acids from SIV Nef are added to the C terminus of HIV-1 Nef to create a chimeric HIV-1/SIV (SHIV) Nef protein, a SHIV with replication kinetics very similar to that of WT SIV is created (6). To help determine if there is a direct relationship between Nef and CyPA, we created HIV-1 chimeric viruses that lack Nef and that have been trans-complemented with either HIV-2 or SIV Nef. HIV-1 virions that contain either HIV-2 or SIV Nef became resistant to treatment by CsA, indicating they were now able to replicate in a CyPA-independent fashion. These studies provide the first evidence of a direct link between the Nef protein and CyPA dependence. MATERIALS AND METHODS Plasmid and viral constructs. Vectors for the eukaryotic expression of HIV-1, HIV-2, and SIV Nef were produced by amplifying the Nef coding regions from pNL4-3 (nucleotides [nt] 8743 to 9632), HIV7312A (nt 8896 to 10085), and SIVMM239 (nt 9137 to 10414), respectively. The following primer pairs were used: HIV-1 forward, 5⬘-CCT AGA AGA ATA AGA CAG GGC, and reverse, 5⬘-CAC TAC TTG AAG CAC TCA AGG C; HIV-2 forward, 5⬘-GAA GAA GGA GGT GGA AAC GAC G, and reverse, 5⬘-AAG TGC TGG TGA GAG TCT AGC; SIV forward, 5⬘-GCT CCT GGC CTT GGC AGA TAG, and reverse, 5⬘-GCT TAC TTC TAA AAT GGC AGC. The resultant PCR products were cloned into the pcDNA3.1/V5-His topo vector (Invitrogen) according to the manufacturer’s instructions. Plasmids containing chimeric HIV-1/HIV-2 or HIV1/SIV Nef reading frames were made by using the flanking primers described above; however, internal primers located at the conserved PPT region were used to amplify either the C-terminal or N-terminal half of each Nef coding region separately (see Fig. 5). The two halves were then used for mutually primed synthesis to complete the reading frame. The sequences of the internal primers were as follows: forward, 5⬘-AGA AAA GGG GGG ACT GGA AGG G; reverse, 5⬘-CCC TTC CAG TCC CCC CTT TTC T. In this manner we could construct a Nef protein containing the N terminus from HIV-1 and the C terminus of HIV-2 or SIV as well as Nef containing the C terminus from HIV-1 and the N terminus from HIV-2 or SIV. Truncations of the C terminus of HIV-2 Nef and SIV Nef were made by using the following primer pairs: HIV-2 forward, 5⬘-GAA GAA GGA GGT GGA AAC GAC G, and reverse, 5⬘-TCA CTA CTG ATA CCC AAA CTC; SIV forward, 5⬘-GCT CCT GGC CTT GGC AGA TAG, and reverse, 5⬘-TCA CTA CTT GCT TCC AAA CTC. Each reverse primer introduced premature stop codons to remove the additional amino acids at the C terminus of HIV-2 and SIV Nef (see Fig. 4). Addition of the C-terminal amino acids of HIV-2 Nef to the C terminus of HIV-1 was done in two steps using the following primers: forward, 5⬘-CCTAGA AGA ATA AGA CAG GGC; first reverse, 5⬘-TAG TCT AGC CTT CCA TTC CTT CTC TGG TAA TCC TGA GCA GTT CTT GAA GTA CTC; second reverse, 5⬘-CTA CTC TGT AGG TAT GCC TCT TGC TTT TAG TCT AGC CTT CCA TTC. The resultant PCR products were cloned into pcDNA3.1/V5-His as described for the WT proteins. Viruses were produced and used in a single-round infectivity assay as previously described (25, 26). Basically, the viral clone pNL4-3 was mutated to delete the env gene by insertion of KpnI linkers into the env reading frame (gift from Eric Freed, National Institutes of Health [NIH]) (17), and the resultant plasmid was designated pNL4-3KFS. A further mutation was made by insertion of tandem stop codons into the nef gene (gift from Judith Levin, NIH), and the resultant plasmid was designated pNL4-3KFS⌬Nef. To produce infectious NL43KFS (WT) and NL4-3KFS⌬Nef (Nef⫺) virus stocks, HEK 293 cells were cotransfected with either the pNL4-3KFS or the pNL4-3KFS⌬Nef plasmid and the envelope plasmid pIIIenv3-1 (gift from Eric Freed (41). Chimeric viruses were produced by a three-way transfection using pNL4-3KFS⌬Nef, pIIIenv3-1, and each of the Nef expression plasmids separately. This allowed us to produce NL4-3 virus stocks containing HIV-1, HIV-2, or SIV Nef. To test for trans dominance of the Nef phenotype, we also made viral stocks using pNL4-3KFS in the three-way transfection described above. These viruses contained both HIV-1 Nef and HIV-2 or SIV Nef. In some cases virions were produced in the presence of 10 ␮M CsA as previously described (25, 26) to block incorporation of CyPA. As an alternative means to produce CyPA-depleted virions, we modified pNL43KFS viral clones to encode mutations (G221A and P222A) in the CyPA binding

J. VIROL. loop of Gag as previously described (26). Since the original mutants have an intact nef reading frame, we replaced the BamHI-NcoI fragments of the G221A and P222A mutants with the same fragment from pNL4-3KFS⌬Nef. This created clones that have the G221A and P222A mutations in a nef-deleted background. Immunoblot analysis of Nef expression. To insure that all Nef constructs could express similar amounts of Nef, we performed immunoblot analysis on cells transfected with the Nef expression plasmids. First, 107 HEK 293 cells were transfected with 6 ␮g of plasmid DNA by using Effectene transfection reagent (Qiagen) according to the manufacturer’s recommendations. The cells were harvested, pelleted by centrifugation at 800 ⫻ g for 10 min, and washed three times in phosphate-buffered saline. The cell pellet was then disrupted in sodium dodecyl sulfate (SDS) loading buffer and heated to 100°C for 5 min. One microgram of each lysate was then resolved by SDS–4 to 20% polyacrylamide gel electrophoresis (PAGE; Bio-Rad). The Nef proteins were detected with a 1:4,000 dilution of an anti-HIV-1 Nef antibody (AIDS Research and Reference Reagent Program catalog no. 2949) and ECL detection (Amersham). As a loading control the blot was stripped and reprobed with a mouse monoclonal antibody against tubulin. Under these conditions the HIV-1 Nef antibody crossreacts with both HIV-2 and SIV Nef proteins. To determine the relative amounts of Nef or CyPA incorporated into virions, we performed immunoblotting on concentrated particles. In each case, virions were harvested from 10 ml of supernatant produced by transfection by centrifugation at 26,000 ⫻ g for 1 h. Each pellet contained approximately 250 ng of p24 as determined by enzyme-linked immunosorbent assay. Fifty nanograms (p24 equivalents) of virus was mixed with SDS loading buffer and heated to 100°C. Proteins were resolved on an SDS–12.5% PAGE gel and probed with either an anti-Nef antibody at a dilution of 1:4,000 (AIDS Research and Reference Reagent Program catalog no. 2949) or anti-CyPA antibody at a dilution of 1:2,000 (Upstate Biotechnology) and detected with ECL (Pierce) according to the manufacturer’s directions. As a loading control the blots were stripped and reprobed with an antibody against HIV-1 p24 at a dilution of 1:5,000 (AIDS Research and Reference Reagent Program catalog no. 6457). To resolve HIV-2 and SIV Nef from HIV-1 Nef, we increased the gel concentration to 15%. Single-round infection assay. The relative infectivity of viral particles was determined by the multinuclear activation of a galactosidase indicator (MAGI) assay (27). HeLa-CD4⫹-LTR-␤gal cells (AIDS Research and Reference Reagent Program catalog no. 3580) were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 0.1 mg of G418/ml, 0.05 mg of hygromycin B/ml, L-glutamine, and 100 U of penicillin–100 ␮g of streptomycin/ml. Assays were done in 24-well plates seeded with 2 ⫻ 104 cells per well the day before the assay. Each well was infected with pseudotyped virus at a concentration of 1 ng of p24/ml and incubated for 48 h at 37°C. Cells were then fixed with 0.2% glutaraldehyde–1% formaldehyde for 5 min at room temperature. The cells were then washed twice with phosphate-buffered saline and stained in X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) solution (0.4 mg of X-Gal/ml dissolved in dimethylformamide–4 mM potassium ferricyanide–4 mM potassium ferrocyanide–2 mM MgCl2 in phosphate-buffered saline). The staining was allowed to continue for 50 min at 37°C, and then cells were washed twice with phosphate-buffered saline. Results were scored as total number of blue cells per nanogram of p24 equivalent of virus added. Alignment of HIV-1, HIV-2, and SIV sequences. Nef protein sequences for HIV-1 (NL4-3), HIV-2 (7312A), and SIV (SMM239) were downloaded from the Entrez protein database. The sequences were aligned with Omiga, version 2.0 (Oxford Molecular), by using the Clustal W alignment algorithm.

RESULTS Expression of Nef constructs. To ensure that all of our Nef plasmids expressed equivalent amounts of protein, we tested cells transfected with the same amount of plasmid DNA. The cells were then washed, and the pellets were lysed and analyzed by Western blotting. The results are shown in Fig. 1A. Each of the plasmid constructs expressed a single protein with a molecular weight similar to that of the recombinant HIV-1 Nef protein (which is His tagged). Supernatants from the same cells also showed equivalent amounts of each Nef (data not shown). To determine if equivalent amounts of Nef were incorporated into viral particles, we also performed Western blotting on virions produced by cotransfection of pNL4-3⌬nef and each of the expression plasmids. Viral particles were har-

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FIG. 1. Immunoblot of Nef expression and incorporation into virions. Eukaryotic expression vectors were constructed to express the HIV-1, HIV-2, or SIV Nef protein. (A) Each expression vector was transfected into 293 cells and allowed to express for 48 h. Cells were then harvested, lysed, and separated on an SDS-PAGE gel. The gel was then blotted, and the Nef protein was detected with an anti-Nef antibody. An antitublin antibody was included as a loading control. (B) The incorporation of each Nef protein into particles was tested by harvesting 50 ng (p24 equivalent) of particles and lysing and separating them on an SDS-PAGE gel. The gel was then blotted, and Nef was detected by using an anti-Nef antibody. The blot was also probed with an anti-p24 antibody as a loading control.

vested and normalized by p24 content before loading. The results are shown in Fig. 1B. All of the particle preparations contained equivalent amounts of Nef (note that HIV-2 and SIV Nef proteins exhibit slightly slower migration). This confirmed that the HIV-2 and SIV Nef proteins could be incorporated into HIV-1 virions at levels equivalent to WT levels. Infectivity of chimeric virions. As has previously been shown (12, 25, 26, 36) virus with nef deleted was approximately fivefold less infectious than NL4-3KFS (WT; Fig. 2A). We used each of the Nef expression vectors to trans-complement NL43KFS⌬Nef (Nef⫺) with HIV-1, HIV-2, or SIV Nef. We then tested each virus preparation by MAGI infectivity assay. Each of the virus preparations had infectivity similar to that of WT virus, confirming that each Nef protein was able to rescue infectivity (Fig. 2A). This confirmed that each Nef protein was fully functional for enhancement of infectivity and that all proteins had roughly equivalent ability to enhance infectivity. When virus was produced in the presence of CsA, the virions containing HIV-1 Nef had approximately a 20-fold decrease in infectivity while the virions containing HIV-2 or SIV Nef were essentially the same as the untreated WT control. This demonstrated that virus which contained HIV-1 Nef was CyPA dependent while the chimeric viruses now replicated in a CyPA-independent fashion. Immunoblot analysis confirmed that virions produced in the presence of CsA contained little or no CyPA (Fig. 2B).

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Another means to deplete virions of CyPA is to mutate residues in the CyPA binding loop of Gag (16). We produced chimeric HIV-1 virions with either G221A or P222A mutations in Gag. These virions fail to incorporate CyPA, as demonstrated by immunoblots of the particles (Fig. 3B). When these virions were tested for infectivity by MAGI assay, it was found that virions that contained HIV-1 Nef had the same infectivity as a control with nef deleted (Fig. 3A). However, virions that contained HIV-2 or SIV Nef showed a two- to threefold increase in infectivity. Neither mutant was restored to WT levels of infectivity. This might be expected if the HIV-2 and SIV proteins can directly interact with the capsid as we have proposed. The changes in capsid could directly affect this interaction and could result in modulation of the effect. However, it is clear that HIV-1 virions that contained HIV-2 or SIV Nef had augmented infectivity in the absence of CyPA, similar to our results for CsA-treated virions. To determine if the CsA resistance phenotype was dominant, we trans-complemented NL4-3KFS, which has an intact nef gene, with HIV-1, HIV-2, and SIV Nef expression plasmids. This allowed us to produce virions containing both HIV-1 Nef and either HIV-2 Nef or SIV Nef. The results are summarized in Fig. 4. When particles were produced in the absence of CsA, each of the trans-complemented viruses showed a modest (approximately 20%) increase in infectivity over WT (Fig. 4A). This was consistent with previous observations that overexpression of Nef can increase infectivity (12, 20, 24, 34, 36). When particles were produced in the presence of CsA, the virions containing only HIV-1 Nef were sensitive to CsA (Fig. 4A). However, the virions containing either HIV-2 or SIV Nef in addition to HIV-1 Nef were partially resistant to CsA (Fig. 4A). These virions had about 30% of the infectivity

FIG. 2. Infectivity of chimeric virions. (A) Equal amounts of virus, as determined with p24, were used to infect P4 MAGI cells. The particles used for the infection were produced either in the presence or absence of CsA (10 ␮M). Each measurement was made in triplicate, and the results shown are from two independent experiments. (B) Virions were tested to confirm the absence of CyPA by Western blotting using an anti-CyPA antibody (ab). The number of virions was normalized by p24 antigen content.

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FIG. 3. The effect of HIV-2 and SIV Nef on G221A and P222A capsid mutants. HIV-1 virions that contained mutations in the CyPA binding loop (G221A and P222A) were trans-complemented with HIV-1, HIV-2, and SIV Nef proteins. (A) The resulting virions were tested for infectivity by the MAGI cell assay. (B) Virions were tested for the presence of CyPA by Western blotting and normalized by p24 antigen content. The results shown are done in triplicate and are representative of two independent experiments. ab, antibody.

of virions containing HIV-2 or SIV Nef alone (compare Fig. 2A and 4A). Curiously, the increase in resistance was similar to the increase in infectivity over that of the WT in the absence of CsA treatment (about 400 cells in each case). Immunoblot analysis of these virions revealed that under these conditions HIV-2 and SIV Nef proteins were present at levels less than 5% of that of HIV-1 Nef (Fig. 4B). This could explain the incomplete rescue of CsA resistance. Immunoblotting using anti-SIV Nef antiserum confirmed the presence of SIV Nef in the SIV/HIV-1 Nef-containing particles (data not shown). Also note that, when both HIV-1 Nef and HIV-2 or SIV Nef were present in the virions, the lower band (which most likely represents Nef cleaved by viral protease) was no longer present (Fig. 4B). We have no explanation for this phenomenon. Mutational analysis of the CsA resistance phenotype. A Clustal alignment of the protein sequences of HIV-1, HIV-2, and SIV Nefs is shown in Fig. 5. Note that HIV-2 and SIV Nefs contain additional amino acids compared to HIV-1 Nef. These additional amino acids are clustered in two regions highlighted in Fig. 5. Since one of the regions is in the N-terminal half of the protein and the other region is at the C-terminal end, we created chimeric Nef molecules in which the N- and C-terminal halves of HIV-1 Nef were mixed with those of HIV-2 and SIV Nef (Fig. 6A). We then used plasmids coding for these chimeric Nef proteins to trans-complement NL4-3⌬nef to produce virions. We tested the infectivity of virions containing each of these chimeric Nefs in the presence and absence of CsA (Fig. 6B). Virions produced in the absence of CsA had essentially the same infectivity, which was approximately 50%

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FIG. 4. Determination of trans-dominance of HIV-2 and SIV Nefs. (A) Infectivity was measured in the same manner as for Fig. 2, but instead of trans-complementing an NL4-3 strain with nef deleted, we trans-complemented a nef-containing strain of NL4-3. In this manner we could produce particles containing both HIV-1 Nef and either more HIV-1 Nef, HIV-2 Nef, or SIV Nef. Each measurement was made in triplicate, and the results shown are from two independent experiments. (B) The relative levels of HIV-1, HIV-2, and SIV Nef were determined by immunoblotting using anti-HIV-1 Nef antiserum. The lower band in the HIV-1 preparations is most likely HIV-1 Nef, which is processed by HIV-1 protease.

of WT. This suggested that none of the chimeric Nefs was fully functional in restoration of infectivity. An immunoblot of the virions revealed that the chimeric Nef proteins were present at approximately 26 to 50% of the amount of WT in virions (Fig. 6C). This could explain the lower infectivity of the viral particles. When virions were produced in the presence of CsA, all

FIG. 5. Clustal alignment of HIV-1, HIV-2, and SIV Nef proteins. The amino acid sequences of HIV-1 Nef (NL4-3), HIV-2 Nef (HIV7312A), and SIV Nef (SMM239) were aligned with the Clustal W algorithm in the Omiga, version 2.0, program (Oxford). Areas of identity are shaded, and the sections of HIV-2 and SIV that contain additional amino acids are boxed.

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FIG. 6. N- and C-terminal chimeras of Nef. PCR was used to fuse the N terminus of HIV-1 Nef to the C terminus of HIV-2 or SIV Nef. Likewise, the N terminus of HIV-2 or SIV Nef was fused to the C terminus of HIV-1 Nef. (A) Schematic of the PCR procedure. The procedure takes advantage of the fact that the PPT sequence is highly conserved in each virus, allowing the same internal primers to be used to create each clone. (B) P4 MAGI cell assays were done to test the infectivity of each chimera. Virions used for the infection were normalized by p24 content, and particles were produced in the presence and absence of CsA (10 ␮M). Each measurement was done in triplicate, and the results shown are from two independent experiments. (C) Immunoblot analysis was performed on virions to determine the relative amount of each chimeric Nef present. Loading was normalized by p24 antigen concentration.

of the chimeric Nef virions were sensitive (Fig. 6B). The chimeras which contained the N-terminal portion from HIV-1 and the C-terminal portion from HIV-2 or SIV showed a slight increase in infectivity. This suggests that the C-terminal portion of the HIV-2 and SIV had a slight effect on CsA resistance. Since the analysis of chimeric Nef proteins was equivocal, we made less-drastic changes in HIV-1, HIV-2, and SIV Nef proteins to help determine the region(s) of Nef that is important for inducing CsA resistance. To accomplish this, we made both loss-of-function and gain-of-function mutations. To check for loss of CsA resistance, we mutated both HIV-2 and SIV Nef proteins to remove the additional amino acids at the C terminus of each protein (Fig. 5) to create proteins with C termini like that of the HIV-1 Nef. To test for gain of function, we added the additional amino acids from HIV-2 onto the C terminus of HIV-1 Nef. Each of these constructs was then used to trans-complement NL4-3⌬nef to produce virions. Each of the mutated Nef proteins was able to restore the infectivity of the strain with nef deleted to nearly WT levels (Fig. 7A). This indicated that the proteins were functional for enhancement of infectivity. An immunoblot of the virions containing the mutated Nef proteins revealed that each of the mutants contained approximately 50% as much Nef as WT (Fig. 7B). This could explain the small reduction in the ability to enhance infectivity. All of the mutated viruses were susceptible to treatment by CsA (Fig. 7A). The HIV-2 and SIV Nef-containing virions lost the ability to induce CsA resistance (compare to Fig. 2A). This suggests that the C terminus of HIV-2 and SIV Nef is important for induction of CsA resistance. However, the addition of the C terminus of HIV-2 Nef to HIV-1 Nef was not sufficient to induce CsA resistance to virions containing this chimeric Nef (Fig. 7A). This demonstrates that, while the C terminus is

necessary to induce CsA resistance in HIV-2 and SIV Nef proteins, it is not sufficient to transfer resistance to HIV-1 Nef. DISCUSSION The results of experiments presented here demonstrate that changes in Nef protein can alter the CyPA dependence of HIV-1. This suggests that the two proteins might either directly interact or interact through a common factor. We have previously proposed that Nef and CyPA could form all or part of a structure called the core envelope linkage (CEL) (25, 26). The CEL is a physical linkage between the small end of the core and the viral envelope (23). If the Nef protein is anchored in the viral envelope via its N-terminal myristylation and CyPA is attached to the core through an interaction with the capsid protein, then the two proteins could form all or part of a linkage between the core and the viral envelope. The CEL could function to keep the core localized to the cell membrane until disassembly has occurred. This could facilitate the attachment of the reverse transcription complex to the microtubule network for transport to the nucleus, as has been proposed (33). The simplest attachment would involve a direct interaction between Nef and CyPA to form the linkage. If HIV-2 and SIV Nef proteins were somehow able to directly interact with CA in the core, they could theoretically form a linkage without the need for CyPA, and this could explain the loss of CyPA dependence seen in this study. Of course, this model predicts that HIV-1 Nef and CyPA should be able to directly (or indirectly) interact and that HIV-2 or SIV Nef should be able to interact with CA in the core. We are currently attempting to determine whether such interactions can actually occur. However, such determinations are complicated in that it is difficult to recreate conditions that are likely to occur inside of virions.

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FIG. 7. Effect of the C-terminal amino acids on CsA sensitivity. The additional amino acids present in the C terminus of HIV-2 and SIV Nef were deleted (HIV-C-term and SIV-C-term), and the Cterminal amino acids of HIV-2 Nef were added to the C terminus of HIV-1 (HIV-1 ⫹ C-term) by PCR mutagenesis. (A) Each mutant was tested for infectivity by a P4 MAGI assay. Particles were normalized by p24 content and were produced either in the presence or absence of CsA (10 ␮M). Each measurement was made in triplicate, and the results shown are from two independent experiments. (B) Immunoblot analysis was performed to determine the relative amount of each mutated Nef protein in each virion preparation. Loading was normalized by p24 antigen content.

For example, binding to purified CA could be different from binding to CA in the mature core. The proposed model would also need to be reconciled with some of the previous findings about Nef and CyPA. For example, it has been reported that purified cores do not appear to contain significant amounts of CyPA (48). CyPA binding to the core is a prerequisite in the proposed model. One possibility is that during the process of isolating cores (which entails treatment with detergent) the CyPA present on the surface is removed. Another possibility is that it may take very little CyPA to form a CEL. The CEL appears to only occur at the small end of the core (23). This could be a result of the unusual conical shape of the HIV-1 core. This could restrict the binding of CyPA to the small end of the core and could result in cores that contain levels of CyPA that are not easily detectable by immunoblotting. Another observation to be reconciled is that Nef has been shown to be associated with the ribonuclear complex that remains after disassembly (15). This suggests a role for Nef following core disassembly, possibly involving reverse transcription. However, we have already seen that Nef performs multiple functions in the viral life cycle. It is entirely

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possible that Nef could function at more that one step in the uncoating process. One of the more intriguing aspects of CyPA dependence is the observation that the transfer of residues 86 to 93 of HIV-1 CA to SIVmac239 confers CsA sensitivity and that the transfer of residues 86 to 90 actually induces CsA dependence in SIV virions (11). In the context of our proposed model one would predict that changes in both Nef and CA could affect CyPA dependence. If we presume that SIV Nef could normally directly interact with HIV-1 or SIV CA to form a CEL, then changes in CA could affect this interaction. It is possible that the transfer of eight amino acids from HIV-1 CA to SIV might disrupt the ability of SIV Nef to directly bind to the core. However, this region is sufficient to allow the binding of CyPA. We show that SIV Nef that lacks its normal C terminus can still enhance infectivity in a CyPA-dependent fashion (Fig. 7A). This may mean that SIV and HIV-2 Nef proteins retain the ability to bind CyPA but in its absence can directly bind to CA via its additional C-terminal residues. In this case, if the SIV Nef were unable to directly bind to the core, it may revert to CyPA dependence as reported. It is possible that, when the substitution is made with only five amino acids from HIV-1, the site for direct binding could be restored. At the same time the CyPA could be bound in a configuration that no longer allows it to bind to Nef. In this case the bound CyPA could actually prevent the formation of a CEL, which could result in CsA dependence. Perhaps an even more intriguing situation is that of HIV-1 group O strains. Group O viruses can incorporate CyPA into virions but do not appear to require it for full infectivity (10, 46). The Nef proteins from group O isolates do not contain additional amino acids at the C terminus like HIV-2 or SIV Nef. While there are some differences in the CyPA binding region in group O viruses, it was found that three of the four group O isolates tested were sensitive to CsA treatment while all four isolates had conserved CyPA binding loops (46). There were, however, changes in CA outside of the CyPA binding region. This suggests that regions outside of the CyPA binding region can influence CyPA dependence. In the context of our proposed model this could be explained if the other changes in CA would allow an HIV-1 Nef to directly bind to CA in the core. Humans and nonhuman primates are known to express inhibitors that can confer resistance to retroviruses (22). Recently it has been shown that HIV-1 sensitivity to restriction factors such as Ref-1 is modulated by CyPA (45). In addition, a single amino acid change in CA (G89V) which alters CyPA binding increases the infectivity of HIV-1 in owl monkey kidney cells by 2 orders of magnitude (45). It seems possible that restriction factors such as Ref-1 could also be involved in some aspect of the uncoating process. However, more data will need to be gathered about both Ref-1 and the normal process of disassembly to determine if such a relationship exists. Several groups have replaced the nef gene of SIV with the nef gene from HIV-1 (5, 28, 32). In two of these studies the SIV containing HIV-1 nef showed delayed replication kinetics when used to infect CEM cells (5, 28). In the third study, the replication kinetics of an SIV containing the HIV-1 nef was actually accelerated when the virus was used to infect macaque peripheral blood mononuclear cells (32). In all of these studies

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animals infected with the chimeric viruses generally had lower viral loads and those few animals that exhibited higher viral loads were associated with mutations in the nef reading frame to increase expression. In general, the results from these studies suggest that SIV containing the HIV-1 nef gene replicated less efficiently and caused lower viral loads in infected animals. In light of the results presented in this study, it might be expected that an SIV which would not incorporate CyPA could be adversely affected by the substitution of the HIV-1 nef gene, which cannot compensate for the lack of CyPA. Chimeric viruses where the nef gene of HIV-1 has been replaced by nef from SIV have also been constructed (49). Unfortunately, in this study only one comparison between WT HIV-1 and a chimeric HIV-1 containing the nef gene from SIV is directly made. In this case both viruses were used to infect CEM cells and the replication kinetics of the WT virus appeared to be modestly accelerated compared to that of the SIV nef-containing chimera. However, the difference was not great and the process of cloning SIV nef into HIV-1 entails an addition of some 550 bp into the HIV-1 genome, which could complicate the comparison of kinetics. In another study HIV-1 Nef containing the additional Cterminal amino acids from SIV Nef was placed in an SIV background (6). The recombinant viruses replicated with essentially the same kinetics in both CEM and macaque peripheral blood mononuclear cells as WT SIV. From the results presented in this study this would not be surprising, given that the addition of the C-terminal amino acids from HIV-2 was not sufficient to induce CsA resistance to HIV-1 in single-round infectivity assays (Fig. 7A). There is evidence to support a role for the C terminus of SIV in increased pathogenicity (30). The clone SIVmacBK28-41 is attenuated. In this study 12 macaques presented various patterns of infection. A prevalent mutation was the extension of the C terminus to contain amino acids similar to those found at the C terminus of SIVmac239. These animals went on to develop disease, whereas the animals with the original strain were nonprogressors. This highlights the importance of the C terminus in pathogenicity. Notably, when a similar mutation was made, the chimeric viruses in this study became CyPA dependent. This study demonstrates that changes in Nef protein can influence CyPA dependence. We have proposed a model in which Nef and CyPA could form all or part of the CEL to account for these observations. However, there remains insufficient information to actually determine how Nef and CyPA act to enhance infectivity. More information will need to be obtained before a complete picture of disassembly can be determined. ACKNOWLEDGMENTS This work was supported by NIH grants G12-RR03034 and S06GM08248. We are grateful to E. Freed and J. Levin (NIH, Bethesda, Md.) for supplying the KFS and KFS⌬Nef plasmids.

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