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Journal of Virology, March 2005, p. 3211-3216, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.3211-3216.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Chimeric Human Immunodeficiency Virus Type 1 Virions That Contain the Simian Immunodeficiency Virus nef Gene Are Cyclosporin A Resistant

Mahfuz Khan, Lingling Jin, Lesa Miles, Vincent C. Bond, and Michael D. Powell^*

Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, Georgia

Received 13 July 2004/ Accepted 15 October 2004

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We have previously shown that human immunodeficiency virus type 1 (HIV-1) virions which have their own nef gene deleted and are trans complemented to contain HIV-2 or simian immunodeficiency virus (SIV) Nef become resistant to treatment with cyclosporin A. To expand and confirm these studies, we have tested an HIV-1 isolate in which the HIV-1 nef gene has been replaced by the nef gene from SIV in a multiround infectivity assay using more physiologically relevant cell types. Our results confirm that HIV-1 virions that contain SIV nef can replicate in a cyclophilin-independent fashion.

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Human immunodeficiency virus type 1 (HIV-1) virions require the incorporation of the host protein cyclophilin A (CyPA) for full infectivity (7, 18, 31). CyPA is incorporated into HIV-1 virions through interactions with the capsid (CA) region of the Gag polyprotein (12, 18), and this interaction can be blocked by the immunosuppressive drug cyclosporin A (CsA) (8, 10, 17). Although CyPA may act on more than one step in replication (27, 32), it clearly has an effect on the accumulation of proviral DNA (7), which suggests that its action is at a step preceding or concurrent with the initiation of reverse transcription. The requirement for CyPA for full infectivity can be eliminated by pseudotyping virions with the envelope glycoprotein from vesicular stomatitis virus, which retargets entry to the endocytic compartment (1). The requirement for CyPA can also be eliminated by treating virions to induce natural endogenous reverse transcription (NERT) prior to infection (20). Since NERT treatment results in partial disassembly of the viral core, this may be an indication of a role for CyPA during disassembly. More recently, CyPA has also been shown to modulate the sensitivity of HIV-1 virus to host restriction factors (32). In human cells, the presence of CyPA appears to modulate restriction by Ref-1. The current evidence suggests that restriction factors bind directly to CA, and this interaction may be blocked by binding CyPA (30, 32).

Many of the effects of CyPA on replication are mirrored by the viral protein Nef. For example, both Nef and CyPA are virion proteins (18, 23). Full infectivity of nef deletion virions can be restored by trans complementation in the viral producer cells but not the target cells (3, 18, 25). This suggests that Nef must be present in the virion to have an effect on infectivity. Likewise, virions produced in the presence of CsA have reduced infectivity (7, 9), suggesting that virion CyPA is important for enhancement of infectivity. However, CyPA has recently been shown to modulate the effect of host restriction factors on infectivity in target cells (32). Along similar lines, full infectivity of both nef deletion and CyPA-depleted virions can be restored by treatment to induce NERT (19, 20) or by pseudotyping virions with the envelope glycoprotein from vesicular stomatitis virus (1). Thus, it appears that both Nef and CyPA could act at the level of disassembly to enhance virion infectivity. It has been previously reported that neither protein appears to directly affect the stability of isolated cores (33, 16). Therefore, if Nef or CyPA plays a role in the disassembly process, it must do so without directly affecting core stability.

We have previously demonstrated that chimeric HIV-1 virions that have their own Nef replaced by HIV-2 or simian immunodeficiency virus (SIV) Nef in trans are no longer CyPA dependent for full infectivity (21). This activity is dependent on amino acids that are present at the C terminus of HIV-2 or SIV Nef proteins which are not present in HIV-1 Nef (21). We have speculated that these additional amino acids may compensate for the lack of CyPA in HIV-2 and SIV virions. Since our original observations were made with trans-complemented virions used to infect MAGI cells in a single-round infectivity assay, we wished to extend these observations to more physiologically relevant cells and include HIV-1 virions that have their nef gene replaced by the SIV nef gene.

We first tested trans-complemented virions that were produced as previously described (21) for relative infectivity on CEM x 174 cells. Briefly, pNL4-3 was mutated to remove the env gene, producing the viral clone NL4-3KFS (gift of Eric Freed, National Institutes of Health [NIH]). The KFS clone was then further mutated to introduce tandem stop codons into the nef reading frame, producing NL4-3KFSnef (gift of Judith Levin, NIH). When either the KFS or KFSnef clones are cotransfected with pIIIenv3-1 plasmid (gift of Eric Freed, NIH) (29), the resultant virions are complete but undergo only a single round of infection. To produce chimeric HIV-1 virions, we carried out three-way transfections separately, including an HIV-1, HIV-2, or SIV nef expression plasmid to trans complement the nef deletion. In this manner we could produce viral stocks of HIV-1 virions that contained either the HIV-1, HIV-2, or SIV Nef protein. The experiments shown here have been done using the same stocks that were used to produce our previously published results (21). In our previous publication we demonstrated that chimeric virions contained equal amounts of each Nef as determined by immunoblot analysis and lacked CyPA after CsA treatment (21). We tested each of these stocks for relative infectivity by infecting 2 x 10⁵ CEM cells with 5 ng (p24) of each stock. After 48 h, tissue culture media was tested for p24 antigen content. The results are summarized in Fig. 1. The infectivity of the KFSnef(–) virus was dramatically reduced from that of the WT (KFS) virus. Trans complementation of the KFSnef(–) virus with HIV-1, HIV-2, or SIV Nef restored infectivity to near-wild-type (WT) levels (Fig. 1). When particles were produced in the presence of 10 µM CsA, the WT virus infectivity was dramatically reduced. Infectivity of the KFSnef(–) particles was reduced essentially to background levels. The KFSnef(–) virus trans complemented with HIV-1 Nef showed a similar background level of infectivity, while the KFSnef(–) virus trans complemented with HIV-2 or SIV Nef was restored to essentially the same level of infectivity as particles produced in the absence of CsA (Fig. 1). These results were similar to those we have previously reported for the same particles tested on MAGI cells (21) and confirm that HIV-1 virions that contain HIV-2 or SIV Nef become resistant to CsA when tested using CEM cells.

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FIG. 1. Single-round infectivity of HIV-1 virions that contain HIV-1, HIV-2, or SIV Nef with CEM cells. Virions were produced in the presence or absence of 10 µM CsA. CEM cells (2 x 105) were infected with 5 ng (p24) of each virus stock. After 48 h, the culture supernatants were tested for p24 content by enzyme-linked immunosorbent assay. Results shown were done in triplicate and are representative of two independent experiments.

As a further test of our original observations, we wished to test an HIV-1 virus with its own nef gene replaced by the SIV nef gene. Sinclair et al. (28) have previously constructed such a virus (R7SIVnef+). We obtained this viral clone (gift of Elizabeth Sinclair, University of California, San Francisco) and the WT R7 virus (gift of Chris Aiken, Vanderbilt University) to test them for relative resistance to CsA. We first tested this chimeric virus using a MAGI infectivity assay for comparison with our previous study (21). One nanogram of each virus was used to infect 2 x 10⁵ MAGI cells. Cells were stained and counted after 48 h, and relative infectivity was determined. R7WT and R7SIVnef viruses showed comparable infectivities in the absence of CsA (Fig. 2). However, in the presence of 2.5 µM CsA, the R7WT virus showed about fivefold-reduced infectivity (Fig. 2), which is similar to what we have previously reported for NL4-3 virus (21).

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FIG. 2. Relative infectivities of R7WT and R7SIVnef were tested in a MAGI infectivity assay. Virions were produced in the presence or absence of 2.5 µM CsA. MAGI cells (2 x 105) were infected with 1 ng (p24) of each virus. After 48 h, the cells were stained and counted.

To demonstrate that the chimeric virus contained a functional nef gene, we compared the infectivity of the WT R7 virus to those of the R7SIVnef+ and R7nef(–) viruses by using a MAGI infectivity assay. One nanogram of each virus was used to infect 2 x 10⁵ MAGI cells. After 48 h the cells were stained and counted as previously described (19-21). The results are summarized in Fig. 3. The results demonstrate that the R7SIVnef+ virus is able to replicate at a level similar to that of WT R7 virus (Fig. 3). The results also demonstrate that the R7nef(–) virus was about fourfold reduced in infectivity, which is similar to the reduction in infectivity we have described in our previous assays (19-21).

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FIG. 3. Relative infectivities of R7WT, R7nef deletion, and R7SIVnef viruses on MAGI cells. MAGI cells (2 x 105) were infected with 1 ng (p24) of each virus. After 48 h, the cells were stained and counted. R esults shown were done in triplicate.

We next wanted to determine if the R7SIVnef+ virus would show resistance to CsA, as we have previously demonstrated with viruses for which the Nef was supplied in trans (21). To determine this, we infected 2 x 10⁶ CEM cells, human peripheral blood mononuclear cells (PBMCs), or activated human PBMCs with 10 ng of WT R7 virus or R7SIVnef+ in either the presence or absence of 2.5 µM CsA. It should be noted that CsA was present both during initial particle formation and during propagation. In each case, samples of tissue culture supernatant were taken at 0, 1, 3, 5, 7, 9, and 12 days, and p24 antigen content was determined by enzyme-linked immunosorbent assay. The results are summarized in Fig. 4. The replication kinetics of the R7 WT virus and the R7SIVnef+ virus were similar in the absence of CsA for all cell types tested. Since the R7SIVnef+ virus contains additional nucleotides, it might be expected to exhibit some delay in kinetics compared to WT R7. In the presence of 2.5 µM CsA, the R7 virus was unable to propagate while the R7SIVnef+ virus was only slightly impaired in its ability to replicate (Fig. 4A, B, and C). This was true with each of the cell types tested. These data are in agreement with our original observation that HIV-1 virions that contain SIV Nef are CsA resistant. In this case, the same is true when nef is replaced in cis and occurs in the context of a multiround infection using both CEM and PBMCs. Immunoblot analysis showed that both R7 WT and R7 SIV virions contained no CyPA after virion production in the presence of 2.5 µM CsA (Fig. 5). Together, these results demonstrate that an HIV-1 virus whose own nef gene is deleted and replaced by the SIV nef gene becomes resistant to CsA treatment.

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FIG. 4. The replication kinetics of R7WT and R7SIVnef were determined on (A) CEM cells, (B) stimulated PBMCs, and (C) nonstimulated PBMCs. Viruses were either treated with 2.5 µM CsA or left untreated. In those samples containing CsA, the drug was present both at the time of particle production and during propagation. Each determination was done in triplicate, and the coefficient of variation was <5% in each case. A representative experiment is shown.

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FIG. 5. Immunoblot analysis of chimeric virions in the presence or absence of CsA. Equal amounts of each virus stock were resolved on a sodium dodecyl sulfate-12.5% polyacrylamide gel, followed by immunoblot analysis using anti-CyPA antibody (ab). The blot was then reprobed with anti-p24 antibody as a loading control.

It has recently become apparent that CyPA also exerts an effect in target cells, most likely through inhibition of Ref-1 restriction (32). Therefore, we wished to determine the effect of CsA addition at the time of infection with our HIV-1 chimeric virus containing SIV nef. We infected 2 x 10⁵ MAGI cells with 1 ng each of KFS (designated WT), a nef-deleted KFS [designated Nef(–)], and KFSnef, which was trans complemented with an SIV nef expression plasmid (designated SIV). At the time of infection, 0, 2.5, or 5 µM CsA was added to the cultured cells. The results are summarized in Fig. 6. The infections done with either no CsA present or 2.5 µM CsA present at the time of infection gave basically the same results. The Nef(–) virus was about fivefold less infectious than the WT. Trans complementation with SIV nef restored infectivity to WT levels (Fig. 6, first two panels). The same experiment done in the presence of 5 µM CsA revealed that infectivity of the WT virus was now reduced to the same levels as that of the Nef(–) virus. This phenomenon is most likely due to the removal of cellular CyPA, which then allows Ref-1 to bind to cores and cause inhibition (32). This results in lower levels of infectivity. Curiously, the level of infectivity of the Nef(–) virus appeared to be unchanged by either concentration of CsA. However, the HIV-1 virus, which contained SIV nef, remained resistant to CsA even at the 5 µM concentration. Unfortunately, higher concentrations of CsA were toxic and could not be tested. These data suggest that the SIV Nef can somehow influence restriction by Ref-1. We have suggested that SIV Nef may be able to directly bind to the core (21; also see discussion below). If this is the case, then SIV Nef may also act to block binding of Ref-1 to the core in a manner similar to that of CyPA. However, the small amount of Nef contained within the virion is not likely to be sufficient to bind all of the surface area of the core particle. Therefore, either the number of sites that need to be saturated must be a small subset of the CA molecules in the core or some other, fundamentally different mechanism is at work.

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FIG. 6. Effect of CsA addition at the time of infection. KFS virions (WT), KFSnef [(Nef (–)], and KFSnef trans complemented with SIV nef (SIV) were tested for infectivity on MAGI cells in the absence of CsA and the presence of 2.5 or 5.0 µM CsA. The CsA was added at the time of infection. One nanogram of virus was used to infect 2 x 105 MAGI cells. The results shown are from triplicate measurements.

Previous studies have investigated chimeric viruses (4, 5, 22, 24, 28, 35) in which either HIV-1 has its nef replaced by SIV or vice versa. The main conclusion from the bulk of these studies is that, at least in tissue culture, the nef genes of HIV-1 and SIV are functionally replaceable. However, none of these studies investigated CyPA dependence. Other studies have shown that when the HIV-1 CA-p2 region is introduced into an SIV virus, the resultant chimera becomes sensitive to the CsA analog SDZ NIM 811 (13). However, the complete virus was described as being made from a ligation of the modified 5' half of the viral clone (p239SpSp5'HIV-CA) to a 3' half (p239SpE3') that contains a truncated Nef which lacks C-terminal amino acids. We have previously shown that these C-terminal amino acids are important in inducing CyPA independence (21). A follow-up study was done in which only a small portion of the HIV-1 CA that contained the CyPA binding region was transferred (13). In this study, the SIV clone was made by ligation of the 5' half of the SIV containing the small HIV-1 insertion to the p239SpE3'/nef-open plasmid, which contains a complete SIV nef gene. The results show that when residues 86 to 93 from HIV-1 CA are placed into the corresponding region of SIV CA, CsA sensitivity is conferred to the resultant SIV virus. Curiously, transfer of residues 86 to 90 from HIV-1 CA resulted in an SIV virus that was actually CsA dependent. These results are more difficult to reconcile with our observations. It is possible that the binding site for SIV Nef to CA could contain residues outside of this small region and that this might affect the induction of CyPA independence in these chimeric viruses. We are currently conducting studies to try to clarify this interesting issue.

In this study we demonstrate that an HIV-1 virus (R7) which has its own nef gene replaced with the nef gene from SIVmac239 can replicate in a CyPA-independent fashion. One of the differences between the Nef proteins from these two viruses is the presence of additional amino acids at the C terminus. We have previously shown that these amino acids are necessary for the induction of CyPA independence (21). Indeed, SIV viruses, such as SIVcpz, which are highly dependent on CyPA for replication, lack these additional amino acids at the C terminus. Likewise, HIV-1 viruses, such as group O viruses, contain these additional amino acids at the C terminus and are CyPA independent. When HIV-1(Nef–) virus is trans complemented with HIV-2 nef, which lacks the extra C-terminal amino acids, the resultant virions are as infectious as the WT in the absence of CsA (21). This suggests that the truncated HIV-2 Nef retains the ability to support CyPA-dependent replication even though it loses the ability to induce CyPA-independent replication to HIV-1 virions.

Exactly why the Nef protein should have an effect on CyPA dependence is a matter for speculation. It has been shown that HIV-1 Nef can directly interact with CyPA (6). Since CyPA can also interact with the HIV-1 CA, it is tempting to speculate that these direct interactions between Nef and CyPA and CyPA and CA could be important. However, several previous studies present results that are at odds with this idea. It has been previously shown that purified cores are associated with Nef (23) but not CyPA (34). One would expect that if a Nef-CyPA interaction were the main association of Nef with the core, the two would be present in equal amounts. However, we know a limited amount regarding how or why Nef might be present in the core. It has been shown that Nef is associated with the ribonuclear complex after disassembly in vitro (16). However, this does not preclude an interaction with CyPA. Since CyPA is known to interact with CA (14), it is possible that at least some of the CyPA is associated with the surface of the viral core and could be free to interact with Nef. However, the bulk of the Nef may remain contained within the core through other means. The core isolation procedure could strip the small amount of Nef-CyPA on the core surface away.

It has also been previously reported that the actions of Nef and CyPA are mechanistically independent (2). Three aspects of this study are important for consideration of the present work. First, the incorporation of CyPA is not dependent on Nef and vice versa. It has been clearly established that CyPA is incorporated into virions via interactions with the CA region of Gag (18). However, as mentioned above, a Nef-CyPA interaction need not be the main method of incorporation of these proteins for their interaction to occur. The second aspect is the observation that Nef(–) virions are reduced in infectivity after CsA treatment. It is has now been reported that treatment with CsA can have an effect on multiple aspects of viral replication (27, 32). This could mean that the residual change in infectivity is due to another aspect of CyPA or CsA regulation of infectivity. Perhaps the most difficult aspect of this study to reconcile is the observation that virions with mutations in CA, which reduce CyPA incorporation, are further reduced in infectivity by deletion of the nef gene (2). This suggests that Nef has an effect on replication which is independent of CyPA incorporation. Nef has many effects on replication and pathogenicity, and it is possible that isolation of individual effects could be difficult.

Finally, one would expect that Nef, CyPA, and CA could form a multiprotein complex. However, to date we have not been able to demonstrate that a stable complex forms between these three purified proteins. It is possible that CA protein in the context of a mature core is somehow different from isolated CA monomers. In this case, a linkage may form between Nef, CyPA, and CA only when CA is present in the mature core. Work is currently under way to attempt to clarify this issue.

How could such an attachment affect infectivity? It is known that Nef can interact with the intracellular sorting machinery (15) and can induce rearrangements of actin microfilaments (26). It could be possible that association of Nef with the viral core allows the intracellular machinery to attach to the core. Indeed, it has recently been suggested that Nef may function to allow the virus to penetrate the cortical actin network, which is a potential barrier, to allow the preintegration complex to reach the nucleus (11). It is possible that the incoming viral core may need to be directed to particular subcellular locations for optimal replication to occur. In the context of comparing HIV-1, HIV-2, and SIV Nef proteins, it could be the case that HIV-1 needs to interact with the core via CyPA interactions. The additional amino acids of HIV-2 and SIV Nef may allow this interaction to occur independently of CyPA. Of course, this hypothesis would predict the direct interaction of HIV-2 and SIV Nef with the viral core and the indirect interaction of HIV-1 Nef with the core. These types of interaction studies are currently under way and should provide additional information on the precise mechanism of Nef-induced CyPA independence.

 
This work was supported by NIH grants G12-RR03034, R21AI60370, and S06GM08248.

We are grateful to E. Freed and J. Levin (NIH, Bethesda, Md.) for supplying the KFS and KFSNef plasmids, E. Sinclair (UCSF, San Francisco, Calif.) for supplying the R7SIVnef+ clone, and C. Aiken (Vanderbilt University, Nashville, Tenn.) for supplying R7 WT and R7nef(–) clones.

 
* Corresponding author. Mailing address: Dept. of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, GA 30310. Phone: (404) 752-1582. Fax: (404) 752-1179. E-mail: powellm{at}msm.edu.

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Journal of Virology, March 2005, p. 3211-3216, Vol. 79, No. 5
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.5.3211-3216.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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