HIV-1 requires the incorporation of the host protein CypA for efficient replication. CypA is packaged into HIV-1 by specifically binding to the CA region of the Gag precursor. Given that CA comprises the shell that surrounds the viral genome and that CypA possesses a cis/trans isomerase activity, it has been proposed for years that CypA acts as an uncoating factor. In this model, CypA destabilizes CA-CA interactions, triggering the rupture of the CA coat, allowing the delivery of the viral genome in the cytosol of host cells. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged in the virus, it was impossible to date to determine if the isomerase activity of CypA was required for HIV-1 replication.
In the present study, we have addressed this issue by taking advantage of a Vpr-CypA fusion protein that bypasses the interaction with the glycine 89-proline 90 packaging signal of CA in Gag and allows the incorporation of CypA into viruses even in the presence of CsA. Using this system, we found that Vpr-CypA rescued HIV-1 replication despite the presence of CsA. We then fused to Vpr a CypA mutant which has no cis/trans prolyl isomerase activity and no capacity to bind to the CA region of Gag or CsA. Importantly, this CypA mutant also rescued HIV-1 replication in the presence of CsA. Altogether, our findings demonstrate that the isomerase activity of CypA is not required for HIV-1 replication and suggest that the interaction of the catalytic site of CypA with glycine 89-proline 90 motif of CA serves no other function than to incorporate CypA into viruses.
Our present data seem to argue against a role for CypA in HIV-1 CA uncoating. However, we cannot exclude the possibility that CypA acts on loci of CA other than the glycine 89-proline 90 packaging signal. We can imagine that, during virus maturation, the processing of the Gag precursor induces major structural changes in cleaved CA that may expose new high-affinity binding sites for CypA. Postassembly actions of CypA, such as uncoating, may be mediated via these new high-affinity binding sites of mature CA proteins. Supporting this hypothesis, several studies suggest that affinities of CypA for Gag and for CA are strikingly different (6
). Specifically, Endrich and colleagues, using recombinant proteins, showed that the processing of immature CA, yielding mature CA, elicited conformational changes in its C-terminal domain. They showed that CypA binds with a higher affinity to mature CA than to immature CA. They identified two new high-affinity binding interaction sites between CypA and mature CA in the C-terminal domain of CA around glycine 156-proline 157 and glycine 223-proline 224 (11
). They proposed that these new binding sites represent loci upon which CypA acts postassembly. However, these sites are located in the C-terminal domain of CA, which is essential for CA oligomerization and CA core formation (7
). Given that Colgan and colleagues demonstrated that Gag multimerization is required for CypA packaging (8
), we cannot exclude the possibility that these sites (glycine 156-proline 157 and glycine 223-proline 224) play only an indirect role in CypA-CA interactions. To clearly demonstrate that these sites represent authentic loci for CypA action, these sites will have to be mutated in the context of whole virus, instead of recombinant proteins, and the resulting mutant viruses will have to be examined for their capacities to assemble, to form regular cores, and to enter and to infect target cells. Corroborating the hypothesis for CA refolding upon maturation, Dietrich and colleagues showed, using recombinant proteins, that CypA binds to cleaved CA in a manner different from its binding to unprocessed Gag (9
). Specifically, they showed that the mutant CypA W121F binds efficiently to Gag but fails to bind to mature CA. Altogether, these observations suggest the occurrence of maturation-dependent conformational changes in CA that may potentiate the influence of CypA on CA uncoating.
Our present findings, however, seem to argue against the hypothesis that CypA-CA contacts are critical for HIV-1 infection at a postassembly step. Specifically, we showed that CsA does not inhibit the infectivity of a virus which has packaged in trans
the Vpr-CypA chimera. Since it has been shown that CsA prevents the binding of CypA to CA (2
), our data suggest that the catalytic site of CypA is not necessary for CypA function in HIV-1 replication after its packaging. Although we cannot rule out the possibility that CypA acts on CA via regions other than its catalytic domain, our present observations strongly suggest that the requirement for CypA in HIV-1 replication does not depend on an action of CypA on CA. Several in vitro studies clearly demonstrated that CsA prevents CypA-CA/Gag interactions (2
); however, one report suggested that a subpopulation of CA molecules may bind CypA even in the presence of CsA (1
). This discrepancy may arise from the different provenance of CA utilized. For example, some studies generated recombinant CA as glutathione S
-transferase fusion proteins (2
), whereas another generated CA from the processing of recombinant Gag by the HIV-1 protease (1
). It is possible that the method used to generate CA may influence its binding properties to CypA. Furthermore, we cannot exclude the possibility that the use of recombinant proteins may not reflect in vivo conditions.
If CypA acts at a postassembly step other than uncoating, what is the function of CypA in the HIV-1 life cycle? Recent reports suggest that incorporated CypA may play an important role in HIV-1 entry into target cells. Specifically, Pushkarsky and colleagues recently showed that HIV-1 entry depends on interactions between virus-associated CypA and cell surface CD147 (29
). Importantly, antibodies directed against CD147 inhibit the entry of HIV-1 into target cells but not that of viruses which do not require CypA for replication, such as simian immunodeficiency virus (29
). In another report, we showed that CypA-deficient viruses attach to target cells less efficiently than do wild-type viruses (31
). Given that recombinant CypA possesses the capacity to bind to target cells via HS and that HIV-1 requires cell surface HS to attach to a large number of adherent cells (31
), we postulated that packaged CypA mediates HIV-1 adsorption to target cells via HS. Corroborating this hypothesis, we and others showed that affinity-purified anti-CypA antibodies or an excess of recombinant CypA decreasees HIV-1 uptake by target cells (31
). Using recombinant CypA, we identified four basic residues in the C terminus of CypA responsible for binding to cell surface HS (31
). We postulated that these residues might play a role in HIV-1 attachment to target cells. To address this possibility using a more physiological system, we fused a mutant CypA lacking these four residues to Vpr and tested the resulting chimera for its ability to rescue HIV-1 replication in the presence of CsA. To our surprise, this mutant also rescued HIV-1 infectivity, suggesting that these residues are not sufficient for HIV-1 attachment. Several possibilities may explain why these residues are not necessary for HIV-1 replication. One possibility is that either additional residues of CypA or residues distinct from the four residues described above are necessary for HIV-1 attachment. We originally assumed that these basic residues of CypA participate in viral attachment arose from experiments using recombinant CypA. However, one could imagine that the use of recombinant protein to demonstrate a role for CypA in HIV-1 attachment is not adequate. Interestingly, there is a close precedent in the literature supporting this hypothesis. Specifically, it has been demonstrated that recombinant gp120 binds to cell surface HS via basic residues located within its V3 loop (30
), suggesting that gp120 may mediate HIV-1 attachment either via gp120-CD4 or via gp120-HS interactions. However, a growing body of evidence indicates that gp120 is not necessary for the initial attachment of HIV-1 to target cells. Indeed, several recent studies showed that viruses with a deletion of gp120 attach to human macrophages or to CD4+
HeLa cells at levels similar to those of wild-type viruses (24
). Thus, these observations strongly suggest that the use of recombinant proteins such as gp120 or CypA to demonstrate their participation in HIV-1 adsorption in the context of whole virus must be regarded with caution. Another possibility to explain why the basic residues of CypA are not necessary for HIV-1 replication using our Vpr chimera system is that, although CypA is required for HIV-1 attachment, it may not mediate virus adsorption directly. One could envision that CypA, only in conjunction with an exposed virus-associated protein, mediates HIV-1 attachment. Alternately, one could also imagine that CypA facilitates the translocation of an attachment factor to the surface of the virus during viral maturation. Nevertheless, our present in-trans
Vpr-CypA incorporation system will permit us to delineate the domains of CypA necessary for both CypA-CD147 and CypA-HS interactions. This will help to better define the respective roles of these interactions in HIV-1 entry.
We recently reported that the two major HIV-1 targets—CD4+
T lymphocytes and macrophages—express opposite patterns of the candidate HIV-1 attachment receptors CD4 and HS (32
). Specifically, we showed that CD4+
T lymphocytes express high CD4 levels but no HS, whereas macrophages express low CD4 levels but high HS levels. We demonstrated that HS serve as the main class of attachment receptors for HIV-1 on macrophages, suggesting that CD4 alone is not sufficient to support the initial adsorption of HIV-1 to these cells. Furthermore, we found that HIV-1 attaches efficiently to CD4+
T lymphocytes despite the absence of cell surface HS (32
), suggesting that high CD4 levels are sufficient to mediate the initial attachment. If CypA is only necessary for HIV-1 attachment, we would expect to observe replication of CypA-deficient viruses in CD4+
T lymphocytes. However, a previous study showed that the nonimmunosuppressive CsA analogue SDZ NIM 811 decreases CypA incorporation and replication in CD4+
T lymphocytes (26
). This observation suggested to us a postentry role for CypA in HIV-1 replication. Corroborating this hypothesis, we recently found that the use of spinoculation (centrifugal inoculation), which bypasses the mechanistic requirement for the initial virus adsorption to target cells (28
), does not fully rescue the infectivity of CypA-deficient viruses pseudotyped or not with the vesicular stomatitis virus envelope (Saphire et al., unpublished). These results strongly suggest that CypA acts at least at two distinct steps in the HIV-1 life cycle: entry and postentry.
In conclusion, we showed that the trans-complementation of CypA restores the infectivity of CypA-deficient viruses even in the presence of CsA. Furthermore, we found that neither the catalytic domain of CypA nor the glycine 89-proline 90 CA-binding site is essential for HIV-1 replication. These results suggest that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses. We are presently using our in-trans incorporation system to identify domains of CypA essential for HIV-1 replication. The identification of these regions will help to delineate the respective roles of CypA in HIV-1 entry and postentry.