Many biological and pathogenetic aspects render FIV an attractive model for AIDS research. Indeed, FIV and is natural host, the domestic cat, have already provided important insights into relevant aspect of HIV infection and pathogenesis (Burkhard and Dean, 2003
; Power et al., 2004
). Moreover, this model has been proposed for testing new specific HIV drugs, for the analysis of resistance mechanisms and for the design of candidate HIV vaccine. The fact that efficacious anti-FIV therapeutic approach would be advantageous also in veterinary medicine, adds value to the system. Finally, the development of safe gene-transfer vectors based on FIV is another interesting application of this non-primate lentivirus.
Despite this great potentiality, some of the experiments carried on in order to test the efficacy of specific HIV-1 drugs and to analyse viral resistance mechanisms (North and LaCasse, 1995
), employing FIV as a model, have clearly brought to light that in many areas our present understanding of FIV lags behind knowledge accumulated on HIV. In this context, a further and deep dissection of FIV structural protein Gag functions could have a significant impact on clarifying different aspects of viral pathogenesis, as well as on drug development and vaccination strategy design.
It has been recently reported that FIV, like other enveloped viruses, exploits the multivesicular body (MVB) biogenesis pathway for its budding (Luttge et al., 2008
). In particular, a four aminoacidic motif (PSAP), present in the carboxy-terminal region of Gag, appears to be crucial in this context (Luttge et al., 2008
). In the present work we intended to contribute to the dissection of the carboxy-terminal region of the FIV Gag protein, in particular focusing on its contribution to VLPs production. Thus, we obtained four different constructs, characterised by the substitution of one or both prolines of the PSAP motif with alanines, in combination or not with the introduction of a STOP codon in place of aminoacid 10 of the protease. Early reports on HIV-1 had demonstrated that mutational inactivation of the viral protease could reverse defects in the L-domain region, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions (Huang et al., 1995
). Here we show that, in contrast with HIV-1, a FIV Gag characterised by an inactive L-domain is impaired in the release from transfected cells also in the absence of the viral protease. In agreement with this finding, we demonstrate that a block of the MVB pathway strongly impairs unprocessed FIV Gag from budding. Interestingly, our data do not rule out the functional linkage between FIV L-domain and protease activity. Indeed, when the L-domain is inactive, in the context of an active viral protease, we do notice the accumulation of intracellular partially processed Gag (, lane 2 “Cells” panel). In particular the MA-CA-p1-NC intermediate seems to accumulate. It is known that in the case of HIV-1, defects in Gag processing, such as the p25 to p24 increased ratio, are typically associated with p6 mutants (Huang et al., 1995
). Thus, even though our data suggest that also in the case of FIV the inactivation of the L-domain is linked to protease activity, we still see a strong impairment in the unprocessed Gag ability to bud, when the L-domain is inactivated. The fact that the carboxy-terminal region of FIV Gag does not overlap with the open reading frame of the viral protease, may contribute to explain this difference between the two lentiviruses. Moreover, the specific properties of the feline protease, that have been identified (Schnölzer et al., 1996
; Lin et al., 2003
), like, for instance, the different order of Gag processing (Lin et al., 2006
), may also contribute to this phenomenon. It is worth to note that an other difference between HIV-1 and FIV emerged form our study. Indeed, we demonstrate that the first proline of the PSAP motif is not involved in the late domain function, while it appears to be essential in the case of HIV-1 (Huang et al., 1995
). It is known that the PSAP finds in the cellular protein Tsg101 the connection with the MVB pathway (Garrus et al., 2001
). Moreover it has been recently reported that knocking down Tsg101 a significant reduction in FIV budding is achieved (Luttge et al., 2008
). Since Sundquist’s group has reported that the first proline of the HIV-1 PT/SAP makes critical contacts with the UEV domain of Tsg101 (Pornillos et al., 2002
) it would be interesting to analyse whether Tsg101 is incorporated in FIV VLPs and to determine whether the FIV ASAP mutant is still able to interact with Tsg101. Moreover, it would also be useful to know whether Tsg101 depletion affects the release of the ASAP mutant.
As mentioned above, FIV may find interesting application in the development of gene-transfer vectors. However, the production of replication-defective lentiviral vectors for a large-scale clinical use is challenging (Lesch et al., 2008
). One of the limiting steps in the process is represented by the low vector titer obtained with the packaging system currently adopted (Segura et al., 2007
). Since our data demonstrated the impact of the L-domain region in FIV VLP production and since it has been reported that L-domains are transferable between unrelated viruses (Parent et al., 1995
), we wanted to investigate the particle release efficiency of a FIV Gag chimera characterised by the Ebola virus L-domain replacing the PSAP motif (WT-Eb). Indeed, it has been shown that HIV-1 based minimal Gag constructs that have been engineered to contain the PTAPPEY motif, which is the one characteristic of Ebola virus, are released very efficiently from transfected cells (Strack et al., 2002
). When we quantified the amount of particles produced by the constructs containing a more or less optimised Ebola virus L-domain (WT-Eb, MUT1, MUT2 and MUT3), we did not notice a significant increase in particle production, with respect to FIV WT. Thus, even though Ebola L-domain is working also in the context of full length FIV Gag, it does not lead to a significant enhancement in particle production. However, our data do not rule out the possibility that carefully manipulating the carboxy-terminal region of FIV Gag the amount of VLP released could be positively influenced. It is worth to mention that by few steps of site specific mutagenesis it is possible to modify the sequence downstream the PSAP motif in order to obtain a Gag construct containing the Ebola virus L-domain combined with the YPxL domain. Thus, it could be interesting to test the impact of this combination between different L-domains on the particle release efficiency.
An other finding of our work is that unprocessed wt FIV Gag is modified by ubiquitination. This is again a peculiar feature of FIV Gag, when compared to HIV-1, which appears to be much less ubiquitinated (Strack et al., 2000
; Martin Serrano et al., 2004
), but it is supported by similar findings published in the case of other lentiviruses, such as SIVmac (Strack et al., 2000
). Moreover, Gag ubiquitination appears to be linked to the functionality of the L-domain. Thus, our data suggest that, in the case of FIV Gag, ubiquitination may be directly linked to budding. We and others have reported that a functional L-domain leads to retroviral Gag ubiquitination and that ubiquitin residues involved in endocytosis are crucial for budding (Strack et al., 2002
; Martin-Serrano et al., 2004
; Gottwein et al., 2006
). However, the role of ubiquitin in the egress of retroviruses, and in particular of lentiviruses, is still under debate. Indeed, even though several findings argue for a functional contribution of the ubiquitin modification to virus release, there are studies suggesting that ubiquitination could be merely a bystander effect (Martin-Serrano, 2007
). In addition, in agreement with our present findings, the enhancement of ubiquitination, does not always correlate with an enhancement in particle release (Strack et al., 2000
; Martin Serrano, 2007
). Finally, while ubiquitination of PPxY-containing Gag is easily explained by the direct interaction of the viral structural protein with Nedd4 family ubiquitin ligases (Harty et al., 2000
, Martin-Serrano et al., 2005
), the involvement of ubiquitin in the egress of viruses lacking such a motif is less clear. However, two recent studies (Chung et al., 2008
; Usami et al., 2008
) have shown that Nedd4-2s, a member of Nedd4-ubiquitin ligases, plays a role in HIV-1 budding. The authors’ conclusion is that Nedd4-2s, even in the absence of a functional PPxY motif, may cooperate with the other L-domains to enhance viral budding, maybe leading to the ubiquitination and consequent activation of MVB components or facilitating Gag interaction with ubiquitin binding protein of the pathway. Moreover, the authors clearly demonstrated that Nedd4-2s is the only ubiquitin ligase showing the ability to rescue L-domain deficient HIV-1 constructs (Usami et al., 2008
). In order to confirm and extend this conclusion to non-primate lentiviruses and since our data suggested that ubiquitin could play a role in FIV egress, we decided to analyse whether Nedd4-2s was involved in this process. We were able to show that indeed Nedd4-2s is involved in FIV Gag ubiquitination and it is capable of rescuing FIV Gag lacking a functional L-domain in its ability to bud from transfected cells. This finding is interesting because it demonstrates that lentiviruses in general respond to Nedd4-2s in the absence of a PPxY motif, in contrast to other retroviruses, like the Mason-Pfizer monkey virus (Usami et al., 2008
Overall our data bring to light peculiar aspects of FIV Gag carboxy-terminal region, that may have an impact when FIV is employed as a model for studying HIV pathogenesis and therapy, or for biotechnological applications of this lentivirus. On the other hand, we extend the recent observation that ubiquitin ligases may cooperate with other late domains to enhance the efficiency of viral release to a non-primate lentivirus, demonstrating that this novel mechanism of connection to the cellular budding machinery is shared between different members of this viral family.