(i) Tsg101 and other components of the MVB pathway.
One of the major breakthroughs in the understanding of the complex interplay between enveloped viruses and the host cell machinery is certainly comprehension of the viral budding process (71
). Recent studies have revealed how enveloped RNA viruses highjack the vesicular cellular machinery for their own purposes (85
). Figure depicts how such viruses can usurp this cellular pathway normally used to create vesicles that bud into late-endosomal compartments, which are better known as multivesicular bodies (MVB).
FIG. 1. Schematicrepresentation of the intimate link between viral components and the human MVB pathway. Cargo proteins to be sorted are first monoubiquitinated and then bound to the Hrs complex in the early endosomes. After fusion of the early endosomes and (more ...)
To take advantage of the MVB pathway, enveloped RNA viruses such as retroviruses, rhabdoviruses, and filoviruses possess conserved sequences in their structural proteins called late domains (L domains) (38
). Each L domain is able to bind to specific cellular factors that redirect the structural proteins of the nascent viruses into the MVB pathway of the infected cell, thereby orchestrating the budding and egress of virions, using the same cellular vesicular machinery as cellular endosomes (85
Enveloped RNA viruses like Ebola viruses, HIV-1, and human T-cell leukemia virus type 1 (HTLV-I) get access to this sorting machinery by binding to the Tsg101 subunit of the human endosomal complex required for transport I (ESCRT-I) via the L domains of their structural proteins. It has been proposed that viral proteins are targeted to the MVB machinery possibly by virtue of a single ubiquitin linked to their L domains, which would explain their binding to Hrs and Tsg101 (38
). Indeed, fairly large amounts of free ubiquitin were detected in purified preparations of simian immunodeficiency virus (SIV), HIV-1, murine leukemia virus (MLV), and Moloney MLV (MMLV) and in avian leucosis virus (41
). While usurping the MVB pathway, these viruses apparently incorporate structures of the cellular sorting machinery within the newly formed entities. Tsg101 incorporation within HIV-1 particles remains the best-known example and has been reported in numerous studies (32
). Tsg101 has been found in purified Mason-Pfizer monkey virus preparations as well, along with the Nedd4 enzyme, an ubiquitin ligase containing a WW sequence with binding capacity to the PPXY L domain. The above proteins have also been found embedded in HTLV-I viral particles (12
). Tal, a novel E3 ubiquitin ligase recently described in yeasts and mammalian cells, has been shown to be incorporated within HIV-1-like particles (3
). This ligase binds to Tsg101 in a bivalent mode and, when bound, mediates multiple monoubiquitination of Tsg101, which disables the sorting activity of Tsg101 and is thought to free the cargo protein.
Other cellular protein subunits of all three ESCRT complexes have been reported to be incorporated intobudding virions. VPS28, a subunit of ESCRT-I that binds to Tsg101, was detected within HIV-1 particles (11
). Tsg101 also interacts with the AIP1/ALIX subunit of the ESCRT-II complex, and the latter is recruited into both HIV-1 and SIV (113
). Of note, equine infectious anemia virus (EIAV) p9 protein is capable of binding AIP1/ALIX, which may presumably be packaged into EIAV virions (102
). The subunit of the ESCRT-III complex,VPS4B, seems to be acquired by HIV-1 (113
). This protein is involved in the very last steps of vesicle formation and has a role in releasing ESCRT complexes from the newly formed vesicles. Nevertheless, not all parts of these large multiprotein complexes are retained in viruses, as exemplified by the reported failure to detect VPS37B in purified HIV-1 preparations (104
(ii) APOBEC3G, a protein of the RNA-editing machinery.
Among the many threats that the RNA viruses must overcome inside cells is the cellular RNA-editing machinery. Products of the mRNA-editing gene family are specialized for deamination of cytidine on RNA, causing a switch from C to U in mRNA. A mechanism of protection exploited by viruses has been unraveled, principally with the discovery of the relationship between APOBEC3G and the HIV-1 Vif protein (49
). The APOBEC3G protein (also termed CEM15) has the ability to get packaged within HIV-1. Being in close proximity to the viral genomic RNA, this editing enzyme therefore has the chance to catalyze the deamination from C to U on the minus strand of the viral nucleic acid. Therefore, upon plus-strand synthesis of the viral genome during the reverse transcription phase, an A nucleotide is inserted into the plus strand at the many spots where the C to U deamination has occurred on the minus strand. As a result, many nonviable mutations are created and the newly synthesized virions are thus inactivated. This G-to-A hypermutation phenomenon occurring throughout the HIV-1 genome has been noticed for quite a while (111
The precise role of the auxiliary Vif protein of HIV-1 in the virus life cycle and/or pathogenesis has long been an enigma. But recently Vif was brought into the spotlight by the discovery that it can associate with APOBEC3G in lymphoid cells. Vif binds to APOBEC3G inside infected cells in order to prevent this protein from being packaged within nascent virions (53
). Convincing evidence seems to indicate that Vif strongly diminishes the APOBEC3G intracellular pool. Here it is interesting to highlight that, in sharp contrast to the usual observation that viruses associate with cellular proteins, in this case the virus evolved to minimize interaction with and incorporation of a specific cellular component. In this regard, we can assume that this phenomenon supports the hypothesis that the incorporation process of host-encoded proteins is specific, at least to a certain extent. It also underlines the fact that association with particular cellular proteins can be highly detrimental to the virus and, therefore, that the process of host cell protein incorporation can be a very critical issue for viruses.
The mechanism of APOBEC3G incorporation within HIV-1 cells is still ill defined, though a few hints are starting to emerge. The first studies reported a potential interaction of the APOBEC3G N terminus with the nucleocapsid protein of HIV-1 (27
). Whether the two zinc coordination motifs contained within the APOBEC3G are directly implicated in this intimate association remains controversial (1
). The same applies to the possible bridging of viral or cellular RNA between APOBEC3G and the Gag protein (27
). Since APOBEC3G can be incorporated into various types of viruses having different Gag sequences (HIV-1, MLV, SIV, and EIAV), it has been proposed that the link between Gag and APOBEC3G may not rely exclusively on sequence but also on some specific structural motifs (27
). Human APOBEC3G activity is not restricted to HIV-1, as it can inactivate HIV-2, SIV, EIAV, and MLV (27
). Furthermore, the effect of Vif on the incorporation of APOBEC3G is species specific. For example, the Vif protein does not prevent the packaging of mouse and African green monkey APOBEC proteins within HIV-1 particles produced by transient transfection in 293T cells, as they do not bind to the Vif protein like their human homolog does (66
). Finally, APOBEC3F, another member of the mRNA-editing enzyme family incorporated into HIV-1, was very recently demonstrated to inhibit viral replication in the same way as APOBEC3G (58
). It will be interesting to evaluate whether other members of this family carry out a similar activity on RNA viruses (10
(iii) UNGs and Staufen.
A variety of viruses encode uracil-DNA glycosylases (UNGs) or dUTPases to block uracil incorporation within the viral DNA (30
). This is the case for herpesviruses, poxviruses, and some nonprimate retroviruses. As for primate lentiviruses that do not encode such enzymes, one of them, HIV-1, does incorporate host cellular UNG within the viral particles (64
). In contrast, HIV-2 and SIVMAC
fail to package this enzyme and may have evolved different, yet-unknown strategies to achieve a similar goal (86
). The mechanism of UNG incorporation is still under investigation, but it appears that it might occur through association with the viral Vpr protein, integrase, and reverse transcriptase enzymes, individually or in cooperation (64
Interactions between host proteins and the genetic material of retroviruses also seem to be necessary for the encapsidation of genomic RNA. Staufen is a double-stranded RNA-binding protein that is enclosed within HIV-1, HIV-2, and MMLV (72
). The amount of genomic RNA included within the released viral particles is correlated with the amount of Staufen protein incorporated, suggesting a role in viral RNA packaging. The Staufen protein was recently shown to cosediment with the viral Pr55Gag
precursor protein and to associate directly with Pr55Gag
. This link involves the Staufen dsRBD3 domain in collaboration with the C-terminal domain (28
(iv) Cyclophilins and other prolyl isomerases.
Expressed in all organisms, from bacteria to primates, cyclophilins catalyze the isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. They also function as chaperones, having a broad subcellular distribution. Cyclophilin A (CypA), an abundant cytosolic protein found in all tissues examined, is best known for its ability to bind cyclosporine A (CsA). CypA is involved in T-cell activation and is thought both to provide a chaperone activity and to maintain proper protein conformation. It has been known for over a decade that CypA is efficiently inserted within HIV-1 at a ratio of 1 CypA molecule to 10 Gag molecules (37
), which represents approximately 250 molecules per virion. The immunosuppressive drug CsA inhibits incorporation of CypA into HIV-1. However, the closely related retroviruses HIV-2 and SIV do not incorporate CypA, with the exception of the chimpanzee-specific SIVcpz
). It has also been detected inside vaccinia virus (VV) and vesicular stomatitis virus (VSV). Interestingly, the ability of HIV-1 to package this host-derived molecule has been investigated for various viral subtypes. Viruses of the outlier O group incorporate CypA in amounts similar to that of viruses belonging to the M group, but their infectivity does not rely on it (18
). Further analyses indicated that all five O-group isolates tested incorporated CypA in a CsA-sensitive way, while their infectivity strongly depends on it in only three cases (116
). CypA is initially packaged inside the virus by a direct interaction between its hydrophobic binding pocket and the proline-rich flexible exposed loop located within the amino-terminal domain of the viral capsid (CA) protein (14
). Nevertheless, its affinity for the CA molecule is weak, and during maturation it has been observed to relocate to the viral surface (95
Several studies have focused on the role of CypA in the HIV-1 life cycle (97
). Most importantly, it is the only incorporated cellular protein shown to be critical for viral infectivity. Moreover, infectivity is finely tuned by host CypA expression levels (121
). It efficiently catalyzes the cis
-isomerization of a peptide bond on CA (15
). As for its exact role in infectivity, some authors have argued for a role in early events of the replication cycle, such as uncoating, and others for a role in late events, such as maturation. Since it has been observed that Gag assembles in the absence of CypA, it has been proposed that CypA is required at a step between Gag assembly and virion morphogenesis, possibly for conformational changes (103
). Moreover, in vitro studies provided evidence that CypA does not efficiently destabilize assembled CA at the molar ratio observed in the virion, and the authors concluded it was unlikely to serve as an uncoating factor (44
). Their data suggest that CypA more likely exerts its effect by facilitating the coordinated rearrangement of CA subunits during the maturation process.
On the other hand, arguing against a role in late events are the facts that assembly occurs in the presence of CsA and that disruption of the Gag-CypA interaction still allows for assembly and budding to give particles with the proper number of Gag proteins. Moreover, it has been shown that the core stability is due to protein-protein contacts between the CA subunits without involvement of CypA, which can bind only to an aggregated form of immature CA and not a dissociated one. Thus, binding of CypA could very well serve only as a means of entry into the virion (14
). Moreover, incorporation of a catalytically inactive form of CypA is sufficient for efficient infection, and thus the isomerase activity is not involved in virus infectivity (96
). Spinoculated CypA-deficient viruses enter target cells efficiently but fail to infect them, which also points to a postentry event (93
Although group O viruses do not require CypA for replication, the fact that they package all CypA into mature HIV-1 particles suggests that they evolved from a virus which was at one time CypA dependent. Thus, interestingly, the study of incorporation of a host protein supports the hypothesis that the group M and O viruses were transmitted to humans on two separate occasions from nonhuman primates, as has previously been suggested (19
In addition to enhancing infectivity, a number of other roles have been suggested for the incorporated CypA. V3 loop peptides derived from HIV-1 macrophage- and T-cell-tropic external envelope gp120 bind with high affinity to the active site of CypA (33
), pointing to a possible role in virus attachment. In other regards, it has been proposed that CypA could be a mediator in the initial attachment of HIV-1 to the host cell plasma membrane (100
) through its interaction with heparans expressed at the cell surface (94
). In fact, at least one CypA isoform has been detected outside the viral membrane (69
). It is yet unclear how the cytosolic protein might penetrate the viral membrane, but the existence of several isoforms differentially located within the virion points to possible posttranslational modifications. Moreover, binding affinity to the CA protein strongly decreases as the CA matures, which could allow dissociation and relocation of the cyclophilin. However, the possible contribution of CypA to virus attachment is difficult to reconcile with the differential dependence of the infectivity of certain O-group viruses on CypA (116
). In other regards, CD147, a transmembrane glycoprotein of the immunoglobulin superfamily, has also been identified as a receptor for CypA, and it would interact with it downstream of the CypA-heparin interaction (87
). A previous study indicated that CypA is also important for the de novo synthesis of the viral protein Vpr, and in the absence of its activity, Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells (122
). Moreover, in human cells it reduces HIV-1 sensitivity to restriction factors present within host cells (108
When packaged in viruses of the Rhabdoviridae
family, such as VSV, CypA seems to act differently. For example, although CypA is important for VSV infection, it acts at the level of primary transcription, helping in the proper folding of the N protein (16
). Interestingly, the prevailing virulent NJ strain of VSV has a critical dependence on CypA, whereas the less virulent and less widespread IND serotype does not. As for VV, a member of the Poxviridae
family, Castro and coworkers have found CypA packaged in viral cores, with approximately 156 molecules per single VV particle (26
). They have speculated that CypA could either mediate the transport of virus proteins to virosomes, catalyze conformational changes in virus proteins important to the assembly process, or participate in the uncoating of viral cores.
Other prolyl isomerases have been studied for their incorporation into viruses, including FK-506-binding proteins (FKBPs), parvulins (e.g., pin1), and other cyclophilins. Among the latter, cyclophilin B (CypB), which is targeted to the endoplasmic reticulum, is not incorporated into HIV-1 in vivo, unlike cytosolic CypA (18
). FKBP12 has been detected inside HIV-1 at an average of 25 molecules per virion (20
). Even though the specificity of FKBPs is much higher than that of CypA and one of their best substrate sequences contains Phe-Pro, which is known as an HIV-1 protease-specific cleavage site (20
), the relevance of FKBP12 to the virus biology remains to be established.