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Restriction factors: a defense against retroviral infection
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Vol.11 No.6 June 2003
Restriction factors: a defense against retroviral infection Paul D. Bieniasz Aaron Diamond AIDS Research Center, 455 First Avenue, New York, NY 10016, USA
Susceptibility to retroviral infection is determined, in part, by host genes with antiviral activity. The Fv1 gene, which inhibits murine leukemia virus infection in mice, encodes one such resistance factor, and was long thought to be unique in that it restricts post-entry, preintegration steps of retroviral replication. However, recent findings suggest the existence of similar restriction factors in primates, including humans. These factors, termed Lv1 and Ref1, can inhibit a range of retroviruses, including human immunodeficiency virus type 1 and its relatives. Fv1, Lv1 and Ref1 target capsid determinants to block infection but can be saturated by incoming virions. Primate- and murine-retrovirus restriction factors have diverse and overlapping specificities, and some variants of Lv1, as well as Ref1, apparently recognize and inhibit infection by widely divergent retroviruses. During mammalian evolution, a variety of mechanisms have arisen to purge the host of viral infectious agents. The adaptive and innate immune responses are primarily responsible for providing protection against viral pathogens, but it is becoming increasingly clear that dominant, inhibitory gene products can also have an important role in controlling host susceptibility. One class of such inhibitors, which for the purpose of this review are termed ‘restriction factors’, block retroviral infection by targeting the incoming capsid and preventing the efficient progression of postentry, pre-integration events. For many years, the existence of retrovirus restriction factors was thought to be unique to the mouse. However, recent evidence indicates that capsid-targeting cellular inhibitors of retroviral infection (Fig. 1) are far more widespread than previously thought, are capable of inhibiting infection by a variety of retroviruses [1– 4], and have probably substantially impacted the prevalence of retroviral infection and disease in mammals.
inoculation of animals. However, two dominant genes – Fv1 and Fv4 – were of particular interest because they were active in vitro: cultured cells derived from mice harboring the genes exhibited substantial resistance to infection [7 –11]. While Fv4 was subsequently shown to encode an endogenous retroviral envelope protein, and
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Fv1: the prototype retrovirus restriction factor The description of a collection of heritable traits in mice that controlled susceptibility to leukemia induced by the Friend strain of murine leukemia virus began more than 30 years ago [5,6]. Many of the implied Friend virus susceptibility (Fv) genes were subsequently shown to modify either immune responses to the virus, or cell proliferation, and only exerted their phenotype upon Corresponding author: Paul D. Bieniasz ([email protected]).
Fig. 1. Retrovirus restriction factors. Restriction factors target the capsid of incoming retroviruses to block infection. Restriction occurs before reverse transcription in the case of lentivirus susceptibility-1 (Lv1) and restriction factor-1 (Ref1), or after reverse transcription in the case of Friend virus susceptibility-1 (Fv1). Fv1, which is present only in mice, has variant alleles that specifically inhibit reciprocal murine leukemia virus (MLV) strains. Lv1, the presumed (but as yet unidentified) restriction factor in non-human primates, exhibits highly variable specificity among monkey species, and some forms can inhibit both certain MLV strains and a range of lentiviruses. Likewise, Ref1, the human restriction factor, might be a humanspecific variant of Lv1, and can block infection by certain MLV strains and at least one lentivirus. Abbreviations: EIAV, equine infectious anemia virus; HIV-1, human immunodeficiency virus type 1; SIV, simian immunodeficiency virus.
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to block infection by the well-known phenomenon of receptor interference [12– 14], the mechanism by which Fv1 restricted post-entry events remained unclear, and, to date, remains poorly understood. Breeding experiments suggested that two major alleles of Fv1, termed Fv1n and Fv1b , existed in inbred laboratory mice, and their ability to inhibit infection by murine leukemia virus (MLV) strains differentially formed one scheme for classifying MLV tropism [10,11]. N-tropic MLV strains (N-MLV, infectious for NIH Swiss mice) efficiently infect cells from Fv1n=n but not Fv1b=b homozygous mice whereas B-tropic strains (B-MLV, infectious for Balb/c mice) show precisely the opposite phenotype. Because Fv1 is co-dominant, neither N- nor B-MLV efficiently infect heterozygotes. Conversely, a third class of MLV strains, which are termed NB-tropic, efficiently infect mouse cells irrespective of the presence of Fv1n or Fv1b [10]. Not all mouse strains carry Fv1n or Fv1b ; some carry a null allele, Fv18, and their cells are equivalently susceptible to infection by N-, B- and NB-tropic MLV strains [15,16]. Others exhibit a phenotype attributable to a fourth Fv1 allele, Fv1nr , which inhibits infection by B-MLV and some N-MLV strains [11,16]. Fv1-mediated resistance is not absolute, but sufficiently strong (10 –1000-fold per replication cycle in vitro) to result in a substantial decrease in the frequency of leukemias in mice carrying restricted virus strains [17,18]. Two crucial observations indicated that the means by which Fv1 blocked infection was entirely unique. First, infection of restricting cells by a particular virus particle is strongly facilitated by the presence of other restricted virus particles, that is, titration curves fit a ‘two-hit’ model whereby the frequency of infection is proportional to the square of the inoculating virus dose [19 – 22]. Moreover, restriction is abrogated by treatment of target cells by inactive virion particles [23,24]. These findings, coupled with its dominant pattern of inheritance, strongly suggested that Fv1 encoded a saturable inhibitor of MLV infection. Second, the viral determinants for N- and B-tropism map to the capsid protein [25]. Residue 110 of the MLV capsid protein specifically determines N-tropism versus B-tropism [26] suggesting that the incoming viral capsid contains the target for restriction by Fv1 (Fig. 1). Fv1 was identified in the mid 90 s by positional cloning [27]. Notably, it encodes a protein that is related (, 60% homology) to the capsid-like domain of the ERV-L family of endogenous retroviral Gag proteins. There are hundreds of copies of these retroviral elements in primates and mice, but substantially fewer in other mammals [28]. In mice, it appears that none of the other ERV-L-like elements contribute to restriction because inheritance of the restricting phenotype follows that expected of a single gene [29]. Moreover, expression of Fv1 in non-murine cells is sufficient to impart a restricting phenotype. Aside from its apparent retroviral origin, Fv1 is almost entirely unrelated to the MLV capsid protein [27]. It is expressed at extremely low levels, which might explain why restriction is saturable by high levels of incoming virions. http://timi.trends.com
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Restriction of murine retroviruses by non-murine restriction factors Non-murine cells are resistant to many murine retroviruses because they lack functional receptors. Nonetheless, cells from several mammalian species, including humans, specifically restrict infection by N-MLV [1]. Although Southern blot and genome sequence analysis indicates that mice are the only species to carry an Fv1 gene [27], human cells behave superficially as if they carry an Fv1b allele. Infection of human cells with N-MLV gives rise to a two-hit titration curve, and restriction can be abrogated by treatment with high does of N-MLV but not B-MLV particles [30]. Because of the remarkable similarity in the restriction profile of human and Fv1b=b murine cells, human cells almost certainly express a factor that has a similar activity to Fv1b, which is termed Restriction factor-1 (Ref1; Fig. 1). Other mammalian species also specifically restrict N-MLV, including cows, dogs, pigs, hamsters and some primates [1,31]. Clearly, retrovirus restriction factors in mammals are widespread and have arisen on more than one occasion during mammalian evolution. Precisely the same amino-acid residue that confers sensitivity and/or resistance to Fv1n and Fv1b (capsid 110) in mice also controls sensitivity to Ref1 in human cells [1]. Thus, restriction factors have not only arisen independently in mice and humans, but also appear to recognize the same viral capsid determinant. Fv1-like restriction of lentiviruses Numerous attempts have been made to derive an animal model for acquired immune deficiency syndrome (AIDS) by inoculation of non-human species with human immunodeficiency virus type 1 (HIV-1) [32 – 34]. Unfortunately, HIV-1 does not replicate efficiently in any non-human species except chimpanzees and gibbon apes [35]. Attempts to adapt HIV-1 to a more practically useful species, such as macaques, have been largely unsuccessful. However, there are numerous related lentiviruses in primates [the simian immunodeficiency viruses (SIVs); see Box 1], and some can replicate efficiently and sometimes cause AIDS in non-human primates. A second group of human AIDS viruses, HIV-2, appear significantly more amenable to adaptation to non-human primates [36 – 38]. The block to HIV-1 replication in macaque cells occurs early in the viral life cycle, before the completion of reverse transcription [39,40]. However, chimeric simian-human immunodeficiency viruses (SHIVs) containing an HIV-1 envelope protein in an otherwise simian immunodeficiency virus from macaque (SIVmac) context replicate to high titer in macaque cells, indicating that the block is unlikely to be at the level of virus entry [41,42]. These observations suggest the presence of an intracellulartropism determinant that distinguishes HIV-1 and SIVmac. Consistent with this hypothesis, HIV-1 and SIVmac vectors pseudotyped with a pan-tropic envelope protein from vesicular stomatitis virus (VSV-G) exhibit distinct and frequently opposing tropism for primate cells [43]. Specifically, most old world (African and Asian) monkey cell lines restrict infection by VSV-G-pseudotyped HIV-1, most new world (South American) monkey cell lines restrict infection by SIVmac, and some primate cell lines
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Box 1. Human and non-human primate lentiviruses African primates harbor a plethora of lentiviruses and phylogenetic evidence indicates that human acquired immune deficiency syndrome (AIDS) epidemics arose as a consequence of transmission of two of these viruses to humans. Human immunodeficiency virus type 1 (HIV-1) is most closely related to simian immunodeficiency virus (SIV) strains found, albeit rarely, in chimpanzees (SIV)cpz [61]. A virus that occurs naturally in sooty mangebeys, SIVsm (simian immunodeficiency virus from sooty mangebeys), was apparently transmitted to humans on multiple occasions [62], giving rise to HIV-2. A very closely related virus also thought to originate in sooty mangebeys [63] was also isolated from Rhesus macaques in a US primate center [64]. This virus is referred to as SIVmac (simian immunodeficiency virus from macaques), and clones of this virus are the most widely used in the rhesus macaque model of human AIDS. SIVmac also forms the basis for the construction of chimeric SIV –HIV-1 constructs, SHIVs, which contain HIV-1 envelopes and are being increasingly used in AIDS research. HIV-1 and HIV-2 are no more closely related to each other than they are to each of the other major primate lentivirus lineages (of which there are at least five; e.g. SIVagm from African green monkeys). Why only the SIVcpz/HIV-1 and SIVsm/SIVmac/HIV-2 lineages have colonized humans is unknown.
restrict both. Importantly, examination of primary cells from various tissues indicates that this phenomenon is determined by the species and not by the tissue of origin of the target cells [43]. Recently, three groups have shown that the resistance of primate cell lines to infection by HIV-1 and/or SIVmac exhibits several of the characteristics that were originally described in the context of murine Fv1-mediated MLV resistance [2 – 4]. In particular, the resistant phenotype is dominant: fusion of HIV-1-susceptible human cells with HIV-1-resistant monkey cells results in heterokaryons that are HIV-1 resistant, suggesting the presence of an inhibitor in monkeys [2,4]. Moreover, infection of restricting monkey cells with high doses of HIV-1 or SIVmac results in titration curves that fit a multi-hit model, and saturation of HIV-1- or SIVmac-resistant monkey cells with restricted virions or virus-like particles can abrogate restriction [2,3]. Together, these findings strongly suggest that primates carry restriction factors that exhibit variable specificity and confer resistance to infection by one or more lentiviruses. Because of shared characteristics with Fv1-mediated MLV restriction, the implied factor that mediates resistance to HIV-1 and/or SIVmac in primates is termed lentivirus susceptibility factor-1 (Lv1; Fig. 1). Like
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Fv1 and Ref1, the viral determinant that governs Lv1 restriction maps to capsid. Specifically, replacement of the capsid in an SIVmac clone with that of HIV-1 results in a virus that behaves largely, in terms of its tropism for multiple primate cell lines, as if it were HIV-1 rather than SIVmac [2,31,44,45]. Moreover, viral constructs containing the SIVmac capsid in an otherwise HIV-1 background behave more like SIVmac than HIV-1. Lv1 and Ref1 can inhibit multiple widely divergent retroviruses Because Lv1- and Ref1-dependent resistance to retroviral infection is saturable, cross-abrogation studies can be used to determine whether two distinct retroviruses are inhibited by the same restriction factor in a given cell. Whereas cell lines from most primate species tested thus far (albeit a very limited sample; see Table 1) restrict either HIV-1 or SIVmac but not both, some cell lines, in particular those derived from African green monkeys (AGM), restrict infection by multiple primate lentiviruses, including HIV-1, HIV-2 and SIVmac [2,3,31]. If AGM cells are saturated with virus-like particles derived from any of these restricted lentiviruses before challenge with another, then restriction is abrogated. This argues that a single form of Lv1 exists in AGM cells, which restricts infection by each of these lentiviruses. Like human cells, several AGM cell lines also potently restrict N-MLV [1,31], suggesting the presence of a Ref1like restriction factor in addition to Lv1. Remarkably, however, saturation of AGM cells with primate lentivirus particles completely abrogates N-MLV restriction, increasing susceptibility by . 100-fold [31]. This suggests that Lv1 alone, rather than two independent restriction factors, is responsible for both lentivirus- and N-MLVrestriction in AGM cells (Fig. 1). Conversely, saturation of human cells with HIV-1, HIV-2 or SIVmac particles only marginally affects N-MLV restriction [31], presumably because these primate lentiviruses are not efficiently recognized by – and therefore cannot saturate – the Ref1 restriction factor in humans. N-MLV is not the only shared retroviral target of Ref1 in humans and Lv1 in AGMs. Equine infectious anemia virus (EIAV), a lentivirus that naturally infects horses, is restricted in both human and AGM cells; in each case, cross-abrogation experiments suggest that the same factors that restrict N-MLV and primate lentiviruses are
Table 1. Retrovirus restricting properties of cell lines from various speciesa,b Target cell (restriction factor)
Retroviruses known to be restricted
Retroviruses known to be unrestricted
Mouse (Fv1n) Mouse (Fv1b) Mouse (Fv18) Human (Ref1) Rhesus monkey (Lv1) African green monkey (Lv1) Owl monkey (Lv1) Squirrel monkey (Lv1)
B-MLV N-MLV
N-MLV, NB-MLV, HIV-1 B-MLV, NB-MLV N-MLV, B-MLV, NB-MLV B-MLV, NB-MLV, HIV-1, HIV-2, SIVmac, SIVagm N-MLV, B-MLV, SIVmac B-MLV, SIVagm N-MLV, B-MLV, SIVmac N-MLV, B-MLV, HIV-1
a
N-MLV, EIAV HIV-1 N-MLV, HIV-1, HIV-2, SIVmac, EIAV HIV-1 SIVmac
Abbreviations: B-MLV, B-tropic MLV; EIAV, equine infectious anemia virus; HIV-1, human immunodeficiency virus type 1; HIV-2, human immunodeficiency virus type 2; MLV, murine leukemia virus; NB-MLV, NB-tropic MLV; N-MLV, N-tropic MLV; SIVagm, simian immunodeficiency virus from African green monkeys; SIVmac, simian immunodeficiency virus from macaques. b ‘Restricted’ is defined as saturable inhibition of the early steps of retroviral infection. ‘Unrestricted’ means that the early post-entry steps of the retroviral life cycle proceed efficiently, not necessarily that a complete cycle is completed or spreading of infection can be achieved. http://timi.trends.com
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responsible [31]. In fact, although surveys have not been exhaustive, saturation of AGM or human cells with any restricted retrovirus at least partially abrogates restriction of any other restricted retrovirus [2,3,31]. Thus, both Lv1 in AGMs and Ref1 in humans appear capable of restricting overlapping subsets of retroviruses. This is surprising given that the capsids encoded by the restricted retroviruses share little sequence homology. Paradoxically, Lv1 in AGMs and Ref1 in humans also share the ability to distinguish between almost identical capsids encoded by N-MLV and B-MLV. Lv1 and Ref1: one and the same, or members of a large family of primate restriction factors? These findings raise the possibility that Ref1 is simply a human-specific variant of Lv1 that lacks the ability to restrict HIV-1, HIV-2 and SIVmac. In fact, it could be that the entire variation in the ability of primate cells to restrict retroviral infection reflects divergence in a single ancestral Lv1 allele. Thus, each primate species or even each individual might carry its own specific Lv1 variant. This hypothesis is the easiest to reconcile with the finding of universal cross-abrogation by restricted retroviruses. Variability in the restriction profiles of cell lines from different primate species, including primates that are closely related to each other, such as AGM subspecies [31], would indicate that Lv1 should be highly polymorphic, if it is indeed a single entity. Conversely, because of the similar specificity (for MLV restriction) of Fv1b in mice, Ref1 in humans and Lv1 in AGMs, it is quite possible that Lv1 and Ref1 are related to Fv1, and are derived from endogenous retroviral Gag or capsid-like proteins. The genomes of all mammals are populated by thousands of endogenous proviruses, providing ample starting material for the genesis of restriction factors; indeed, endogenous retroviral envelope-mediated Fv4-like activities have arisen on multiple occasions. It is not difficult to imagine that restriction factors could have arisen on multiple occasions and that the variable restricting properties of primate cells reflects the action of several independent genes. It is even possible that a single cell could express multiple restriction factors that are responsible for its overall restriction properties. The occurrence of restriction factors and retroviruses in contemporary primate species does not fit with any simple evolutionary model that would illuminate this argument and, clearly, the identification of Lv1 and Ref1 is required to resolve these issues. Mechanism of action of restriction factors Fv1 is the only restriction factor to have been identified and the domains that function to mediate restriction have been partly delineated [46,47] (Fig. 2). The Fv1 protein is either 440 or 459 residues in length and shares ,60% sequence identity with the capsid-like domains of the human and murine ERV-L family of endogenous retroviruses [27]. It contains a recognizable major homology region (MHR), characteristic of all ortho-retroviral capsid proteins (Fig. 1). N- and B- variants of Fv1 differ at three positions towards the C-terminus, and each of these contribute to restriction specificity in a complex combinatorial manner [47]. By combining variants at each of these http://timi.trends.com
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MHR --Q---E----ΦX-RX---Fv1 NRQKAKEHARKWILRVWDNG
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Fig. 2. Elements of Fv1 required for inhibitory activity. The Fv1 protein is homologous to human and murine endogenous retroviral Gag-like proteins and encodes a central sequence motif that is similar to the major homology region (MHR) region found in all ortho-retroviral Gag proteins (dark green shading). An alignment of the consensus MHR and the analogous Fv1 sequence is shown. (F ¼ aromatic residues, X ¼ non-aromatic hydrophobic residues). No single residue or motif in the N-terminal Fv1 domain has been identified that is absolutely required for restriction, and a significant fraction is dispensable for function. Nonetheless, large deletions in this domain inhibit or ablate restriction activity [47]. The originally identified Fv1n and Fv1b alleles are identical except for three positions in the C-terminal domain (at positions 358, 399 and the extreme C-terminus); each of these differences contributes to the restriction specificity of Fv1 [46,47]. It is worth noting that Fv1 alleles in other mouse species harbor numerous additional polymorphisms [65], and non-synonymous mutations are found more frequently than expected suggesting selection for change. However, the impact of these polymorphisms on restriction of retroviruses has not been determined.
positions it is relatively straightforward to derive artificial Fv1 alleles with expanded specificity. Indeed, some recombinant Fv1 proteins can restrict both N-MLV and B-MLV, as well as NB-tropic viruses that are not ordinarily restricted by either parent allele [46,47]. Almost nothing is known about the mechanism by which restriction factors actually inhibit retroviral infection. Based in part on the specificity of Fv1 restriction, a favored model for inhibition involves direct binding of the incoming viral capsid by the restriction factor [48,49] (Fig. 1). However, no form of physical interaction between Fv1- and MLV-capsid has been demonstrated thus far. One surprising property of Fv1 is that overexpression of a particular Fv1 protein inhibits the inhibitory activity of another that is co-expressed at a lower level [46]. This suggests that Fv1 acts as a component of a complex to effect restriction, perhaps as a homomultimer. Gag proteins from all retroviruses multimerize, so it would be unsurprising if this is true of Fv1. Consistent with this, the integrity of the MHR is required for restriction activity [47]. Alternatively, it is possible that Fv1 complexes with other host-cell proteins that are required for restriction, and that overexpression of one form of Fv1 saturates other required factors. Fv1 acts predominantly after reverse transcription but before integration [50,51], whereas Ref1 and Lv1 apparently act to inhibit reverse transcription [1– 4] (Fig. 1). The inhibition of distinct (albeit temporally proximal) steps of the viral life cycle by different restriction factors seems at odds with the targeting of almost precisely the same viral determinant (at least in the case of MLV) [1,26]. It could be that the distinction between apparently pre- and postreverse transcription blocks is artificial. The kinetics of reverse-transcript accumulation have not been accurately measured under restricting conditions and it might be that a consequence of Lv1 and Ref1 (but not Fv1) restriction is destabilization of nascent viral DNA rather than inhibition of its synthesis. A second possibility is that restriction factor expression levels, which are extremely
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low in Fv1, could impact the kinetics of restriction. Perhaps initial recognition of the incoming viral reverse transcription complex occurs at the same point for all restriction factors, but inhibition requires additional events or factors that differ for each restriction factor or in the context of primate versus murine cells. Although the mechanism of action of restriction factors remains to be defined, their shared properties might give some insight into the retroviral life cycle. In particular, although capsid association with the pre-integration complex has been demonstrated biochemically for MLV [52,53], purification of HIV-1 pre-integration complexes has revealed an apparent lack of capsid protein association, and it was thought that lentivirus cores rapidly disassemble after entry [54– 56]. That capsid encodes the target for restriction suggests it might have a hitherto unappreciated role in the post-entry, pre-integration steps of the lentivirus life cycle. At a minimum, these findings indicate that some fraction of capsid must, at least temporarily, remain associated with the viral reverse transcription complex after virus entry. Recent fluorescent microscopic analyses of HIV-1 reverse-transcription complexes during infection also suggest that this is the case [57]. Concluding remarks Much remains to be learned about host genes that inhibit retroviral replication. This review has focused on a particular class of Fv1-like endogenous inhibitors, but these are by no means the sole cellular defense against retroviral infection, and it is becoming increasingly evident that a variety of non-immunological antiretroviral activities have evolved in vertebrates. Other recently described inhibitors of retroviruses include CEM15, which acts during the late-stage of the lentivirus life cycle to attenuate the infectivity of progeny virions [58], and ZAP, a gene that appears to destabilize or inhibit the nuclear export of retroviral mRNAs [59]. Intuitively, it seems surprising that hosts could evolve inhibitors that are effective in blocking retroviral infection because exogenous retroviruses evolve at a much greater pace than do the hosts. An endogenous, slowly evolving inhibitor would be predicted to be of limited benefit, in much the same way as antiretroviral drugs that are only temporarily effective against a constantly mutating swarm of retroviral sequences. Perhaps endogenous inhibitors originally evolved as a defense against pathogenic effects of endogenous retroviruses, which evolve at a slower, more host-like pace than their exogenous cousins. The recently described activities that apparently target certain exogenous viruses might simply be fortuitous. Alternatively, endogenous cellular inhibitors might function in a way that is less affected by viral sequence variation, or they might selectively target essential functions in the retroviral life cycle that are less tolerant of sequence variation. As such, endogenous inhibitors might provide clues as to what constitutes useful targets for drug development. Indeed, it appears that the inhibitory effects of CEM15 on HIV-SIV replication cannot be escaped by simple viral mutations because these viruses have undergone the much more complex adaptation of acquiring an additional http://timi.trends.com
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gene whose sole function is to block the effect of this cellular inhibitor [58]. Although recent reports have documented several very surprising and interesting phenomena, we are little closer to understanding how restriction factors actually protect the cell from retroviral infection, and whether such innate inhibitors could be usefully manipulated or mimicked in the context of therapeutic intervention. Perhaps one day it will be possible to exploit the inhibitory properties of Fv1-like restriction factors in the context of gene therapy to provide protection from retroviral disease. Thus, the identification of Lv1 and Ref1 and the elucidation of the mechanism of restriction are clear research priorities. Given that there is an ongoing risk of zoonosis, particularly from African primate lentiviruses [60], it might also be important to document precisely which retroviruses are blocked by restriction factors and other inhibitory genes in humans. Finally, an important and probably achievable goal is the engineering of HIV-1 strains that avoid Lv1 restriction in monkeys. These could form the basis for a viable HIV-1 animal model that would substantially impact the way in which AIDS vaccine research is conducted, and perhaps facilitate the pre-clinical evaluation of new therapies. Clearly, the far-reaching implications of a curious hereditary resistance to murine leukemia virus, first observed over three decades ago, are only beginning to be appreciated. Acknowledgements I thank Theodora Hatziioannou for critical review of the manuscript. Studies in the laboratory of P.D.B. are supported by the NIH, AmFAR and an Elizabeth Glaser Scientist Award from the Elizabeth Glaser Pediatric AIDS Foundation.
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with Fv-4 gene-controlled resistance to Friend leukemia virus infection. Virology 128, 127– 139 Kozak, C.A. et al. (1984) A unique sequence related to the ecotropic murine leukemia virus is associated with the Fv-4 resistance gene. Proc. Natl. Acad. Sci. U. S. A. 81, 834 – 837 Ikeda, H. and Sugimura, H. (1989) Fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties. J. Virol. 63, 5405– 5412 Hartley, J.W. and Rowe, W.P. (1975) Clonal cells lines from a feral mouse embryo which lack host-range restrictions for murine leukemia viruses. Virology 65, 128 – 134 Kozak, C.A. (1985) Analysis of wild-derived mice for Fv-1 and Fv-2 murine leukemia virus restriction loci: a novel wild mouse Fv-1 allele responsible for lack of host range restriction. J. Virol. 55, 281 – 285 Rowe, W.P. (1972) Studies of genetic transmission of murine leukemia virus by AKR mice. I. Crosses with Fv-1 n strains of mice. J. Exp. Med. 136, 1272 – 1285 Rowe, W.P. and Hartley, J.W. (1972) Studies of genetic transmission of murine leukemia virus by AKR mice. II. Crosses with Fv-1 b strains of mice. J. Exp. Med. 136, 1286 – 1301 Decleve, A. et al. (1975) Replication kinetics of N- and B-tropic murine leukemia viruses on permissive and nonpermissive cells in vitro. Virology 65, 320 – 332 Duran-Troise, G. et al. (1977) Loss of Fv-1 restriction in Balb/3T3 cells following infection with a single N tropic murine leukemia virus particle. Cell 10, 479 – 488 Pincus, T. et al. (1975) A major genetic locus affecting resistance to infection with murine leukemia viruses. IV. Dose-response relationships in Fv-1-sensitive and resistant cell cultures. Virology 65, 333 – 342 Tennant, R.W. et al. (1979) Characterization of Fv-1 host range strains of murine retroviruses by titration and p30 protein characteristics. Virology 99, 349 – 357 Boone, L.R. et al. (1990) Abrogation of Fv-1 restriction by genomedeficient virions produced by a retrovirus packaging cell line. J. Virol. 64, 3376 – 3381 Bassin, R.H. et al. (1978) Abrogation of Fv-1b restriction with murine leukemia viruses inactivated by heat or by gamma irradiation. J. Virol. 26, 306 – 315 DesGroseillers, L. and Jolicoeur, P. (1983) Physical mapping of the Fv1 tropism host range determinant of BALB/c murine leukemia viruses. J. Virol. 48, 685 – 696 Kozak, C.A. and Chakraborti, A. (1996) Single amino acid changes in the murine leukemia virus capsid protein gene define the target of Fv1 resistance. Virology 225, 300 – 305 Best, S. et al. (1996) Positional cloning of the mouse retrovirus restriction gene Fv1. Nature 382, 826– 829 Benit, L. et al. (1999) ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals. J. Virol. 73, 3301 – 3308 Pincus, T. et al. (1971) A major genetic locus affecting resistance to infection with murine leukemia viruses. II. Apparent identity to a major locus described for resistance to friend murine leukemia virus. J. Exp. Med. 133, 1234– 1241 Towers, G. et al. (2002) Abrogation of Ref1 retrovirus restriction in human cells. J. Virol. 76, 2548 – 2550 Hatziioannou, T. et al. (2003) Restriction of multiple divergent retroviruses by Lv1 and Ref1. EMBO J. 22, 385– 394 Gartner, S. et al. (1994) Adaptation of HIV-1 to pigtailed macaques. J. Med. Primatol. 23, 155– 163 Levy, J.A. et al. (1985) AIDS-associated retroviruses (ARV) can productively infect other cells besides human T helper cells. Virology 147, 441 – 448 McClure, M.O. et al. (1987) HIV infection of primate lymphocytes and conservation of the CD4 receptor. Nature 330, 487 – 489 Gardner, M.B. and Luciw, P.A. (1989) Animal models of AIDS. FASEB J. 3, 2593 – 2606 Otten, R.A. et al. (1994) Differential replication and pathogenic effects of HIV-1 and HIV-2 in Macaca nemestrina. AIDS 8, 297– 306 McClure, J. et al. (2000) Derivation and characterization of a highly pathogenic isolate of human immunodeficiency virus type 2 that
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Restriction factors: a defense against retroviral infection Paul D. Bieniasz Aaron Diamond AIDS Research Center, 455 First Avenue, New York, NY 10016, USA
Susceptibility to retroviral infection is determined, in part, by host genes with antiviral activity. The Fv1 gene, which inhibits murine leukemia virus infection in mice, encodes one such resistance factor, and was long thought to be unique in that it restricts post-entry, preintegration steps of retroviral replication. However, recent findings suggest the existence of similar restriction factors in primates, including humans. These factors, termed Lv1 and Ref1, can inhibit a range of retroviruses, including human immunodeficiency virus type 1 and its relatives. Fv1, Lv1 and Ref1 target capsid determinants to block infection but can be saturated by incoming virions. Primate- and murine-retrovirus restriction factors have diverse and overlapping specificities, and some variants of Lv1, as well as Ref1, apparently recognize and inhibit infection by widely divergent retroviruses. During mammalian evolution, a variety of mechanisms have arisen to purge the host of viral infectious agents. The adaptive and innate immune responses are primarily responsible for providing protection against viral pathogens, but it is becoming increasingly clear that dominant, inhibitory gene products can also have an important role in controlling host susceptibility. One class of such inhibitors, which for the purpose of this review are termed ‘restriction factors’, block retroviral infection by targeting the incoming capsid and preventing the efficient progression of postentry, pre-integration events. For many years, the existence of retrovirus restriction factors was thought to be unique to the mouse. However, recent evidence indicates that capsid-targeting cellular inhibitors of retroviral infection (Fig. 1) are far more widespread than previously thought, are capable of inhibiting infection by a variety of retroviruses [1– 4], and have probably substantially impacted the prevalence of retroviral infection and disease in mammals.
inoculation of animals. However, two dominant genes – Fv1 and Fv4 – were of particular interest because they were active in vitro: cultured cells derived from mice harboring the genes exhibited substantial resistance to infection [7 –11]. While Fv4 was subsequently shown to encode an endogenous retroviral envelope protein, and
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Fv1: the prototype retrovirus restriction factor The description of a collection of heritable traits in mice that controlled susceptibility to leukemia induced by the Friend strain of murine leukemia virus began more than 30 years ago [5,6]. Many of the implied Friend virus susceptibility (Fv) genes were subsequently shown to modify either immune responses to the virus, or cell proliferation, and only exerted their phenotype upon Corresponding author: Paul D. Bieniasz ([email protected]).
Fig. 1. Retrovirus restriction factors. Restriction factors target the capsid of incoming retroviruses to block infection. Restriction occurs before reverse transcription in the case of lentivirus susceptibility-1 (Lv1) and restriction factor-1 (Ref1), or after reverse transcription in the case of Friend virus susceptibility-1 (Fv1). Fv1, which is present only in mice, has variant alleles that specifically inhibit reciprocal murine leukemia virus (MLV) strains. Lv1, the presumed (but as yet unidentified) restriction factor in non-human primates, exhibits highly variable specificity among monkey species, and some forms can inhibit both certain MLV strains and a range of lentiviruses. Likewise, Ref1, the human restriction factor, might be a humanspecific variant of Lv1, and can block infection by certain MLV strains and at least one lentivirus. Abbreviations: EIAV, equine infectious anemia virus; HIV-1, human immunodeficiency virus type 1; SIV, simian immunodeficiency virus.
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to block infection by the well-known phenomenon of receptor interference [12– 14], the mechanism by which Fv1 restricted post-entry events remained unclear, and, to date, remains poorly understood. Breeding experiments suggested that two major alleles of Fv1, termed Fv1n and Fv1b , existed in inbred laboratory mice, and their ability to inhibit infection by murine leukemia virus (MLV) strains differentially formed one scheme for classifying MLV tropism [10,11]. N-tropic MLV strains (N-MLV, infectious for NIH Swiss mice) efficiently infect cells from Fv1n=n but not Fv1b=b homozygous mice whereas B-tropic strains (B-MLV, infectious for Balb/c mice) show precisely the opposite phenotype. Because Fv1 is co-dominant, neither N- nor B-MLV efficiently infect heterozygotes. Conversely, a third class of MLV strains, which are termed NB-tropic, efficiently infect mouse cells irrespective of the presence of Fv1n or Fv1b [10]. Not all mouse strains carry Fv1n or Fv1b ; some carry a null allele, Fv18, and their cells are equivalently susceptible to infection by N-, B- and NB-tropic MLV strains [15,16]. Others exhibit a phenotype attributable to a fourth Fv1 allele, Fv1nr , which inhibits infection by B-MLV and some N-MLV strains [11,16]. Fv1-mediated resistance is not absolute, but sufficiently strong (10 –1000-fold per replication cycle in vitro) to result in a substantial decrease in the frequency of leukemias in mice carrying restricted virus strains [17,18]. Two crucial observations indicated that the means by which Fv1 blocked infection was entirely unique. First, infection of restricting cells by a particular virus particle is strongly facilitated by the presence of other restricted virus particles, that is, titration curves fit a ‘two-hit’ model whereby the frequency of infection is proportional to the square of the inoculating virus dose [19 – 22]. Moreover, restriction is abrogated by treatment of target cells by inactive virion particles [23,24]. These findings, coupled with its dominant pattern of inheritance, strongly suggested that Fv1 encoded a saturable inhibitor of MLV infection. Second, the viral determinants for N- and B-tropism map to the capsid protein [25]. Residue 110 of the MLV capsid protein specifically determines N-tropism versus B-tropism [26] suggesting that the incoming viral capsid contains the target for restriction by Fv1 (Fig. 1). Fv1 was identified in the mid 90 s by positional cloning [27]. Notably, it encodes a protein that is related (, 60% homology) to the capsid-like domain of the ERV-L family of endogenous retroviral Gag proteins. There are hundreds of copies of these retroviral elements in primates and mice, but substantially fewer in other mammals [28]. In mice, it appears that none of the other ERV-L-like elements contribute to restriction because inheritance of the restricting phenotype follows that expected of a single gene [29]. Moreover, expression of Fv1 in non-murine cells is sufficient to impart a restricting phenotype. Aside from its apparent retroviral origin, Fv1 is almost entirely unrelated to the MLV capsid protein [27]. It is expressed at extremely low levels, which might explain why restriction is saturable by high levels of incoming virions. http://timi.trends.com
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Restriction of murine retroviruses by non-murine restriction factors Non-murine cells are resistant to many murine retroviruses because they lack functional receptors. Nonetheless, cells from several mammalian species, including humans, specifically restrict infection by N-MLV [1]. Although Southern blot and genome sequence analysis indicates that mice are the only species to carry an Fv1 gene [27], human cells behave superficially as if they carry an Fv1b allele. Infection of human cells with N-MLV gives rise to a two-hit titration curve, and restriction can be abrogated by treatment with high does of N-MLV but not B-MLV particles [30]. Because of the remarkable similarity in the restriction profile of human and Fv1b=b murine cells, human cells almost certainly express a factor that has a similar activity to Fv1b, which is termed Restriction factor-1 (Ref1; Fig. 1). Other mammalian species also specifically restrict N-MLV, including cows, dogs, pigs, hamsters and some primates [1,31]. Clearly, retrovirus restriction factors in mammals are widespread and have arisen on more than one occasion during mammalian evolution. Precisely the same amino-acid residue that confers sensitivity and/or resistance to Fv1n and Fv1b (capsid 110) in mice also controls sensitivity to Ref1 in human cells [1]. Thus, restriction factors have not only arisen independently in mice and humans, but also appear to recognize the same viral capsid determinant. Fv1-like restriction of lentiviruses Numerous attempts have been made to derive an animal model for acquired immune deficiency syndrome (AIDS) by inoculation of non-human species with human immunodeficiency virus type 1 (HIV-1) [32 – 34]. Unfortunately, HIV-1 does not replicate efficiently in any non-human species except chimpanzees and gibbon apes [35]. Attempts to adapt HIV-1 to a more practically useful species, such as macaques, have been largely unsuccessful. However, there are numerous related lentiviruses in primates [the simian immunodeficiency viruses (SIVs); see Box 1], and some can replicate efficiently and sometimes cause AIDS in non-human primates. A second group of human AIDS viruses, HIV-2, appear significantly more amenable to adaptation to non-human primates [36 – 38]. The block to HIV-1 replication in macaque cells occurs early in the viral life cycle, before the completion of reverse transcription [39,40]. However, chimeric simian-human immunodeficiency viruses (SHIVs) containing an HIV-1 envelope protein in an otherwise simian immunodeficiency virus from macaque (SIVmac) context replicate to high titer in macaque cells, indicating that the block is unlikely to be at the level of virus entry [41,42]. These observations suggest the presence of an intracellulartropism determinant that distinguishes HIV-1 and SIVmac. Consistent with this hypothesis, HIV-1 and SIVmac vectors pseudotyped with a pan-tropic envelope protein from vesicular stomatitis virus (VSV-G) exhibit distinct and frequently opposing tropism for primate cells [43]. Specifically, most old world (African and Asian) monkey cell lines restrict infection by VSV-G-pseudotyped HIV-1, most new world (South American) monkey cell lines restrict infection by SIVmac, and some primate cell lines
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Box 1. Human and non-human primate lentiviruses African primates harbor a plethora of lentiviruses and phylogenetic evidence indicates that human acquired immune deficiency syndrome (AIDS) epidemics arose as a consequence of transmission of two of these viruses to humans. Human immunodeficiency virus type 1 (HIV-1) is most closely related to simian immunodeficiency virus (SIV) strains found, albeit rarely, in chimpanzees (SIV)cpz [61]. A virus that occurs naturally in sooty mangebeys, SIVsm (simian immunodeficiency virus from sooty mangebeys), was apparently transmitted to humans on multiple occasions [62], giving rise to HIV-2. A very closely related virus also thought to originate in sooty mangebeys [63] was also isolated from Rhesus macaques in a US primate center [64]. This virus is referred to as SIVmac (simian immunodeficiency virus from macaques), and clones of this virus are the most widely used in the rhesus macaque model of human AIDS. SIVmac also forms the basis for the construction of chimeric SIV –HIV-1 constructs, SHIVs, which contain HIV-1 envelopes and are being increasingly used in AIDS research. HIV-1 and HIV-2 are no more closely related to each other than they are to each of the other major primate lentivirus lineages (of which there are at least five; e.g. SIVagm from African green monkeys). Why only the SIVcpz/HIV-1 and SIVsm/SIVmac/HIV-2 lineages have colonized humans is unknown.
restrict both. Importantly, examination of primary cells from various tissues indicates that this phenomenon is determined by the species and not by the tissue of origin of the target cells [43]. Recently, three groups have shown that the resistance of primate cell lines to infection by HIV-1 and/or SIVmac exhibits several of the characteristics that were originally described in the context of murine Fv1-mediated MLV resistance [2 – 4]. In particular, the resistant phenotype is dominant: fusion of HIV-1-susceptible human cells with HIV-1-resistant monkey cells results in heterokaryons that are HIV-1 resistant, suggesting the presence of an inhibitor in monkeys [2,4]. Moreover, infection of restricting monkey cells with high doses of HIV-1 or SIVmac results in titration curves that fit a multi-hit model, and saturation of HIV-1- or SIVmac-resistant monkey cells with restricted virions or virus-like particles can abrogate restriction [2,3]. Together, these findings strongly suggest that primates carry restriction factors that exhibit variable specificity and confer resistance to infection by one or more lentiviruses. Because of shared characteristics with Fv1-mediated MLV restriction, the implied factor that mediates resistance to HIV-1 and/or SIVmac in primates is termed lentivirus susceptibility factor-1 (Lv1; Fig. 1). Like
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Fv1 and Ref1, the viral determinant that governs Lv1 restriction maps to capsid. Specifically, replacement of the capsid in an SIVmac clone with that of HIV-1 results in a virus that behaves largely, in terms of its tropism for multiple primate cell lines, as if it were HIV-1 rather than SIVmac [2,31,44,45]. Moreover, viral constructs containing the SIVmac capsid in an otherwise HIV-1 background behave more like SIVmac than HIV-1. Lv1 and Ref1 can inhibit multiple widely divergent retroviruses Because Lv1- and Ref1-dependent resistance to retroviral infection is saturable, cross-abrogation studies can be used to determine whether two distinct retroviruses are inhibited by the same restriction factor in a given cell. Whereas cell lines from most primate species tested thus far (albeit a very limited sample; see Table 1) restrict either HIV-1 or SIVmac but not both, some cell lines, in particular those derived from African green monkeys (AGM), restrict infection by multiple primate lentiviruses, including HIV-1, HIV-2 and SIVmac [2,3,31]. If AGM cells are saturated with virus-like particles derived from any of these restricted lentiviruses before challenge with another, then restriction is abrogated. This argues that a single form of Lv1 exists in AGM cells, which restricts infection by each of these lentiviruses. Like human cells, several AGM cell lines also potently restrict N-MLV [1,31], suggesting the presence of a Ref1like restriction factor in addition to Lv1. Remarkably, however, saturation of AGM cells with primate lentivirus particles completely abrogates N-MLV restriction, increasing susceptibility by . 100-fold [31]. This suggests that Lv1 alone, rather than two independent restriction factors, is responsible for both lentivirus- and N-MLVrestriction in AGM cells (Fig. 1). Conversely, saturation of human cells with HIV-1, HIV-2 or SIVmac particles only marginally affects N-MLV restriction [31], presumably because these primate lentiviruses are not efficiently recognized by – and therefore cannot saturate – the Ref1 restriction factor in humans. N-MLV is not the only shared retroviral target of Ref1 in humans and Lv1 in AGMs. Equine infectious anemia virus (EIAV), a lentivirus that naturally infects horses, is restricted in both human and AGM cells; in each case, cross-abrogation experiments suggest that the same factors that restrict N-MLV and primate lentiviruses are
Table 1. Retrovirus restricting properties of cell lines from various speciesa,b Target cell (restriction factor)
Retroviruses known to be restricted
Retroviruses known to be unrestricted
Mouse (Fv1n) Mouse (Fv1b) Mouse (Fv18) Human (Ref1) Rhesus monkey (Lv1) African green monkey (Lv1) Owl monkey (Lv1) Squirrel monkey (Lv1)
B-MLV N-MLV
N-MLV, NB-MLV, HIV-1 B-MLV, NB-MLV N-MLV, B-MLV, NB-MLV B-MLV, NB-MLV, HIV-1, HIV-2, SIVmac, SIVagm N-MLV, B-MLV, SIVmac B-MLV, SIVagm N-MLV, B-MLV, SIVmac N-MLV, B-MLV, HIV-1
a
N-MLV, EIAV HIV-1 N-MLV, HIV-1, HIV-2, SIVmac, EIAV HIV-1 SIVmac
Abbreviations: B-MLV, B-tropic MLV; EIAV, equine infectious anemia virus; HIV-1, human immunodeficiency virus type 1; HIV-2, human immunodeficiency virus type 2; MLV, murine leukemia virus; NB-MLV, NB-tropic MLV; N-MLV, N-tropic MLV; SIVagm, simian immunodeficiency virus from African green monkeys; SIVmac, simian immunodeficiency virus from macaques. b ‘Restricted’ is defined as saturable inhibition of the early steps of retroviral infection. ‘Unrestricted’ means that the early post-entry steps of the retroviral life cycle proceed efficiently, not necessarily that a complete cycle is completed or spreading of infection can be achieved. http://timi.trends.com
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responsible [31]. In fact, although surveys have not been exhaustive, saturation of AGM or human cells with any restricted retrovirus at least partially abrogates restriction of any other restricted retrovirus [2,3,31]. Thus, both Lv1 in AGMs and Ref1 in humans appear capable of restricting overlapping subsets of retroviruses. This is surprising given that the capsids encoded by the restricted retroviruses share little sequence homology. Paradoxically, Lv1 in AGMs and Ref1 in humans also share the ability to distinguish between almost identical capsids encoded by N-MLV and B-MLV. Lv1 and Ref1: one and the same, or members of a large family of primate restriction factors? These findings raise the possibility that Ref1 is simply a human-specific variant of Lv1 that lacks the ability to restrict HIV-1, HIV-2 and SIVmac. In fact, it could be that the entire variation in the ability of primate cells to restrict retroviral infection reflects divergence in a single ancestral Lv1 allele. Thus, each primate species or even each individual might carry its own specific Lv1 variant. This hypothesis is the easiest to reconcile with the finding of universal cross-abrogation by restricted retroviruses. Variability in the restriction profiles of cell lines from different primate species, including primates that are closely related to each other, such as AGM subspecies [31], would indicate that Lv1 should be highly polymorphic, if it is indeed a single entity. Conversely, because of the similar specificity (for MLV restriction) of Fv1b in mice, Ref1 in humans and Lv1 in AGMs, it is quite possible that Lv1 and Ref1 are related to Fv1, and are derived from endogenous retroviral Gag or capsid-like proteins. The genomes of all mammals are populated by thousands of endogenous proviruses, providing ample starting material for the genesis of restriction factors; indeed, endogenous retroviral envelope-mediated Fv4-like activities have arisen on multiple occasions. It is not difficult to imagine that restriction factors could have arisen on multiple occasions and that the variable restricting properties of primate cells reflects the action of several independent genes. It is even possible that a single cell could express multiple restriction factors that are responsible for its overall restriction properties. The occurrence of restriction factors and retroviruses in contemporary primate species does not fit with any simple evolutionary model that would illuminate this argument and, clearly, the identification of Lv1 and Ref1 is required to resolve these issues. Mechanism of action of restriction factors Fv1 is the only restriction factor to have been identified and the domains that function to mediate restriction have been partly delineated [46,47] (Fig. 2). The Fv1 protein is either 440 or 459 residues in length and shares ,60% sequence identity with the capsid-like domains of the human and murine ERV-L family of endogenous retroviruses [27]. It contains a recognizable major homology region (MHR), characteristic of all ortho-retroviral capsid proteins (Fig. 1). N- and B- variants of Fv1 differ at three positions towards the C-terminus, and each of these contribute to restriction specificity in a complex combinatorial manner [47]. By combining variants at each of these http://timi.trends.com
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MHR --Q---E----ΦX-RX---Fv1 NRQKAKEHARKWILRVWDNG
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Fig. 2. Elements of Fv1 required for inhibitory activity. The Fv1 protein is homologous to human and murine endogenous retroviral Gag-like proteins and encodes a central sequence motif that is similar to the major homology region (MHR) region found in all ortho-retroviral Gag proteins (dark green shading). An alignment of the consensus MHR and the analogous Fv1 sequence is shown. (F ¼ aromatic residues, X ¼ non-aromatic hydrophobic residues). No single residue or motif in the N-terminal Fv1 domain has been identified that is absolutely required for restriction, and a significant fraction is dispensable for function. Nonetheless, large deletions in this domain inhibit or ablate restriction activity [47]. The originally identified Fv1n and Fv1b alleles are identical except for three positions in the C-terminal domain (at positions 358, 399 and the extreme C-terminus); each of these differences contributes to the restriction specificity of Fv1 [46,47]. It is worth noting that Fv1 alleles in other mouse species harbor numerous additional polymorphisms [65], and non-synonymous mutations are found more frequently than expected suggesting selection for change. However, the impact of these polymorphisms on restriction of retroviruses has not been determined.
positions it is relatively straightforward to derive artificial Fv1 alleles with expanded specificity. Indeed, some recombinant Fv1 proteins can restrict both N-MLV and B-MLV, as well as NB-tropic viruses that are not ordinarily restricted by either parent allele [46,47]. Almost nothing is known about the mechanism by which restriction factors actually inhibit retroviral infection. Based in part on the specificity of Fv1 restriction, a favored model for inhibition involves direct binding of the incoming viral capsid by the restriction factor [48,49] (Fig. 1). However, no form of physical interaction between Fv1- and MLV-capsid has been demonstrated thus far. One surprising property of Fv1 is that overexpression of a particular Fv1 protein inhibits the inhibitory activity of another that is co-expressed at a lower level [46]. This suggests that Fv1 acts as a component of a complex to effect restriction, perhaps as a homomultimer. Gag proteins from all retroviruses multimerize, so it would be unsurprising if this is true of Fv1. Consistent with this, the integrity of the MHR is required for restriction activity [47]. Alternatively, it is possible that Fv1 complexes with other host-cell proteins that are required for restriction, and that overexpression of one form of Fv1 saturates other required factors. Fv1 acts predominantly after reverse transcription but before integration [50,51], whereas Ref1 and Lv1 apparently act to inhibit reverse transcription [1– 4] (Fig. 1). The inhibition of distinct (albeit temporally proximal) steps of the viral life cycle by different restriction factors seems at odds with the targeting of almost precisely the same viral determinant (at least in the case of MLV) [1,26]. It could be that the distinction between apparently pre- and postreverse transcription blocks is artificial. The kinetics of reverse-transcript accumulation have not been accurately measured under restricting conditions and it might be that a consequence of Lv1 and Ref1 (but not Fv1) restriction is destabilization of nascent viral DNA rather than inhibition of its synthesis. A second possibility is that restriction factor expression levels, which are extremely
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low in Fv1, could impact the kinetics of restriction. Perhaps initial recognition of the incoming viral reverse transcription complex occurs at the same point for all restriction factors, but inhibition requires additional events or factors that differ for each restriction factor or in the context of primate versus murine cells. Although the mechanism of action of restriction factors remains to be defined, their shared properties might give some insight into the retroviral life cycle. In particular, although capsid association with the pre-integration complex has been demonstrated biochemically for MLV [52,53], purification of HIV-1 pre-integration complexes has revealed an apparent lack of capsid protein association, and it was thought that lentivirus cores rapidly disassemble after entry [54– 56]. That capsid encodes the target for restriction suggests it might have a hitherto unappreciated role in the post-entry, pre-integration steps of the lentivirus life cycle. At a minimum, these findings indicate that some fraction of capsid must, at least temporarily, remain associated with the viral reverse transcription complex after virus entry. Recent fluorescent microscopic analyses of HIV-1 reverse-transcription complexes during infection also suggest that this is the case [57]. Concluding remarks Much remains to be learned about host genes that inhibit retroviral replication. This review has focused on a particular class of Fv1-like endogenous inhibitors, but these are by no means the sole cellular defense against retroviral infection, and it is becoming increasingly evident that a variety of non-immunological antiretroviral activities have evolved in vertebrates. Other recently described inhibitors of retroviruses include CEM15, which acts during the late-stage of the lentivirus life cycle to attenuate the infectivity of progeny virions [58], and ZAP, a gene that appears to destabilize or inhibit the nuclear export of retroviral mRNAs [59]. Intuitively, it seems surprising that hosts could evolve inhibitors that are effective in blocking retroviral infection because exogenous retroviruses evolve at a much greater pace than do the hosts. An endogenous, slowly evolving inhibitor would be predicted to be of limited benefit, in much the same way as antiretroviral drugs that are only temporarily effective against a constantly mutating swarm of retroviral sequences. Perhaps endogenous inhibitors originally evolved as a defense against pathogenic effects of endogenous retroviruses, which evolve at a slower, more host-like pace than their exogenous cousins. The recently described activities that apparently target certain exogenous viruses might simply be fortuitous. Alternatively, endogenous cellular inhibitors might function in a way that is less affected by viral sequence variation, or they might selectively target essential functions in the retroviral life cycle that are less tolerant of sequence variation. As such, endogenous inhibitors might provide clues as to what constitutes useful targets for drug development. Indeed, it appears that the inhibitory effects of CEM15 on HIV-SIV replication cannot be escaped by simple viral mutations because these viruses have undergone the much more complex adaptation of acquiring an additional http://timi.trends.com
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gene whose sole function is to block the effect of this cellular inhibitor [58]. Although recent reports have documented several very surprising and interesting phenomena, we are little closer to understanding how restriction factors actually protect the cell from retroviral infection, and whether such innate inhibitors could be usefully manipulated or mimicked in the context of therapeutic intervention. Perhaps one day it will be possible to exploit the inhibitory properties of Fv1-like restriction factors in the context of gene therapy to provide protection from retroviral disease. Thus, the identification of Lv1 and Ref1 and the elucidation of the mechanism of restriction are clear research priorities. Given that there is an ongoing risk of zoonosis, particularly from African primate lentiviruses [60], it might also be important to document precisely which retroviruses are blocked by restriction factors and other inhibitory genes in humans. Finally, an important and probably achievable goal is the engineering of HIV-1 strains that avoid Lv1 restriction in monkeys. These could form the basis for a viable HIV-1 animal model that would substantially impact the way in which AIDS vaccine research is conducted, and perhaps facilitate the pre-clinical evaluation of new therapies. Clearly, the far-reaching implications of a curious hereditary resistance to murine leukemia virus, first observed over three decades ago, are only beginning to be appreciated. Acknowledgements I thank Theodora Hatziioannou for critical review of the manuscript. Studies in the laboratory of P.D.B. are supported by the NIH, AmFAR and an Elizabeth Glaser Scientist Award from the Elizabeth Glaser Pediatric AIDS Foundation.
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