N-tropic Murine Leukemia Virus Research Articles

The small-molecule 3G11 inhibits HIV-1 reverse transcription.

The small-molecule 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (3G11) inhibits HIV-1 replication in the human T cell line MT-2. Here, we showed that 3G11 specifically and potently blocks HIV-1 infection. By contrast, 3G11 did not block other retroviruses such as HIV-2, simian immunodeficiency virus (SIVmac ), bovine immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, N-tropic murine leukemia virus, B-tropic murine leukemia virus, and Moloney murine leukemia virus. Analysis of DNA metabolism by real-time PCR revealed that 3G11 blocks the formation of HIV-1 late reverse transcripts during infection prior to the first-strand transfer step. In agreement, an in vitro assay revealed that 3G11 blocks the enzymatic activity of HIV-1 reverse transcriptase as strong as nevirapine. Docking of 3G11 to the HIV-1 reverse transcriptase enzyme suggested a direct interaction between residue L100 and 3G11. In agreement, an HIV-1 virus bearing the reverse transcriptase change L100I renders HIV-1 resistant to 3G11, which suggested that the reverse transcriptase enzyme is the viral determinant for HIV-1 sensitivity to 3G11. Although NMR experiments revealed that 3G11 binds to the HIV-1 capsid, functional experiments suggested that capsid is not the viral determinant for sensitivity to 3G11. Overall, we described a novel non-nucleoside reverse transcription inhibitor that blocks HIV-1 infection.

Functional Evidence for the Involvement of Microtubules and Dynein Motor Complexes in TRIM5α-Mediated Restriction of Retroviruses

The tripartite motif (TRIM) family of proteins includes the TRIM5α antiretroviral restriction factor. TRIM5α from many Old World and some New World monkeys can restrict the human immunodeficiency virus type 1 (HIV-1), while human TRIM5α restricts N-tropic murine leukemia virus (N-MLV). TRIM5α forms highly dynamic cytoplasmic bodies (CBs) that associate with and translocate on microtubules. However, the functional involvement of microtubules or other cytoskeleton-associated factors in the viral restriction process had not been shown. Here, we demonstrate the dependency of TRIM5α-mediated restriction on microtubule-mediated transport. Pharmacological disruption of the microtubule network using nocodazole or disabling it using paclitaxel (originally named taxol) decreased the restriction of N-MLV and HIV-1 by human or simian alleles of TRIM5α, respectively. In addition, pharmacological inhibition of dynein motor complexes using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and small interfering RNA-mediated depletion of the dynein heavy chain (DHC) similarly decreased TRIM5α-mediated restriction. The loss in restriction resulting from either the disassembly of microtubules or the disruption of dynein motor activity was seen for both endogenous and overexpressed TRIM5α and was not due to differences in protein stability or cell viability. Both nocodazole treatment and DHC depletion interfered with the dynamics of TRIM5α CBs, increasing their size and altering their intracellular localization. In addition, nocodazole, paclitaxel, and DHC depletion were all found to increase the stability of HIV-1 cores in infected cells, providing an alternative explanation for the decreased restriction. In conclusion, association with microtubules and the translocation activity of dynein motor complexes are required to achieve efficient restriction by TRIM5α. The primate innate cellular defenses against infection by retroviruses include a protein named TRIM5α, belonging to the family of restriction factors. TRIM5α is present in the cytoplasm, where it can intercept incoming retroviruses shortly after their entry. How TRIM5α manages to be present at the appropriate subcytoplasmic location to interact with its target is unknown. We hypothesized that TRIM5α, either as a soluble protein or a high-molecular-weight complex (the cytoplasmic body), is transported within the cytoplasm by a molecular motor called the dynein complex, which is known to interact with and move along microtubules. Our results show that destructuring microtubules or crippling their function decreased the capacity of human or simian TRIM5α to restrict their retroviral targets. Inhibiting dynein motor activity, or reducing the expression of a key component of this complex, similarly affected TRIM5α-mediated restriction. Thus, we have identified specific cytoskeleton structures involved in innate antiretroviral defenses.

Virus-Specific Effects of TRIM5α rh RING Domain Functions on Restriction of Retroviruses

The tripartite motif protein TRIM5α restricts particular retrovirus infections by binding to the incoming capsid and inhibiting the early stage of virus infection. The TRIM5α RING domain exhibits E3 ubiquitin ligase activity and assists the higher-order association of TRIM5α dimers, which promotes capsid binding. We characterized a panel of RING domain mutants of the rhesus monkey TRIM5α (TRIM5α(rh)) protein. The RING domain function that significantly contributed to retroviral restriction depended upon the restricted virus. The E3 ubiquitin ligase activity of the RING domain contributes to the potency of HIV-1 restriction. Nonetheless, TRIM5α(rh) mutants without detectable E3 ubiquitin ligase activity still blocked reverse transcription and inhibited HIV-1 infection at a moderate level. When TRIM5α(rh) capsid binding was weakened by substitution with a less efficient B30.2/SPRY domain, the promotion of higher-order association by the RING domain was more important to HIV-1 restriction than its E3 ubiquitin ligase activity. For the restriction of N-tropic murine leukemia virus (N-MLV) and equine infectious anemia virus (EIAV) infection, promotion of higher-order association represented the major contribution of the RING domain. Thus, both identity of the target virus and the B30.2/SPRY domain-mediated affinity for the viral capsid determine the relative contribution of the two known RING domain functions to TRIM5α restriction of retrovirus infection.

A Comparison of Murine Leukemia Viruses That Escape from Human and Rhesus Macaque TRIM5αs

To better understand the binding mechanism of TRIM5α to retrovirus capsid, we had previously selected N-tropic murine leukemia virus (N-MLV) mutants escaping from rhesus macaque TRIM5α (rhTRIM5α) by passaging the virus in rhTRIM5α-expressing cells and selecting for nonrestricted variants. To test the commonality of the findings from the rhTRIM5α study, we have now employed a similar genetic approach using human TRIM5α (huTRIM5α). Consistent with the rhTRIM5α study, the mapped huTRIM5α escape mutations were distributed across the capsid exterior, confirming the extended binding surface between virus and restriction factor. Compared to the results of the previous study, fewer escape mutations were identified, with particular mutants being repeatedly selected. Three out four huTRIM5α escape variants showed resistance to all primate TRIM5αs tested, but two of them sacrificed viral fitness, observations that were not made in the rhTRIM5α study. Moreover, differences in amino acid changes associated with escape from hu- and rhTRIM5αs suggested a charge dependence of the restriction by different TRIM5αs. Taken together, these results suggest that the recognition of the entire capsid surface is a general strategy for TRIM5α to restrict MLV but that significantly different specific interactions are involved in the binding of TRIM5α from different species to the MLV capsid core.

Role of SUMO-1 and SUMO interacting motifs in rhesus TRIM5α-mediated restriction

BackgroundTRIM5α is a member of the tripartite motif family of proteins that restricts retroviral infection in a species-specific manner. The restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5α. Previously, we determined that two SUMO interacting motifs (SIMs) present in the B30.2/SPRY domain of human TRIM5α (huTRIM5α) were important for the restriction of N-tropic Murine Leukemia Virus. Here, we examined whether SUMO expression and the SIM1 and SIM2 motifs in rhesus monkey TRIM5α (rhTRIM5α) are similarly important for Human Immunodeficiency Type 1 (HIV-) restriction.ResultsWe found that mutation of SIM1 and SIM2 of rhTRIM5α abolished the restriction of HIV-1 virus. Further, knockdown of SUMO-1 in rhTRIM5α expressing cells abolished restriction of HIV-1. These results may be due, in part, to the ability of SUMO-1 to stabilize rhTRIM5α protein expression, as SUMO-1 knockdown increased rhTRIM5α turnover and the mutations in SIM1 and SIM2 led to more rapid degradation than the wild type protein. The NF-κB signaling ability of rhTRIM5α was also attenuated by SUMO-1 knockdown. Finally, upon inhibition of CRM1-dependent nuclear export with Leptomycin B (LMB), wild type rhTRIM5α localized to SUMO-1 bodies in the nucleus, while the SIM1 and SIM2 mutants did not localize to SUMO-1.ConclusionsOur results suggest that the rhTRIM5α B30.2/SPRY domain is not only important for the recognition of the HIV-1 CA, but it is also important for its association with SUMO-1 or SUMO-1 modified proteins. These interactions help to maintain TRIM5α protein levels and its nuclear localization into specific nuclear bodies.

Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1

The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to block HIV-1 and SIVmac infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine–aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIVmac. We demonstrated that the HD domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active purified SAMHD1 protein from insect cells. Interestingly, we showed that double-stranded RNA inhibits the enzymatic activity of SAMHD1 in vitro suggesting the possibility that RNA from a pathogen might modulate the enzymatic activity of SAMHD1 in cells. By contrast, we found that the SAM domain is dispensable for retroviral restriction, oligomerization and RNA binding. Finally we tested the ability of SAMHD1 to block the infection of retroviruses other than HIV-1 and SIVmac. These results showed that SAMHD1 blocks infection of HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Equine infectious anemia virus (EIAV), N-tropic murine leukemia virus (N-MLV), and B-tropic murine leukemia virus (B-MLV).

Role of Human TRIM5α in Intrinsic Immunity

Human immunodeficiency virus (HIV) has a very narrow host range. HIV type 1 (HIV-1) does not infect Old World monkeys, such as the rhesus monkey (Rh). Rh TRIM5α was identified as a factor that confers resistance, intrinsic immunity, to HIV-1 infection. Unfortunately, human TRIM5α is almost powerless to restrict HIV-1. However, human TRIM5α potently restricts N-tropic murine leukemia viruses (MLV) but not B-tropic MLV, indicating that human TRIM5α represents the restriction factor previously designated as Ref1. African green monkey TRIM5α represents another restriction factor previously designated as Lv1, which restricts both HIV-1 and simian immunodeficiency virus isolated from macaque (SIVmac) infection. TRIM5 is a member of the tripartite motif family containing RING, B-box2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase, and TRIM5α is thought to degrade viral core via ubiquitin–proteasome-dependent and -independent pathways. The alpha isoform of TRIM5 has an additional C-terminal PRYSPRY domain, which is a determinant of species-specific retrovirus restriction by TRIM5α. On the other hand, the target regions of viral capsid protein (CA) are scattered on the surface of core. A single amino acid difference in the surface-exposed loop between α-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA affects viral sensitivity to human TRIM5α and was also shown to be associated with viral load in West African HIV-2 patients, indicating that human TRIM5α is a critical modulator of HIV-2 replication in vivo. Interestingly, L6/7 of CA corresponds to the MLV determinant of sensitivity to mouse factor Fv1, which potently restricts N-tropic MLV. In addition, human genetic polymorphisms also affect antiviral activity of human TRIM5α. Recently, human TRIM5α was shown to activate signaling pathways that lead to activation of NF-κB and AP-1 by interacting with TAK1 complex. TRIM5α is thus involved in control of viral infection in multiple ways.

Productive replication and evolution of HIV-1 in ferret cells.

A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1 animal model.

RING domain mutations uncouple TRIM5α restriction of HIV-1 from inhibition of reverse transcription and acceleration of uncoating.

Rhesus TRIM5α (TRIM5α(rh)) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5α(rh) to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5α(rh) RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5α(rh) RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5α(rh) RING domain variants also prevented the degradation of TRIM5α(rh) that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5α to accelerate uncoating. Collectively, these results suggest that TRIM5α(rh) blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells.

Reduction of N-Tropic Mutant Porcine Endogenous Retrovirus Infectivity by Human Tripartite Motif-Containing 5-Isoform Alpha

In cases of retroviral infection, the host cell deploys antiviral proteins as a type of innate immunity. Tripartite motif-containing 5-isoform alpha (TRIM5α) is a potent antiviral protein. TRIM5α has been reported to restrict human immunodeficiency virus (HIV) 1 infection in rhesus monkey cells by targeting the incoming viral capsid at the postentry or preintegration stage of the viral life cycle. As a consequence, virus replication and reverse transcription are interrupted. TRIM5α of human origin has also been shown to inhibit N-tropic murine leukemia virus infection. To investigate the inhibitory effect of TRIM5α on porcine endogenous retrovirus (PERV) infection in humans, we constructed a 293T cell line stably expressing human TRIM5α (293T-huTRIM5α) and tested the infectivity of vesicular stomatitis virus glycoprotein envelope pseudotyped viruses (wild-type PERV [wt-PERV], N-tropic mutant PERV, N-tropic murine leukemia virus, and MoMLV). Infectivity of N-tropic mutant PERV was reduced by 43.3% in 293T-huTRIM5α cells, a decrease in efficiency that was more than 3-fold greater than that of wt-PERV in 293T-huTRIM5α cells. Human TRIM5α exhibited inhibitory activity against N-tropic MLV and N-tropic mutant PERV, but showed no antiviral activity against Moloney murine leukemia virus or wt-PERV.

Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

Many tripartite motif (TRIM) proteins self-associate, forming dimers and higher order complexes. For example, dimers of TRIM5α, a host factor that restricts retrovirus infection, assemble into higher order arrays on the surface of the viral capsid, resulting in an increase in avidity. Here we show that the higher order association of different TRIM proteins exhibits a wide range of efficiencies. Homologous association (self-association) was more efficient than the heterologous association of different TRIM proteins, indicating that specificity determinants of higher order self-association exist. To investigate the structural determinants of higher order self-association, we studied TRIM mutants and chimeras. These studies revealed the following: 1) the RING domain contributes to the efficiency of higher order self-association, which enhances the binding of TRIM5α to the human immunodeficiency virus (HIV-1) capsid; 2) the RING and B-box 2 domains work together as a homologous unit to promote higher order association of dimers; 3) dimerization is probably required for efficient higher order self-association; 4) the Linker 2 region contributes to higher order self-association, independently of effects of Linker 2 changes on TRIM dimerization; and 5) for efficiently self-associating TRIM proteins, the B30.2(SPRY) domain is not required for higher order self-association. These results support a model in which both ends of the core TRIM dimer (RING-B-box 2 at one end and Linker 2 at the other) contribute to the formation of higher order arrays.

Homology-based Identification of Capsid Determinants That Protect HIV1 from Human TRIM5α Restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.

Unique Spectrum of Activity of Prosimian TRIM5α against Exogenous and Endogenous Retroviruses

Lentiviruses, the genus of retrovirus that includes HIV-1, rarely endogenize. Some lemurs uniquely possess an endogenous lentivirus called PSIV ("prosimian immunodeficiency virus"). Thus, lemurs provide the opportunity to study the activity of host defense factors, such as TRIM5α, in the setting of germ line invasion. We characterized the activities of TRIM5α proteins from two distant lemurs against exogenous retroviruses and a chimeric PSIV. TRIM5α from gray mouse lemur, which carries PSIV in its genome, exhibited the narrowest restriction activity. One allelic variant of gray mouse lemur TRIM5α restricted only N-tropic murine leukemia virus (N-MLV), while a second variant restricted N-MLV and, uniquely, B-tropic MLV (B-MLV); both variants poorly blocked PSIV. In contrast, TRIM5α from ring-tailed lemur, which does not contain PSIV in its genome, revealed one of the broadest antiviral activities reported to date against lentiviruses, including PSIV. Investigation into the antiviral specificity of ring-tailed lemur TRIM5α demonstrated a major contribution of a 32-amino-acid expansion in variable region 2 (v2) of the B30.2/SPRY domain to the breadth of restriction. Data on lemur TRIM5α and the prediction of ancestral simian sequences hint at an evolutionary scenario where antiretroviral specificity is prominently defined by the lineage-specific expansion of the variable loops of B30.2/SPRY.

Inhibitory effect of human TRIM5α on HIV-1 production

Tripartite motif-containing 5 isoform-α (TRIM5α), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5α, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5α limits HIV-1 production. Transient expression of TRIM5α decreased HIV-1 production, whereas knockdown of TRIM5α in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5α and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5α had no effect on the restriction ability. Our findings suggest human TRIM5α is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.

An Invariant Surface Patch on the TRIM5α PRYSPRY Domain Is Required for Retroviral Restriction but Dispensable for Capsid Binding

TRIM5alpha is a retrovirus restriction factor in the host cell cytoplasm that blocks infection before provirus establishment. Restriction activity requires capsid (CA)-specific recognition by the PRYSPRY domain of TRIM5alpha. To better understand the restriction mechanism, nine charge-cluster-to-triple-alanine mutants in the TRIM5alpha PRYSPRY domain were assessed for CA-specific restriction activity. Five mutants distributed along the TRIM5alpha PRYSPRY primary sequence disrupted restriction activity against N-tropic murine leukemia virus and equine infectious anemia virus. Modeling of the TRIM5alpha PRYSPRY domain based on the crystal structures of PRYSPRY-19q13.4.1, GUSTAVUS, and TRIM21 identified a surface patch where disruptive mutants clustered. All mutants in this patch retained CA-binding activity, a reticular distribution in the cytoplasm, and steady-state protein levels comparable to those of the wild type. Residues in the essential patch are conserved in TRIM5alpha orthologues and in closely related paralogues. The same surface patch in the TRIM18 and TRIM20 PRYSPRY domains is the site of mutants causing Opitz syndrome and familial Mediterranean fever. These results indicate that, in addition to CA-specific binding, the PRYSPRY domain possesses a second function, possibly binding of a cofactor, that is essential for retroviral restriction activity by TRIM5alpha.

A human TRIM5α B30.2/SPRY domain mutant gains the ability to restrict and prematurely uncoat B-tropic murine leukemia virus

Human TRIM5α restricts N-tropic murine leukemia virus (N-MLV) but not B-tropic MLV (B-MLV) infection. Here we study B30.2/SPRY domain mutants of human TRIM5α that acquire the ability to inhibit B-MLV infection prior to reverse transcription without losing the ability to restrict N-MLV infection. Remarkably, these mutants gain the ability to decrease the amount of particulate B-MLV capsids in the cytosol of infected cells. In addition, these mutants gain the ability to restrict SIVmac and HIV-2 infection. B-MLV and SIVmac infections were blocked by the mutant TRIM5α proteins prior to reverse transcription. Thus, the range of retroviruses restricted by human TRIM5α can be increased by changes in the B30.2/SPRY domain, which also result in the ability to cause premature uncoating of the restricted retroviral capsid.

Proteasomal Degradation of TRIM5α during Retrovirus Restriction

The host protein TRIM5α inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5α. Here, we show that TRIM5α is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5α-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5α protein but not the nonrestrictive human TRIM5α protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5α was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV) but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5α and TRIMCyp proteins. We also detected degradation of endogenous TRIM5α in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5α degradation by a proteasome-dependent mechanism.

Both TRIM5α and TRIMCyp have only weak antiviral activity in canine D17 cells

BackgroundTRIM5α, which is expressed in most primates and the related TRIMCyp, which has been found in one of the New World monkey species, are antiviral proteins of the TRIM5 family that are able to intercept incoming retroviruses early after their entry into cells. The mechanism of action has been partially elucidated for TRIM5α, which seems to promote premature decapsidation of the restricted retroviruses. In addition, through its N-terminal RING domain, TRIM5α may sensitize retroviruses to proteasome-mediated degradation. TRIM5α-mediated restriction requires a physical interaction with the capsid protein of targeted retroviruses. It is unclear whether other cellular proteins are involved in the inhibition mediated by TRIM5α and TRIMCyp. A previous report suggested that the inhibition of HIV-1 by the rhesus macaque orthologue of TRIM5α was inefficient in the D17a canine cell line, suggesting that the cellular environment was important for the restriction mechanism. Here we investigated further the behavior of TRIM5α and TRIMCyp in the D17 cells.ResultsWe found that the various TRIM5α orthologues studied (human, rhesus macaque, African green monkey) as well as TRIMCyp had poor antiviral activity in the D17 cells, despite seemingly normal expression levels and subcellular distribution. Restriction of both HIV-1 and the distantly related N-tropic murine leukemia virus (N-MLV) was low in D17 cells. Both TRIM5αrh and TRIMCyp promoted early HIV-1 decapsidation in murine cells, but weak levels of restriction in D17 cells correlated with the absence of accelerated decapsidation in these cells and also correlated with normal levels of cDNA synthesis. Fv1, a murine restriction factor structurally unrelated to TRIM5α, was fully functional in D17 cells, showing that the loss of activity was specific to TRIM5α/TRIMCyp.ConclusionWe show that D17 cells provide a poor environment for the inhibition of retroviral replication by proteins of the TRIM5 family. Because both TRIM5α and TRIMCyp are poorly active in these cells, despite having quite different viral target recognition domains, we conclude that a step either upstream or downstream of target recognition is impaired. We speculate that an unknown factor required for TRIM5α and TRIMCyp activity is missing or inadequately expressed in D17 cells.

Alpha Interferon Enhances TRIM5α-Mediated Antiviral Activities in Human and Rhesus Monkey Cells

Dominant, constitutively expressed antiretroviral factors, including TRIM5alpha and APOBEC3 proteins, are distinguished from the conventional innate immune systems and are classified as intrinsic immunity factors. Here, we demonstrate that interferon alpha (IFN-alpha) treatment upregulates TRIM5alpha mRNA in rhesus monkey cells, which correlates with the enhanced TRIM5alpha-mediated pre- and postintegration blocks of human immunodeficiency virus replication. In human cells, IFN-alpha increases the levels of TRIM5alpha mRNA, resulting in enhanced antiviral activity against N-tropic murine leukemia virus infection. These observations indicate that the TRIM5alpha-mediated antiviral effects can be orchestrated by the conventional innate immune response. It is conceivable that TRIM5alpha plays an essential role in controlling both the initial retroviral exposure and the subsequent viral dissemination in vivo.

The human TRIM5alpha restriction factor mediates accelerated uncoating of the N-tropic murine leukemia virus capsid.

The host cell factors TRIM5alpha(hu) and Fv-1 restrict N-tropic murine leukemia virus (N-MLV) infection at an early postentry step before or after reverse transcription, respectively. Interestingly, the identity of residue 110 of the MLV capsid determines susceptibility to both TRIM5alpha(hu) and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5alpha(hu) or Fv-1 restriction factor. The expression of TRIM5alpha(hu), but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5alpha(hu)-mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5alpha(hu) domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5alpha(hu). These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5alpha(hu), but not by Fv-1.