Biomedical subjects

Hassan Javanbakht

Publications and source records attributed to Hassan Javanbakht.

At least 19 recordsLinked to original sources

PubMed2006

Unique features of TRIM5alpha among closely related human TRIM family members.

The tripartite motif (TRIM) protein, TRIM5alpha, restricts some retroviruses, including human immunodeficiency virus (HIV-1), from infecting the cells of particular species. TRIM proteins contain RING, B-box, coiled-coil and, in some cases, B30.2(SPRY) domains. We investigated the properties of human TRIM family members closely related to TRIM5. These TRIM proteins, like TRIM5alpha, assembled into homotrimers and co-localized in the cytoplasm with TRIM5alpha. TRIM5alpha turned over more rapidly than related TRIM proteins. TRIM5alpha, TRIM34 and TRIM6 associated with HIV-1 capsid-nucleocapsid complexes assembled in vitro; the TRIM5alpha and TRIM34 interactions with these complexes were dependent on their B30.2(SPRY) domains. Only TRIM5alpha potently restricted infection by the retroviruses studied; overexpression of TRIM34 resulted in modest inhibition of simian immunodeficiency virus (SIV(mac)) infection. In contrast to the other TRIM genes examined, TRIM5 exhibited evidence of positive selection. The unique features of TRIM5alpha among its TRIM relatives underscore its special status as an antiviral factor.

Adaptor Proteins, Signal Transducing↗

PubMed2006

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.

Amino Acid Substitution↗

PubMed2006

Effects of human TRIM5alpha polymorphisms on antiretroviral function and susceptibility to human immunodeficiency virus infection.

TRIM5alpha acts on several retroviruses, including human immunodeficiency virus (HIV-1), to restrict cross-species transmission. Using natural history cohorts and tissue culture systems, we examined the effect of polymorphism in human TRIM5alpha on HIV-1 infection. In African Americans, the frequencies of two non-coding SNP variant alleles in exon 1 and intron 1 of TRIM5 were elevated in HIV-1-infected persons compared with uninfected subjects. By contrast, the frequency of the variant allele encoding TRIM5alpha 136Q was relatively elevated in uninfected individuals, suggesting a possible protective effect. TRIM5alpha 136Q protein exhibited slightly better anti-HIV-1 activity in tissue culture than the TRIM5alpha R136 protein. The 43Y variant of TRIM5alpha was less efficient than the H43 variant at restricting HIV-1 and murine leukemia virus infections in cultured cells. The ancestral TRIM5 haplotype specifying no observed variant alleles appeared to be protective against infection, and the corresponding wild-type protein partially restricted HIV-1 replication in vitro. A single logistic regression model with a permutation test indicated the global corrected P value of <0.05 for both SNPs and haplotypes. Thus, polymorphism in human TRIM5 may influence susceptibility to HIV-1 infection, a possibility that merits additional evaluation in independent cohorts.

Adolescent↗

PubMed2006

Characterization of TRIM5alpha trimerization and its contribution to human immunodeficiency virus capsid binding.

The coiled-coil domain of the tripartite motif (TRIM) family protein TRIM5alpha is required for trimerization and function as an antiretroviral restriction factor. Unlike the coiled-coil regions of other related TRIM proteins, the coiled coil of TRIM5alpha is not sufficient for multimerization. The linker region between the coiled-coil and B30.2 domains is necessary for efficient TRIM5alpha trimerization. Most of the hydrophilic residues predicted to be located on the surface-exposed face of the coiled coil can be altered without compromising TRIM5alpha antiviral activity against human immunodeficiency virus (HIV-1). However, changes that disrupt TRIM5alpha trimerization proportionately affect the ability of TRIM5alpha to bind HIV-1 capsid complexes. Therefore, TRIM5alpha trimerization makes a major contribution to its avidity for the retroviral capsid, and to the ability to restrict virus infection.

Antiviral Restriction Factors↗

PubMed2006

Cyclophilin A: an auxiliary but not necessary cofactor for TRIM5alpha restriction of HIV-1.

Cyclophilin A (Cyp A) binds the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein and contributes to the early events in virus replication in some cells. The retroviral restriction factor TRIM5alpha can inhibit the early, post-entry phase of infection by associating with the incoming viral capsid. Cyp A has been proposed to prevent restriction factor binding in human cells, thus enhancing HIV-1 infectivity, and to potentiate restriction of HIV-1 in monkey cells. Here we show that the positive effects of Cyp A-CA binding on HIV-1 infectivity do not depend on human TRIM5alpha. Disruption of Cyp A binding to CA partially relieved the block to HIV-1 infection imposed by several TRIM5alpha variants, but Cyp A-CA binding was not absolutely required for TRIM5alpha antiviral activity. Inhibition of Cyp A function by cyclosporine significantly decreased the efficiency of TRIM5alpha-mediated restriction only when the restricted virus capsid interacted with Cyp A.

Animals↗

PubMed2006

Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor.

The host restriction factor TRIM5alpha mediates species-specific, early blocks to retrovirus infection; susceptibility to these blocks is determined by viral capsid sequences. Here we demonstrate that TRIM5alpha variants from Old World monkeys specifically associate with the HIV type 1 (HIV-1) capsid and that this interaction depends on the TRIM5alpha B30.2 domain. Human and New World monkey TRIM5alpha proteins associated less efficiently with the HIV-1 capsid, accounting for the lack of restriction in cells of these species. After infection, the expression of a restricting TRIM5alpha in the target cells correlated with a decrease in the amount of particulate capsid in the cytosol. In some cases, this loss of particulate capsid was accompanied by a detectable increase in soluble capsid protein. Inhibiting the proteasome did not abrogate restriction. Thus, TRIM5alpha restricts retroviral infection by specifically recognizing the capsid and promoting its rapid, premature disassembly.

Animals↗

PubMed2006

Rapid turnover and polyubiquitylation of the retroviral restriction factor TRIM5.

TRIM5alpha and TRIMCyp are retroviral restriction factors that, like other members of the tripartite motif (TRIM) family, contain RING, B-box 2 and coiled-coil domains. We found that both proteins are rapidly turned over, with half-lives of 50-60 min. Polyubiquitylation and rapid degradation of TRIM5alpha depended upon intact RING and B-box 2 domains. A chimera consisting of monkey TRIM5alpha with a RING domain of human TRIM21 exhibited a half-life of 210 min, yet potently restricted human immunodeficiency virus; therefore, rapid turnover of TRIM5alpha is not required for its antiretroviral activity. TRIM5alpha forms cytoplasmic bodies that contain other polyubiquitylated proteins, heat shock proteins and dynein, and thus resemble aggresome precursors. Consistent with this interpretation, proteasomal inhibitors triggered the formation of TRIM5alpha(rh)-containing aggresomes in a microtubule-dependent manner. Thus, TRIM5alpha levels in the cell are maintained by continuous synthesis and rapid proteasome-mediated degradation, imbalances in which result in the formation of pre-aggresomal cytoplasmic bodies.

Antiviral Restriction Factors↗

PubMed2005

The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha.

TRIM5alpha is a cytoplasmic protein that mediates a post-entry block to infection by some retroviruses. TRIM5alpha contains a tripartite motif (TRIM), which includes RING, B-box 2, and coiled-coil domains, and a C-terminal B30.2 (SPRY) domain. We investigated the contribution of the RING and B-box 2 domains to the antiretroviral activity of rhesus monkey TRIM5alpha (TRIM5alpharh), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIVagm). Disruption of the RING domain caused mislocalization of TRIM5alpharh so that the cytoplasmic level of the protein was decreased compared with that of the wild-type protein. Nonetheless, partial ability to restrict HIV-1 and SIVagm was retained by the RING domain mutants. By contrast, although TRIM5alpharh mutants with disrupted B-box 2 domains were efficiently expressed and correctly localized to the cytoplasm, antiretroviral activity was absent. The B-box 2 mutants colocalized and associated with wild-type TRIM5alpharh and exerted dominant-negative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5alpharh molecules that retain a coiled coil can act as dominant-negative inhibitors of wild-type TRIM5alpharh function. The RING domain of TRIM5alpharh is not absolutely required for retrovirus restriction but can influence cytoplasmic levels of the protein and thus indirectly alter function. The B-box 2 domain, by contrast, appears to be essential for efficient retrovirus restriction.

Animals↗

PubMed2005

The B30.2(SPRY) domain of the retroviral restriction factor TRIM5alpha exhibits lineage-specific length and sequence variation in primates.

Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5alpha, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5alpha has recently been shown to mediate innate intracellular resistance to retroviruses, an activity dependent on the integrity of the B30.2 domain, in particular primate species. An examination of the sequences of several TRIM proteins related to TRIM5 revealed the existence of four variable regions (v1, v2, v3, and v4) in the B30.2 domain. Species-specific variation in TRIM5alpha was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5alpha B30.2 v1 region in Old World primates and in v3 in New World monkeys. We observed substitution patterns indicative of selection bordering these particular B30.2 domain variable elements. These results suggest that occasional, complex changes were incorporated into the TRIM5alpha B30.2 domain at discrete time points during the evolution of primates. Some of these time points correspond to periods during which primates were exposed to retroviral infections, based on the appearance of particular endogenous retroviruses in primate genomes. The results are consistent with a role for TRIM5alpha in innate immunity against retroviruses.

Amino Acid Sequence↗

PubMed2005

Retroviral restriction factor TRIM5alpha is a trimer.

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.

Animals↗

PubMed2005

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates.

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.

Amino Acid Sequence↗

PubMed2004

Cellular distribution of Lysyl-tRNA synthetase and its interaction with Gag during human immunodeficiency virus type 1 assembly.

Lysyl-tRNA synthetase (LysRS) is packaged into human immunodeficiency virus type 1 (HIV-1) via its interaction with Gag, and this enzyme facilitates the selective packaging of tRNA(3)(Lys), the primer for initiating reverse transcription, into HIV-1. The Gag/LysRS interaction is detected at detergent-resistant membrane but not in membrane-free cell compartments that contain Gag and LysRS. LysRS is found (i). in the nucleus, (ii). in a cytoplasmic high-molecular-weight aminoacyl-tRNA synthetase complex (HMW aaRS complex), (iii). in mitochondria, and (iv). associated with plasma membrane. The cytoplasmic form of LysRS lacking the mitochondrial import signal was previously shown to be efficiently packaged into virions, and in this report we also show that LysRS compartments in nuclei, in the HMW aaRS complex, and at the membrane are also not required as a primary source for viral LysRS. Exogenous mutant LysRS species unable to either enter the nucleus or bind to the cell membrane are still incorporated into virions. Many HMW aaRS components are not packaged into the virion along with LysRS, and the interaction of LysRS with p38, a protein that binds tightly to LysRS in the HMW aaRS complex, is not required for the incorporation of LysRS into virions. These data indicate that newly synthesized LysRS may interact rapidly with Gag before the enzyme has the opportunity to move to the above-mentioned cellular compartments. In confirmation of this idea, we found that newly synthesized LysRS is associated with Gag after a 10-min pulse with [(35)S]cysteine/methionine. This observation is also supported by previous work indicating that the incorporation of LysRS into HIV-1 is very sensitive to the inhibition of new synthesis of LysRS.

Cell Line↗

PubMed2004

Ability of wild-type and mutant lysyl-tRNA synthetase to facilitate tRNA(Lys) incorporation into human immunodeficiency virus type 1.

The major human tRNA(Lys) isoacceptors, tRNA1,2Lys and tRNA3Lys, are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where tRNA3Lys acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNA(Lys), via its interaction with Gag, and it is a strong candidate for being the signal that specifically targets tRNA(Lys) for viral incorporation. Expression of exogenous wild-type LysRS in cells results in an approximately twofold increase in the viral packaging of both LysRS and tRNA(Lys). Herein, we show that this increase in tRNA(Lys) incorporation into virions is dependent upon the ability of LysRS to bind to tRNA(Lys) but not upon its ability to aminoacylate the tRNA(Lys). COS7 cells were cotransfected with plasmids coding for both HIV-1 and either wild-type or mutant human LysRS, all of which are incorporated into virions with similar efficiency. However, N-terminally truncated LysRS, which binds poorly to tRNA(Lys), does not increase tRNA(Lys) packaging into viruses, while C-terminally truncated LysRS, which binds to but does not aminoacylate tRNA(Lys), still facilitates an increase in tRNA(Lys) packaging into virions.

Animals↗

PubMed2004

The selective packaging and annealing of primer tRNALys3 in HIV-1.

In HIV-1, tRNA(Lys3) serves as the primer for reverse transcriptase, and during viral assembly, the tRNA(Lys) isoacceptors, tRNA(Lys1,2) and tRNA(Lys3), are selectively packaged into the virion. In this review, we shall first discuss the evidence for the formation of a tRNA(Lys) packaging complex, whose components include Gag, GagPol, genomic RNA, lysyl-tRNA synthetase (LysRS), and tRNA(Lys). Evidence suggests that the formation of this complex involves a Gag/GagPol/viral genomic RNA complex interacting with a tRNA(Lys)/ LysRS complex, with Gag interacting with both GagPol and LysRS, and GagPol interacting with the tRNA(Lys). The interaction of Gag with LysRS is quite specific, does not require the presence of tRNA(Lys), and LysRS is believed to be the target that allows the specific packaging of tRNA(Lys) into the virion. The parameters influencing these interactions, and the molecular sites of interaction, will be discussed. The selective packaging of tRNA(Lys3) into HIV-1 facilitates annealing of tRNA(Lys) to the 5' region of viral RNA genome. This region contains a series of stem loops, and there exists several regions in both the viral RNA and the tRNA(Lys) that are believed to be important for tRNA(Lys) annealing. The annealing is facilitated by viral proteins such as unprocessed Gag, nucleocapsid, and reverse transcriptase.

Base Pairing↗

PubMed2003

The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly.

Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag.LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208-259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys.

Binding Sites↗

PubMed2003

Specific inhibition of the synthesis of human lysyl-tRNA synthetase results in decreases in tRNA(Lys) incorporation, tRNA(3)(Lys) annealing to viral RNA, and viral infectivity in human immunodeficiency virus type 1.

The major human tRNA(Lys) isoacceptors, and, are selectively packaged into human immunodeficiency virus type 1 (HIV-1) during assembly, where acts as a primer for reverse transcription. Lysyl-tRNA synthetase (LysRS) is also incorporated into HIV-1, independently of tRNA(Lys), via its interaction with Gag, and is a strong candidate for being the signal that specifically targets tRNA(Lys) for viral incorporation. We have transfected 293T cells with HIV-1 proviral DNA and short interfering RNA (siRNA) specific for LysRS to study the effect of diminished cellular LysRS upon tRNA(Lys) packaging, annealing to viral genomic RNA, and viral production and infectivity. At early time points after siRNA transfection, an 80% inhibition of LysRS incorporation into viruses reflects an 80% reduction of newly synthesized LysRS, rather than a more limited 20 to 25% decrease in the concentration of total cell LysRS, indicating that newly synthesized LysRS in the cell may be the main source of viral LysRS. Viruses produced from cells transfected with siRNA show reduced tRNA(Lys) packaging, reduced annealing to viral RNA, and reduced viral infectivity.

HIV-1↗

PubMed2002

Correlation between tRNALys3 aminoacylation and its incorporation into HIV-1.

During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) is selectively packaged into the virus, where tRNA(Lys3) serves as the primer for reverse transcription. Lysyl-tRNA synthetase is also selectively incorporated into HIV-1 and is therefore a strong candidate for being the signal by which viral proteins interact with tRNA(Lys) isoacceptors. Previously, mutations in the tRNA(Lys3) anticodon have been shown to strongly inhibit the charging of tRNA(Lys3) by lysyl-tRNA synthetase in vitro, and we show here that in vivo aminoacylation is also inhibited by anticodon changes. The order of decreasing in vivo aminoacylation for tRNA(Lys3) anticodon mutants is: wild-type SUU (where S = mcm(5)S(2)U) 100%) --> SGU (49%) --> CGU (40%) --> SGA (0%) and CGA (0%). We found that the ability of these tRNA(Lys3) anticodon variants to be aminoacylated in vivo is directly correlated with their ability to be packaged into HIV-1. These data showed that the anticodon is a major determinant for tRNA(Lys3) packaging and support the conclusion that its productive interaction with lysyl-tRNA synthetase is important for tRNA(Lys3) incorporation into HIV-1.

Animals↗

PubMed2002

Effect of altering the tRNA(Lys)(3) concentration in human immunodeficiency virus type 1 upon its annealing to viral RNA, GagPol incorporation, and viral infectivity.

Human immunodeficiency virus type 1 (HIV-1) uses tRNA(Lys)(3) as a primer for reverse transcription and, during viral assembly, this tRNA is selectively packaged into the virus along with the other major tRNA(Lys), tRNA(Lys)(3). Increasing the cytoplasmic concentration of tRNA(Lys)(3) through transfection of cells with a plasmid containing both HIV-1 proviral DNA and a tRNA(Lys)(3) gene results in a greater incorporation of tRNA(Lys)(3) into virions, which is accompanied by increased annealing of tRNA(Lys)(3) to the viral genome and increased infectivity of the viral population. Increased viral tRNA(Lys)(3) is accompanied by decreased viral tRNA(Lys)(3), with the total tRNA(Lys)/virion and the GagPol/Gag ratios remaining unchanged. Viral tRNA(Lys) can be doubled, with increases in both tRNA(Lys)(3) and tRNA(Lys)(1,2) concentrations, by overexpressing lysyl tRNA synthetase. This also results in increased tRNA(Lys)(3) annealing to the viral RNA and increased viral infectivity but, again, no change in the GagPol/Gag ratio was observed. This result indicates that GagPol, whose interaction is required during packaging, is not a limiting factor during tRNA(Lys) incorporation into HIV-1, whereas LysRS is.

Animals↗