Unique features of TRIM5α among closely related human TRIM family members

AbstractThe tripartite motif (TRIM) protein, TRIM5α, 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 TRIM5α, assembled into homotrimers and co-localized in the cytoplasm with TRIM5α. TRIM5α turned over more rapidly than related TRIM proteins. TRIM5α, TRIM34 and TRIM6 associated with HIV-1 capsid–nucleocapsid complexes assembled in vitro; the TRIM5α and TRIM34 interactions with these complexes were dependent on their B30.2(SPRY) domains. Only TRIM5α potently restricted infection by the retroviruses studied; overexpression of TRIM34 resulted in modest inhibition of simian immunodeficiency virus (SIVmac) infection. In contrast to the other TRIM genes examined, TRIM5 exhibited evidence of positive selection. The unique features of TRIM5α among its TRIM relatives underscore its special status as an antiviral factor

Effects of human TRIM5α polymorphisms on antiretroviral function and susceptibility to human immunodeficiency virus infection

AbstractTRIM5α 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 TRIM5α 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 TRIM5α 136Q was relatively elevated in uninfected individuals, suggesting a possible protective effect. TRIM5α 136Q protein exhibited slightly better anti-HIV-1 activity in tissue culture than the TRIM5α R136 protein. The 43Y variant of TRIM5α 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

Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

We have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. Our method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. We have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0519-7) contains supplementary material, which is available to authorized users

Two Surface-Exposed Elements of the B30.2/SPRY Domain as Potency Determinants of N-Tropic Murine Leukemia Virus Restriction by Human TRIM5α

Human TRIM5α (TRIM5α(hu)) potently restricts N-tropic (N-MLV), but not B-tropic, murine leukemia virus in a manner dependent upon residue 110 of the viral capsid. Rhesus monkey TRIM5α (TRIM5α(rh)) inhibits N-MLV only weakly. The study of human-monkey TRIM5α chimerae revealed that both the v1 and v3 variable regions of the B30.2/SPRY domain contain potency determinants for N-MLV restriction. These variable regions are predicted to be surface-exposed elements on one face of the B30.2 domain. Acidic residues in v3 complement basic residue 110 of the N-MLV capsid. The results support recognition of the retroviral capsid by the TRIM5α B30.2 domain

Species-Specific Variation in the B30.2(SPRY) Domain of TRIM5α Determines the Potency of Human Immunodeficiency Virus Restriction

Retroviruses encounter dominant postentry restrictions in cells of particular species. Human immunodeficiency virus type 1 (HIV-1) is blocked in the cells of Old World monkeys by TRIM5α, a tripartite motif (TRIM) protein composed of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. Rhesus monkey TRIM5α (TRIM5α(rh)) more potently blocks HIV-1 infection than human TRIM5α (TRIM5α(hu)). Here, by studying chimeric TRIM5α proteins, we demonstrate that the major determinant of anti-HIV-1 potency is the B30.2(SPRY) domain. Analysis of species-specific variation in TRIM5α has identified three variable regions (v1, v2, and v3) within the B30.2 domain. The TRIM5α proteins of Old World primates exhibit expansion, duplication, and residue variation specifically in the v1 region. Replacement of three amino acids in the N terminus of the TRIM5α(hu) B30.2 v1 region with the corresponding TRIM5α(rh) residues resulted in a TRIM5α molecule that restricted HIV-1 nearly as efficiently as wild-type TRIM5α(rh). Surprisingly, a single-amino-acid change in this region of TRIM5α(hu) allowed potent restriction of simian immunodeficiency virus, a phenotype not observed for either wild-type TRIM5α(hu) or TRIM5α(rh). Some of the chimeric TRIM5α proteins that are >98% identical to the human protein yet mediate a strong restriction of HIV-1 infection may have therapeutic utility. These observations implicate the v1 variable region of the B30.2(SPRY) domain in TRIM5α(rh) antiviral potency

Removal of Arginine 332 Allows Human TRIM5α To Bind Human Immunodeficiency Virus Capsids and To Restrict Infection

Human TRIM5α (TRIM5α(hu)) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIV(mac)). Alteration of arginine 332 in the TRIM5α(hu) B30.2 domain to proline, the residue found in rhesus monkey TRIM5α, has been shown to create a potent restricting factor for both HIV-1 and SIV(mac.) Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5α(hu.) The increase in restricting activity correlated with an increase in the ability of TRIM5α(hu) mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5α(hu) R332P binding and allowed escape from restriction. The ability of TRIM5α(hu) to restrict SIV(mac) could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5α(hu) B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5α(hu) might potentiate the innate resistance of human cells to HIV-1 infection

Effects of Combined Anti-Hypertensive and Statin Treatment on Memory, Fear Extinction, Adult Neurogenesis, and Angiogenesis in Adult and Middle-Aged Mice

Hyperlipidemia and hypertension are modifiable risk factors for cognitive decline. About 25% of adults over age 65 use both antihypertensives (AHTs) and statins to treat these conditions. Recent research in humans suggests that their combined use may delay or prevent dementia onset. However, it is not clear whether and how combination treatment may benefit brain function. To begin to address this question, we examined effects of atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and Captopril, an angiotensin-converting enzyme inhibitor (ACEI), administration on memory function, anxiety-like behavior, adult hippocampal neurogenesis and angiogenesis in adult and middle-aged male C57Bl/6J mice. In adult mice (3-months-old) combination (combo) treatment, as well as administration of each compound individually, for six weeks, accelerated memory extinction in contextual fear conditioning. However, pattern separation in the touchscreen-based location discrimination test, a behavior linked to adult hippocampal neurogenesis, was unchanged. In addition, dentate gyrus (DG) neurogenesis and vascularization were unaffected. In middle-aged mice (10-months-old) combo treatment had no effect on spatial memory in the Morris water maze, but did reduce anxiety in the open field test. A potential underlying mechanism may be the modest increase in new hippocampal neurons (~20%) in the combo as compared to the control group. DG vascularization was not altered. Overall, our findings suggest that statin and anti-hypertensive treatment may serve as a potential pharmacotherapeutic approach for anxiety, in particular for post-traumatic stress disorder (PTSD) patients who have impairments in extinction of aversive memories

Rapid turnover and polyubiquitylation of the retroviral restriction factor TRIM5

AbstractTRIM5α 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 TRIM5α depended upon intact RING and B-box 2 domains. A chimera consisting of monkey TRIM5α with a RING domain of human TRIM21 exhibited a half-life of 210 min, yet potently restricted human immunodeficiency virus; therefore, rapid turnover of TRIM5α is not required for its antiretroviral activity. TRIM5α 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 TRIM5αrh-containing aggresomes in a microtubule-dependent manner. Thus, TRIM5α 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

Binding and Susceptibility to Postentry Restriction Factors in Monkey Cells Are Specified by Distinct Regions of the Human Immunodeficiency Virus Type 1 Capsid

In cells of Old World and some New World monkeys, dominant factors restrict human immunodeficiency virus type 1 (HIV-1) infections after virus entry. The simian immunodeficiency virus SIV(mac) is less susceptible to these restrictions, a property that is determined largely by the viral capsid protein. For this study, we altered exposed amino acid residues on the surface of the HIV-1 capsid, changing them to the corresponding residues found on the SIV(mac) capsid. We identified two distinct pathways of escape from early, postentry restriction in monkey cells. One set of mutants that were altered near the base of the cyclophilin A-binding loop of the N-terminal capsid domain or in the interdomain linker exhibited a decreased ability to bind the restricting factor(s). Consistent with the location of this putative factor-binding site, cyclophilin A and the restricting factor(s) cooperated to achieve the postentry block. A second set of mutants that were altered in the ridge formed by helices 3 and 6 of the N-terminal capsid domain efficiently bound the restricting factor(s) but were resistant to the consequences of factor binding. These results imply that binding of the simian restricting factor(s) is not sufficient to mediate the postentry block to HIV-1 and that SIV(mac) capsids escape the block by decreases in both factor binding and susceptibility to the effects of the factor(s)

The B30.2(SPRY) Domain of the Retroviral Restriction Factor TRIM5α 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 TRIM5α, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5α 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 TRIM5α was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5α 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 TRIM5α 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 TRIM5α in innate immunity against retroviruses