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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.

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE—detected in the Yoruba population of Nigeria—conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the “emerging” nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz’s critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.

Exercise has profound benefits for brain function in animals and humans. In rodents, voluntary wheel running increases the production of new neurons and upregulates neurotrophin levels in the hippocampus, as well as improving synaptic plasticity, memory function and mood. The underlying cellular mechanisms, however, remain unresolved. Recent research indicates that peripheral organs such as skeletal muscle, liver and adipose tissue secrete factors during physical activity that may influence neuronal function. Here we used an in vitro cell assay and proteomic analysis to investigate the effects of proteins secreted from skeletal muscle cells on adult hippocampal neural progenitor cell (aNPC) differentiation. We also sought to identify the relevant molecules driving these effects. Specifically, we treated rat L6 skeletal muscle cells with the AMP-kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or vehicle (distilled water). We then collected the conditioned media (CM) and fractionated it using high-performance liquid chromatography (HPLC). Treatment of aNPCs with a specific fraction of the AICAR-CM upregulated expression of doublecortin (DCX) and Tuj1, markers of immature neurons. Proteomic analysis of this fraction identified proteins known to be involved in energy metabolism, cell migration, adhesion and neurogenesis. Culturing differentiating aNPCs in the presence of one of the factors, glycolytic enzyme glucose-6-phosphate isomerase (GPI), or AICAR-CM, increased the proportion of neuronal (Tuj1+) and astrocytic, glial fibrillary acidic protein (GFAP+) cells. Our study provides further evidence that proteins secreted from skeletal muscle cells may serve as a critical communication link to the brain through factors that enhance neural differentiation.

The cross-species transmission of retroviruses is limited by host restriction factors that exhibit inter-species diversity. For example, the TRIM5α proteins of Old World monkeys block the early, post-entry steps in human immunodeficiency virus (HIV-1) infection. We adapted an HIV-1 isolate to replicate in cells expressing TRIM5αrh from rhesus monkeys, an Old World species. A single amino acid change in the cyclophilin-binding loop of the HIV-1 capsid protein allowed virus replication in cells expressing TRIM5αrh. The capsid of the escape virus exhibited a reduced affinity for TRIM5αrh, but retained the ability to bind cyclophilin A efficiently. Thus, a preferred HIV-1 escape pathway involves decreased binding to TRIM5α, a capsid-destabilizing factor, and retention of binding to cyclophilin A, a capsid-stabilizing factor.

Rhesus TRIM5alpha (rhTRIM5alpha), but not human TRIM5alpha (huTRIM5alpha), potently inhibits human immunodeficiency virus (HIV) infection and is thus a potentially valuable therapeutic tool. Primary human CD4 T cells engineered to express rhTRIM5alpha were highly resistant to cell-free HIV type 1 (HIV-1) infection. However, when cocultured with unmodified T cells, rhTRIM5alpha-expressing cells became highly permissive to HIV-1 infection. Physical separation of rhTRIM5alpha-expressing cells and unmodified cells revealed that rhTRIM5alpha efficiently restricts cell-free but not cell-associated HIV transmission. Furthermore, we observed that HIV-infected human cells could infect rhesus CD4 T cells by cell-to-cell contact, but the infection was self-limiting. Subsequently, we noted that a spreading infection ensued when HIV-1-infected rhTRIM5alpha-expressing human cells were cultured with huTRIM5alpha- but not rhTRIM5alpha-expressing cells. Our results suggest that cell-associated HIV transmission in humans is blocked only when both donor and recipient cells express rhTRIM5alpha. These studies further define the role of rhTRIM5alpha in cell-free and cell-associated HIV transmission and delineate the utility of rhTRIM5alpha in anti-HIV therapy.

The 2013–2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013–2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics., National Institutes of Health (U.S.), United States. Dept. of Health and Human Services (Award 1DP2OD006514-01), National Institute of Allergy and Infectious Diseases (U.S.) (Contracts HHSN272200900049C, HHSN272201000022C, HHSN272200900018C, and U19AI110818), United States Army Medical Acquisition Activity (Grant W81XWH-10-1-0098), David & Lucile Packard Foundation (Fellowship for Science and Engineering), Broad Institute of MIT and Harvard (SPARC Award), German Research Foundation (Grant GU 883/1-1), National Science Foundation (U.S.). Graduate Research Fellowship Program

Next-generation sequencing (NGS) has the potential to transform the discovery of viruses causing unexplained acute febrile illness (UAFI) because it does not depend on culturing the pathogen or a priori knowledge of the pathogen’s nucleic acid sequence. More generally, it has the potential to elucidate the complete human virome, including viruses that cause no overt symptoms of disease, but may have unrecognized immunological or developmental consequences. We have used NGS to identify RNA viruses in the blood of 195 patients with UAFI and compared them with those found in 328 apparently healthy (i.e., no overt signs of illness) control individuals, all from communities in southeastern Nigeria. Among UAFI patients, we identified the presence of nucleic acids from several well-characterized pathogenic viruses, such as HIV-1, hepatitis, and Lassa virus. In our cohort of healthy individuals, however, we detected the nucleic acids of two novel rhabdoviruses. These viruses, which we call Ekpoma virus-1 (EKV-1) and Ekpoma virus-2 (EKV-2), are highly divergent, with little identity to each other or other known viruses. The most closely related rhabdoviruses are members of the genus Tibrovirus and Bas-Congo virus (BASV), which was recently identified in an individual with symptoms resembling hemorrhagic fever. Furthermore, by conducting a serosurvey of our study cohort, we find evidence for remarkably high exposure rates to the identified rhabdoviruses. The recent discoveries of novel rhabdoviruses by multiple research groups suggest that human infection with rhabdoviruses might be common. While the prevalence and clinical significance of these viruses are currently unknown, these viruses could have previously unrecognized impacts on human health; further research to understand the immunological and developmental impact of these viruses should be explored. More generally, the identification of similar novel viruses in individuals with and without overt symptoms of disease highlights the need for a broader understanding of the human virome as efforts for viral detection and discovery advance., Author Summary Next-generation sequencing, a high-throughput method for sequencing DNA and RNA, has the potential to transform virus discovery because it does not depend on culturing the pathogen or a priori knowledge of the pathogen’s nucleic acid sequence. We used next-generation sequencing to identify RNA viruses present in the blood of patients with unexplained fever, as well as apparently healthy individuals in a peri-urban community in Nigeria. We found several well-characterized viruses in the blood of the febrile patients, including HIV-1, hepatitis B and C, as well as Lassa virus. We also discovered two novel rhabdoviruses in the blood of two apparently healthy (afebrile) females, which we named Ekpoma virus-1 and Ekpoma virus-2. Rhabdoviruses are distributed globally and include several human pathogens from the genera lyssavirus and vesiculovirus (e.g., rabies, Chandipura and vesicular stomatitis virus). The novel rhabdoviruses identified in this study are most similar to Bas-Congo virus, which was recently identified in an individual with an acute febrile illness. Furthermore, we demonstrate evidence of high levels of previous exposure to the two rhabdoviruses among our larger study population. Our results suggest that such rhabdovirus infections could be common, and may not necessarily cause overt disease. The identification of viral nucleic acid sequences in apparently healthy individuals highlights the need for a broader understanding of all viruses infecting humans as we increase efforts to identify viruses causing human disease.

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.

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.

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.

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.

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.

Murine leukemia viruses (MLVs) have been classified as N-tropic (N-MLV) or B-tropic (B-MLV), depending on their ability to infect particular mouse strains. The early phase of N-MLV infection is blocked in the cells of several mammalian species, including humans. This block is mediated by a dominant host factor that targets the viral capsid soon after virus entry into the cell has been achieved. A similar block to HIV-1 in rhesus monkey cells is mediated by TRIM5alpha. Here we show that human TRIM5alpha is both necessary and sufficient for the restriction of N-MLV in human cells. Rhesus monkey TRIM5alpha, which potently blocks HIV-1 infection, exhibited only modest inhibition of N-MLV infection. B-MLV was resistant to the antiviral effects of both human and rhesus monkey TRIM5alpha; susceptibility to TRIM5alpha-mediated restriction was conferred by alteration of residue 110 of the B-MLV capsid protein to the amino acid found in the N-MLV capsid. Our results demonstrate that species-specific variation in TRIM5alpha governs its ability to block infection by diverse retroviruses.

Host cell barriers to the early phase of immunodeficiency virus replication explain the current distribution of these viruses among human and non-human primate species. Human immunodeficiency virus type 1 (HIV-1), the cause of acquired immunodeficiency syndrome (AIDS) in humans, efficiently enters the cells of Old World monkeys but encounters a block before reverse transcription. This species-specific restriction acts on the incoming HIV-1 capsid and is mediated by a dominant repressive factor. Here we identify TRIM5alpha, a component of cytoplasmic bodies, as the blocking factor. HIV-1 infection is restricted more efficiently by rhesus monkey TRIM5alpha than by human TRIM5alpha. The simian immunodeficiency virus, which naturally infects Old World monkeys, is less susceptible to the TRIM5alpha-mediated block than is HIV-1, and this difference in susceptibility is due to the viral capsid. The early block to HIV-1 infection in monkey cells is relieved by interference with TRIM5alpha expression. Our studies identify TRIM5alpha as a species-specific mediator of innate cellular resistance to HIV-1 and reveal host cell components that modulate the uncoating of a retroviral capsid.

Outbreaks this year of the deadly and highly contagious Ebola and Marburg viruses in the Democratic Republic of Congo and Uganda and Lassa virus in Nigeria raised concerns about possible epidemic spread of these hemorrhagic fevers. These pathogens seemed to appear out of nowhere around the middle of the 20th century: Marburg virus in 1967, Lassa virus in 1969, and Ebola virus in 1976. By the early 1990s, public health concerns were crystallized in a landmark report ( 1 ) that was the first to popularize the concept of “emerging pathogens” (fig. S1). But could “emerging diagnosis” explain the rise in appearance of hemorrhagic fevers caused by these pathogens? Recent epidemiologic and genetic studies of Lassa and Ebola fevers suggest that these diseases may have widespread prevalence and ancient origins. They raise the possibility that some viral infections may reflect “emerging diagnoses” of diseases that are circulating more widely than thought, with an emerging character primarily a matter of improved detection of the culprit pathogens.

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.

The host cell factors TRIM5α 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 TRIM5α hu and Fv-1. In this study, we investigate the fate of the MLV capsid in cells expressing either the TRIM5α hu or Fv-1 restriction factor. The expression of TRIM5α hu , but not Fv-1, specifically promoted the premature conversion of particulate N-MLV capsids within infected cells to soluble capsid proteins. The TRIM5α hu -mediated disassembly of particulate N-MLV capsids was dependent upon residue 110 of the viral capsid. Furthermore, the deletion or disruption of TRIM5α hu domains necessary for potent N-MLV restriction completely abrogated the disappearance of particulate N-MLV capsids observed with wild-type TRIM5α hu . These results suggest that premature disassembly of the viral capsid contributes to the restriction of N-MLV infection by TRIM5α hu , but not by Fv-1.

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.

Tripartite motif 5α (TRIM5α) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting the cells of particular species. TRIM5α is a member of the TRIM family of proteins, which contain RING, B-box, coiled-coil (CC), and, in some cases, B30.2(SPRY) domains. Here we investigated the abilities of domains from TRIM proteins (TRIM6, TRIM34, and TRIM21) that do not restrict HIV-1 infection to substitute for the domains of rhesus monkey TRIM5α (TRIM5α rh ). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5α rh domains, allowing HIV-1 restriction. By contrast, similar chimeras containing the components of TRIM21, a slightly more distant relative of TRIM5, did not restrict HIV-1 infection. The TRIM21 B-box 2 domain and its flanking linker regions contributed to the functional defectiveness of these chimeras. All of the chimeric proteins formed trimers. All of the chimeras that restricted HIV-1 infection bound the assembled HIV-1 capsid complexes. These results indicate that heterologous RING, B-box 2, and CC domains from related TRIM proteins can functionally substitute for TRIM5α rh domains.

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.

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

The retrovirus restriction factor TRIM5α targets the viral capsid soon after entry. Here we show that the TRIM5α protein oligomerizes into trimers. The TRIM5α coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5α mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5α, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5α to interact with threefold pseudosymmetrical structures on retroviral capsids.

The tripartite motif (TRIM) protein, TRIM5alpha, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5alpha. In addition to diffuse cytoplasmic staining, the TRIM5alpha proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5alpha from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5alpha proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5alpha-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5alpha fusion proteins indicated that no TRIM5alpha domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5alpha.

The TRIM5α 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 (SIV mac ) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5α orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5α proteins functionally resembled human TRIM5α, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5α proteins from several New World monkey species restricted infection by SIV mac and the SIV of African green monkeys, SIV agm . Spider monkey TRIM5α, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIV mac , SIV agm , HIV-1, and N-MLV. Tandem duplications in the TRIM5α B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5α proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5α proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.

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.

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).

Premature disassembly of the HIV-1 capsid is caused by the rhesus monkey protein, TRIM5, and a single amino acid change in human TRIM5confers similar anti-HIV-1 activity.

The restriction factors, TRIM5alpha in most primates and TRIMCyp in owl monkeys, block infection of various retroviruses soon after virus entry into the host cell. Rhesus monkey TRIM5alpha (TRIM5alpha rh) inhibits human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV) more potently than human TRIM5alpha (TRIM5alpha hu). TRIMCyp restricts infection of HIV-1, simian immunodeficiency virus of African green monkeys (SIV agm) and FIV. Early after infection, TRIMCyp, like TRIM5alpha rh and TRIM5alpha hu, decreased the amount of particulate viral capsid in the cytosol of infected cells. The requirements for the TRIMCyp and TRIM5alpha domains in restricting different retroviruses were investigated. Potent restriction of FIV by TRIMCyp occurred in the complete absence of RING and B-box 2 domains; by contrast, efficient FIV restriction by TRIM5alpha rh required these domains. Variable region 1 of the TRIM5alpha rh B30.2 domain contributed to the potency of HIV-1, FIV and equine infectious anemia virus restriction. Thus, although differences exist in the requirements of TRIMCyp and TRIM5alpha for RING/B-box 2 domains, both restriction factors exhibit mechanistic similarities.

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.