David Perez-Caballero

Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins

The release of enveloped viruses from infected cells often requires a virally encoded activity, termed a late-budding domain (L domain), encoded by essential PTAP, PPXY, or YPDL sequence motifs. PTAP-type L domains recruit one of three endosomal sorting complexes required for transport (ESCRT-I). However, subsequent events in viral budding are poorly defined, and neither YPDL nor PPXY-type L domains require ESCRT-I. Here, we show that ESCRT-I and other class E vacuolar protein sorting (VPS) factors are linked by a complex series of protein–protein interactions. In particular, interactions between ESCRT-I and ESCRT-III are bridged by AIP-1/ALIX, a mammalian orthologue of the yeast class E VPS factor, Bro1. Expression of certain ESCRT-III components as fusion proteins induces a late budding defect that afflicts all three L-domain types, suggesting that ESCRT-III integrity is required in a general manner. Notably, the prototype YPDL-type L domain encoded by equine infectious anemia virus (EIAV) acts by recruiting AIP-1/ALIX and expression of a truncated form of AIP-1/ALIX or small interfering RNA-induced AIP-1/ALIX depletion specifically inhibits EIAV YPDL-type L-domain function. Overall, these findings indicate that L domains subvert a subset of class E VPS factors to mediate viral budding, some of which are required for each of the L-domain types, whereas others apparently act as adaptors to physically link specific L-domain types to the class E VPS machinery.

Human Tripartite Motif 5α Domains Responsible for Retrovirus Restriction Activity and Specificity

ABSTRACT The tripartite motif 5α protein (TRIM5α) is one of several factors expressed by mammalian cells that inhibit retrovirus replication. Human TRIM5α (huTRIM5α) inhibits infection by N-tropic murine leukemia virus (N-MLV) but is inactive against human immunodeficiency virus type 1 (HIV-1). However, we show that replacement of a small segment in the carboxy-terminal B30.2/SPRY domain of huTRIM5α with its rhesus macaque counterpart (rhTRIM5α) endows it with the ability to potently inhibit HIV-1 infection. The B30.2/SPRY domain and an additional domain in huTRIM5α, comprising the amino-terminal RING and B-box components of the TRIM motif, are required for N-MLV restriction activity, while the intervening coiled-coil domain is necessary and sufficient for huTRIM5α multimerization. Truncated huTRIM5α proteins that lack either or both the N-terminal RING/B-Box or the C-terminal B30.2/SPRY domain form heteromultimers with full-length huTRIM5α and are dominant inhibitors of its N-MLV restricting activity, suggesting that homomultimerization of intact huTRIM5α monomers is necessary for N-MLV restriction. However, localization in large cytoplasmic bodies is not required for inhibition of N-MLV by huTRIM5α or for inhibition of HIV-1 by chimeric or rhTRIM5α.

APOBEC3G Incorporation into Human Immunodeficiency Virus Type 1 Particles

ABSTRACT APOBEC3G is promiscuous with respect to its antiretroviral effect, requiring that it be packaged into diverse retrovirus particles. Here, we show that most virally encoded human immunodeficiency virus type 1 particle components are dispensable for APOPEC3G incorporation. However, replacement of the nucleocapsid (NC) Gag domain with a leucine zipper abolished APOBEC3G incorporation. Moreover, coprecipitation analysis showed that APOBEC3G-Gag interaction requires NC and nonspecific RNA. These observations suggest that APOBEC3G exploits an essential property of retroviruses, namely, RNA packaging, to infiltrate particles. Because it is, therefore, difficult to evolve specific sequences that confer escape from APOBEC3G, these findings may explain why lentiviruses evolved an activity that induces its destruction.

Cyclophilin Interactions with Incoming Human Immunodeficiency Virus Type 1 Capsids with Opposing Effects on Infectivity in Human Cells

ABSTRACT Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that binds to the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1) and by doing so facilitates HIV-1 replication. Although CypA is incorporated into HIV-1 virions by virtue of CypA-Gag interactions that occur during virion assembly, in this study we show that the CypA-CA interaction that occurs following the entry of the viral capsid into target cells is the major determinant of CypAs effects on HIV-1 replication. Specifically, by using normal and CypA-deficient Jurkat cells, we demonstrate that the presence of CypA in the target and not the virus-producing cell enhances HIV-1 infectivity. Moreover, disruption of the CypA-CA interaction with cyclosporine A (CsA) inhibits HIV-1 infectivity only if the target cell expresses CypA. The effect of CsA on HIV-1 infection of human cells varies according to which particular cell line is used as a target, and CA mutations that confer CsA resistance and dependence exert their effects only if target cells, and not if virus-producing cells, are treated with CsA. The differential effects of CsA on HIV-1 infection in different human cells appear not to be caused by polymorphisms in the recently described retrovirus restriction factor TRIM5α. We speculate that CypA and/or CypA-related proteins affect the fate of incoming HIV-1 capsid either directly or by modulating interactions with unidentified host cell factors.

Restriction of Human Immunodeficiency Virus Type 1 by TRIM-CypA Occurs with Rapid Kinetics and Independently of Cytoplasmic Bodies, Ubiquitin, and Proteasome Activity

ABSTRACT TRIM-CypA is an owl monkey-specific variant of the retrovirus restriction factor TRIM5α. Here, we exploit its modular domain organization and cyclosporine sensitivity to probe the kinetics and mechanism of TRIM5-mediated restriction. Time of addition/withdrawal experiments reveal that inhibition of incoming human immunodeficiency virus type 1 capsids by TRIM-CypA occurs within minutes of their delivery to the target cell cytoplasm. However, while TRIM-CypA restriction is partly dependent on a RING domain, restriction occurs independently of the ubiquitin/proteasome system. Moreover, tagged TRIM-CypA proteins can be fully active as restriction factors without forming cytoplasmic bodies.

Context-dependent effects of L domains and ubiquitination on viral budding

ABSTRACT Many enveloped viruses encode late assembly domains, or L domains, that facilitate virion egress. PTAP-type L domains act by recruiting the ESCRT-I (endosomal sorting complex required for transport I) component Tsg101, and YPXL/LXXLF-type L domains recruit AIP-1/ALIX, both of which are class E vacuolar protein sorting (VPS) factors, normally required for the generation of vesicles within endosomes. The binding cofactors for PPXY-type L domains have not been unambiguously resolved but may include Nedd4-like ubiquitin ligases. Largely because they act as autonomous binding sites for host factors, L domains are generally transferable and active in a context-independent manner. Ebola virus matrix protein (EbVP40) contains two overlapping L-domain motifs within the sequence ILPTAPPEYMEA. Here, we show that both motifs are required for efficient EbVP40 budding. However, upon transplantation into two different retroviral contexts, the relative contributions of the PTAP and PPEY motifs differ markedly. In a murine leukemia virus carrying the EbVP40 sequence, both motifs contributed to overall L domain activity, and budding proceeded in a partly Tsg101-independent manner. Conversely, when transplanted into the context of human immunodeficiency virus type 1 (HIV-1), EbVP40 L-domain activity was entirely due to a PTAP-Tsg101 interaction. In fact, a number of PPXY-type L domains were inactive in the context of HIV-1. Surprisingly, PTAP and YPXL-type L domains that simulated HIV-1 budding reduced the amount of ubiquitin conjugated to Gag, while inactive PPXY-type L domains increased Gag ubiquitination. These observations suggest that active L domains recruit deubiquitinating enzymes as a consequence of class E VPS factor recruitment. Moreover, context-dependent L-domain function may reflect distinct requirements for host functions during the morphogenesis of different viral particles or the underlying presence of additional, as yet undiscovered L domains.

Human Immunodeficiency Virus Type 1 Matrix Inhibits and Confers Cooperativity on Gag Precursor-Membrane Interactions

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Gag multimerization and membrane binding are required for particle formation. However, it is unclear what constitutes a minimal plasma membrane-specific targeting signal and what role the matrix (MA) globular head and other Gag domains play in membrane targeting. Here, we use membrane flotation and microscopic analysis of Gag deletion mutants to demonstrate that the HIV-1 MA globular head inhibits a plasma membrane-specific targeting signal contained within the six amino-terminal MA residues. MA-mediated inhibition is relieved by concentration-dependent Gag multimerization and imparts a high degree of cooperativity on Gag-membrane association. This cooperativity may confer temporal and spatial regulation on HIV-1 assembly.

Fates of retroviral core components during unrestricted and TRIM5-restricted infection.

TRIM5 proteins can restrict retroviral infection soon after delivery of the viral core into the cytoplasm. However, the molecular mechanisms by which TRIM5α inhibits infection have been elusive, in part due to the difficulty of developing and executing biochemical assays that examine this stage of the retroviral life cycle. Prevailing models suggest that TRIM5α causes premature disassembly of retroviral capsids and/or degradation of capsids by proteasomes, but whether one of these events leads to the other is unclear. Furthermore, how TRIM5α affects the essential components of the viral core, other than capsid, is unknown. To address these questions, we devised a biochemical assay in which the fate of multiple components of retroviral cores during infection can be determined. We utilized cells that can be efficiently infected by VSV-G-pseudotyped retroviruses, and fractionated the cytosolic proteins on linear gradients following synchronized infection. The fates of capsid and integrase proteins, as well as viral genomic RNA and reverse transcription products were then monitored. We found that components of MLV and HIV-1 cores formed a large complex under non-restrictive conditions. In contrast, when MLV infection was restricted by human TRIM5α, the integrase protein and reverse transcription products were lost from infected cells, while capsid and viral RNA were both solubilized. Similarly, when HIV-1 infection was restricted by rhesus TRIM5α or owl monkey TRIMCyp, the integrase protein and reverse transcription products were lost. However, viral RNA was also lost, and high levels of preexisting soluble CA prevented the determination of whether CA was solubilized. Notably, proteasome inhibition blocked all of the aforementioned biochemical consequences of TRIM5α-mediated restriction but had no effect on its antiviral potency. Together, our results show how TRIM5α affects various retroviral core components and indicate that proteasomes are required for TRIM5α-induced core disruption but not for TRIM5α-induced restriction.

No effect of endogenous TRIM5α on HIV-1 production

To the editor: The TRIM5α protein forms part of an intrinsic host defense against retrovirus infection in mammalian cells, and many monkey species encode TRIM5α variants that recognize and inactivate incoming HIV-1 capsids1,2. Whereas this mode of inhibition by TRIM5α is well established, Sakuma et al.3 have reported a second, previously undescribed antiretroviral activity that is present in the rhesus macaque TRIM5α protein and absent in human TRIM5α. Specifically, they reported that rhesus TRIM5α inhibits the generation of new HIV-1 particles from infected cells by inducing the degradation of the HIV-1 Gag protein3. Rhesus macaque simian immunodeficiency virus (SIVMAC) was reported to be resistant to this activity. HIV-1 cannot replicate in rhesus cells owing to the action of rhesus TRIM5α and, sometimes, rhesus APOBE3G cytidine deaminase proteins4. However, HIV-1 strains carrying capsid and Vif proteins from SIVMAC can overcome this effect and replicate as well as SIVMAC in rhesus macaque cells 5,6. Notably, we found that, in contrast to wild-type (WT) HIV-1, an HIV-1 strain in which only the capsid protein was replaced by that of SIVMAC, termed HIV(SCA)5, can replicate robustly in a spreading infection assay in a human T cell line engineered to express rhesus TRIM5α (Fig. 1a). This means that substitution of the HIV-1 capsid with its SIVMAC counterpart provides resistance to the anti–HIV-1 effects of rhesus TRIM5α, regardless of whether they occur early or late in the HIV-1 life cycle (Fig. 1a). Because this capsid substitution allows for HIV-1 replication in the presence of rhesus TRIM5α, it should facilitate Gag expression and HIV-1 production in rhesus cells (as compared to WT HIV-1 capsid) if the model proposed by Sakuma et al.3 is correct. To test this, we generated virions by transfecting WT HIV-1 and HIV(SCA) viral genomes and HIV(SCA) Gag-Pol and vesicular stomatitis virus G protein expression plasmids into 293T cells and used the virions to infect rhesus monkey FRhK4 cells. After extensive washing, analysis of the progeny virus production by HIV-1– and HIV(SCA)-infected FRhK4 cells revealed that nearly identical amounts of HIV-1 and HIV(SCA) Gag No effect of endogenous TRIM5α on HIV-1 production

Evidence for Restriction of Ancient Primate Gammaretroviruses by APOBEC3 but Not TRIM5α Proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5α and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a ‘fossil record’ of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5α proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species–dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.