Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly.

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer.

Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesis of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine.

Insights into vaccine development for acquired immune deficiency syndrome from crystal structures of human immunodeficiency virus-1 gp41 and equine infectious anemia virus gp45.

An effective vaccine against acquired immune deficiency syndrome is still unavailable after dozens of years of striving. The glycoprotein gp41 of human immunodeficiency virus is a good candidate as potential immunogen because of its conservation and relatively low glycosylation. As a reference of human immunodeficiency virus gp41, gp45 from equine infectious anemia virus (EIAV) could be used for comparison because both wild-type and vaccine strain of EIAV have been extensively studied. From structural studies of these proteins, the conformational changes during viral invasion could be unveiled, and a more effective acquired immune deficiency syndrome vaccine immunogen might be designed based on this information.

Structural and biochemical insights into the V/I505T mutation found in the EIAV gp45 vaccine strain.

BACKGROUND: The equine infectious anemia virus (EIAV) is a lentivirus of the Retrovirus family, which causes persistent infection in horses often characterized by recurrent episodes of high fever. It has a similar morphology and life cycle to the human immunodeficiency virus (HIV). Its transmembrane glycoprotein, gp45 (analogous to gp41 in HIV), mediates membrane fusion during the infection. However, the post-fusion conformation of EIAV gp45 has not yet been determined. EIAV is the first member of the lentiviruses for which an effective vaccine has been successfully developed. The attenuated vaccine strain, FDDV, has been produced from a pathogenic strain by a series of passages in donkey dermal cells. We have previously reported that a V/I505T mutation in gp45, in combination with other mutations in gp90, may potentially contribute to the success of the vaccine strain. To this end, we now report on our structural and biochemical studies of the gp45 protein from both wide type and vaccine strain, providing a valuable structural model for the advancement of the EIAV vaccine. RESULTS: We resolved crystal structures of the ecto-domain of gp45 from both the wild-type EIAV and the vaccine strain FDDV. We found that the V/I505T mutation in gp45 was located in a highly conserved d position within the heptad repeat, which protruded into a 3-fold symmetry axis within the six-helix bundle. Our crystal structure analyses revealed a shift of a hydrophobic to hydrophilic interaction due to this specific mutation, and further biochemical and virological studies confirmed that the mutation reduced the overall stability of the six-helix bundle in post-fusion conformation. Moreover, we found that altering the temperatures drastically affected the viral infectivity. CONCLUSIONS: Our high-resolution crystal structures of gp45 exhibited high conservation between the gp45/gp41 structures of lentiviruses. In addition, a hydrophobic to hydrophilic interaction change in the EIAV vaccine strain was found to modulate the stability and thermal-sensitivity of the overall gp45 structure. Our observations suggest that lowering the stability of the six-helix bundle (post-fusion), which may stabilizes the pre-fusion conformation, might be one of the reasons of acquired dominance for FDDV in viral attenuation.

Unclosed HIV-1 capsids suggest a curled sheet model of assembly.

The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that ~25% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms.

The maturational refolding of the ß-hairpin motif of equine infectious anemia virus capsid protein extends its helix α1 at capsid assembly locus.

A retroviral capsid (CA) protein consists of two helical domains, CA(N) and CA(C), which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a ß-hairpin motif upon processing from its precursor polyprotein Gag. The ß-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the ß-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the ß-hairpin structure demonstrated that folding of the ß-hairpin specifically extended the N terminus of helix α1 from Tyr(20) to Pro(17). This coil to helix transition involves the conserved sequence of Thr(16)-Pro(17)-Arg(18) (Ser(16)-Pro(17)-Arg(18) in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA(N) oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg(18), helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the ß-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA(N) oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA(N) domain oligomerization, independent to the faster EIAV-CA(C) domain dimerization.

Development of a high throughput, semi-automated, infectious center cell-based ELISA for equine infectious anemia virus.

A faster semi-automated 96-well microtiter plate assay to determine viral infectivity titers, or viral focal units (vfu), of equine infectious anemia virus (EIAV) stocks is described. Optimization of the existing method modernizes a classic virological technique for viral titer determination by quantitating EIAV in experimentally infected cells via a cell-based ELISA. To allow for automation, multiple parameters of the current assay procedures were modified resulting in an assay that required only one quarter the original amount of virus and/or serum for infectivity or neutralization assays, respectively. Equivalent reductions in the required volumes of tissue culture, cell processing, and protein detection reagents were also achieved. Additionally, the new assay decreased the time required from start to finish from 10 days to 6 days (viral titer) or 7 days (viral neutralization), while increasing the number of samples that can be processed concurrently by transition to a 96-well microtiter plate format and by automated counting.

Expression, refolding and preliminary X-ray crystallographic analysis of equine MHC class I molecule complexed with an EIAV-Env CTL epitope.

In order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (MHC) class I molecule, a complex of equine MHC class I molecule (ELA-A1 haplotype, 7-6 allele) with mouse ß(2)-microglobulin and the cytotoxic T lymphocyte (CTL) epitope Env-RW12 (RVEDVTNTAEYW) derived from equine infectious anaemia virus (EIAV) envelope protein (residues 195-206) was refolded and crystallized. The crystal, which belonged to space group P2(1), diffracted to 2.3 Å resolution and had unit-cell parameters a = 82.5, b = 71.4, c = 99.8 Å, ß = 102.9°. The crystal structure contained two molecules in the asymmetric unit. These results should help to determine the first equine MHC class I molecule structure presenting an EIAV CTL epitope.

Cloning, expression and preliminary crystallographic analysis of the equine infectious anaemia virus (EIAV) gp45 ectodomain.

Like human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV) belongs to the lentivirus genus. The first successful lentiviral vaccine was developed for EIAV. Thus, EIAV may serve as a valuable model for HIV vaccine research. EIAV glycoprotein 45 (gp45) plays a similar role to gp41 in HIV by mediating virus-host membrane fusion. The gp45 ectodomain was constructed according to the structure of HIV gp41, with removal of the disulfide-bond loop region. The protein was expressed in Escherichia coli and crystallized following purification. However, most of the crystals grew as aggregates and could not be used for data collection. By extensively screening hundreds of crystals, a 2.7 Šresolution data set was collected from a single crystal. The crystal belonged to space group P6(3), with unit-cell parameters a = b = 46.84, c = 101.61 Å, α = ß = 90, γ = 120°. Molecular replacement was performed using the coordinates of various lengths of HIV gp41 as search models. A long bent helix was identified and a well defined electron-density map around the long helix was obtained. This primary model provided the starting point for further refinement.

Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein.

BACKGROUND: The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell. RESULTS: In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this alpha-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein. CONCLUSIONS: These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

Structural model of the Rev regulatory protein from equine infectious anemia virus.

Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to generate and evaluate a structural model of the Rev protein. The modeled EIAV Rev (ERev) structure includes a total of 6 helices, four of which form an anti-parallel four-helix bundle. The first helix contains the leucine-rich nuclear export signal (NES). An arginine-rich RNA binding motif, RRDRW, is located in a solvent-exposed loop region. An ERLE motif required for Rev activity is predicted to be buried in the core of modeled structure where it plays an essential role in stabilization of the Rev fold. This structural model is supported by existing genetic and functional data as well as by targeted mutagenesis of residues predicted to be essential for overall structural integrity. Our predicted structure should increase understanding of structure-function relationships in Rev and may provide a basis for the design of new therapies for lentiviral diseases.

Structural insights into the cyclin T1-Tat-TAR RNA transcription activation complex from EIAV.

The replication of many retroviruses is mediated by a transcriptional activator protein, Tat, which activates RNA polymerase II at the level of transcription elongation. Tat interacts with Cyclin T1 of the positive transcription-elongation factor P-TEFb to recruit the transactivation-response TAR RNA, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. Here we present the structure of the cyclin box domain of Cyclin T1 in complex with the Tat protein from the equine infectious anemia virus and its corresponding TAR RNA. The basic RNA-recognition motif of Tat adopts a helical structure whose flanking regions interact with a cyclin T-specific loop in the first cyclin box repeat. Together, both proteins coordinate the stem-loop structure of TAR. Our findings show that Tat binds to a surface on Cyclin T1 similar to where recognition motifs from substrate and inhibitor peptides were previously found to interact within Cdk-cyclin pairs.

A comparative analysis of viral matrix proteins using disorder predictors.

BACKGROUND: A previous study (Goh G.K.-M., Dunker A.K., Uversky V.N. (2008) Protein intrinsic disorder toolbox for comparative analysis of viral proteins. BMC Genomics. 9 (Suppl. 2), S4) revealed that HIV matrix protein p17 possesses especially high levels of predicted intrinsic disorder (PID). In this study, we analyzed the PID patterns in matrix proteins of viruses related and unrelated to HIV-1. RESULTS: Both SIVmac and HIV-1 p17 proteins were predicted by PONDR VLXT to be highly disordered with subtle differences containing 50% and 60% disordered residues, respectively. SIVmac is very closely related to HIV-2. A specific region that is predicted to be disordered in HIV-1 is missing in SIVmac. The distributions of PID patterns seem to differ in SIVmac and HIV-1 p17 proteins. A high level of PID for the matrix does not seem to be mandatory for retroviruses, since Equine Infectious Anemia Virus (EIAV), an HIV cousin, has been predicted to have low PID level for the matrix; i.e. its matrix protein p15 contains only 21% PID residues. Surprisingly, the PID percentage and the pattern of predicted disorder distribution for p15 resemble those of the influenza matrix protein M1 (25%). CONCLUSION: Our data might have important implications in the search for HIV vaccines since disorder in the matrix protein might provide a mechanism for immune evasion.

[Comparison of proviral genomes between the Chinese EIAV donkey leukocyte-attenuated vaccine and its parental virulent strain].

The donkey leukocyte-attenuated vaccine of equine infectious anemia virus (EIAV) was the first lentiviral vaccine that induced solid protection from the infection of virulent strains. To elucidate the mechanism of increased immunogenicity and attenuated virulence of the vaccine, the proviral genomic DNA of an EIAV vaccine strain, EIAV(DLV121) was analyzed and compared with the genome of a parental virulent strain EIAV(DV117). Full length viral genomic DNAs were amplified as two segments by LA-PCR and were cloned. Because of the genomic diversity of retroviral quasispecies, 10 full-length sequences of EIAV(DLV121) and 4 full-length sequences of EIAV(DV117) from randomly picked clones were analyzed. Results showed that the average length of the complete nucleotide sequence of EIAV(DLV121) was 8,236bp and EIAV(DV117) was 8,249bp, with the inter-strain diversity of 2.8%. As for individual genes between the vaccine and virulent strains, the differences in nucleotide sequence of S2, LTR and env were significantly higher than the other genes with the diversity of 4.1%, 3.7% and 3.1%, respectively. Considerable variations in deduced amino acid sequences were found in S2, S3 and env. The diversities were 10.4%, 5.6% and 4.8%, respectively. Furthermore, the LTR of EIAV(DLV121) consisted of at least 5 subtypes grouped by their nucleotide sequences. There were two additional N-linked glycosylation sites in the deduced amino acid sequence of EIAV(DV117) gp90 than that of EIAV(DLV121). Among glycosylation sites in the gp90 of virulent strain, 3 were found unique only in EIAV(DV117), of which 2 were located in the principle neutralizing domain (PND). In addition, there was one EIAV(DLV121) -specific glycosylation site, which was positioned in the PND, too. Taken together, it is clear that greatly increased genomic diversity exists in the EIAV vaccine strain, which provides important information for the further study on biological characters of the Chinese EIAV attenuated vaccine.

Structural and biochemical studies of ALIX/AIP1 and its role in retrovirus budding.

ALIX/AIP1 functions in enveloped virus budding, endosomal protein sorting, and many other cellular processes. Retroviruses, including HIV-1, SIV, and EIAV, bind and recruit ALIX through YPX(n)L late-domain motifs (X = any residue; n = 1-3). Crystal structures reveal that human ALIX is composed of an N-terminal Bro1 domain and a central domain that is composed of two extended three-helix bundles that form elongated arms that fold back into a "V." The structures also reveal conformational flexibility in the arms that suggests that the V domain may act as a flexible hinge in response to ligand binding. YPX(n)L late domains bind in a conserved hydrophobic pocket on the second arm near the apex of the V, whereas CHMP4/ESCRT-III proteins bind a conserved hydrophobic patch on the Bro1 domain, and both interactions are required for virus budding. ALIX therefore serves as a flexible, extended scaffold that connects retroviral Gag proteins to ESCRT-III and other cellular-budding machinery.

A single amino acid difference within the alpha-2 domain of two naturally occurring equine MHC class I molecules alters the recognition of Gag and Rev epitopes by equine infectious anemia virus-specific CTL.

Although CTL are critical for control of lentiviruses, including equine infectious anemia virus, relatively little is known regarding the MHC class I molecules that present important epitopes to equine infectious anemia virus-specific CTL. The equine class I molecule 7-6 is associated with the equine leukocyte Ag (ELA)-A1 haplotype and presents the Env-RW12 and Gag-GW12 CTL epitopes. Some ELA-A1 target cells present both epitopes, whereas others are not recognized by Gag-GW12-specific CTL, suggesting that the ELA-A1 haplotype comprises functionally distinct alleles. The Rev-QW11 CTL epitope is also ELA-A1-restricted, but the molecule that presents Rev-QW11 is unknown. To determine whether functionally distinct class I molecules present ELA-A1-restricted CTL epitopes, we sequenced and expressed MHC class I genes from three ELA-A1 horses. Two horses had the 7-6 allele, which when expressed, presented Env-RW12, Gag-GW12, and Rev-QW11 to CTL. The other horse had a distinct allele, designated 141, encoding a molecule that differed from 7-6 by a single amino acid within the alpha-2 domain. This substitution did not affect recognition of Env-RW12, but resulted in more efficient recognition of Rev-QW11. Significantly, CTL recognition of Gag-GW12 was abrogated, despite Gag-GW12 binding to 141. Molecular modeling suggested that conformational changes in the 141/Gag-GW12 complex led to a loss of TCR recognition. These results confirmed that the ELA-A1 haplotype is comprised of functionally distinct alleles, and demonstrated for the first time that naturally occurring MHC class I molecules that vary by only a single amino acid can result in significantly different patterns of epitope recognition by lentivirus-specific CTL.

Structural features in EIAV NCp11: a lentivirus nucleocapsid protein with a short linker.

Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential role in RNA packaging and viral infectivity. They contain two CX(2)CX(4)HX(4)C zinc binding motifs connected by a basic linker of variable length. The 3D structure of a 37-aa peptide corresponding to sequence 22-58 from lentiviral EIAV nucleocapsid protein NCp11, complexed with zinc, has been determined by 2D (1)H NMR spectroscopy, simulated annealing, and molecular dynamics. The solution structure consists of two zinc binding domains held together by a five-residue basic linker Arg(38)-Ala-Pro-Lys-Val(42) that allows for spatial proximity between the two finger domains. Observed linker folding is stabilized by H bonded secondary structure elements, resulting in an Omega-shaped central region, asymmetrically centered on the linker. The conformational differences and similarities with other NC zinc binding knuckles have been systematically analyzed. The two CCHC motifs, both characterized by a peculiar Pro-Gly sequence preceding the His residue, although preserving Zn-binding geometry and chirality of other known NC proteins, exhibit local fold differences both between each other and in comparison with other previously characterized retroviral CCHC motifs.

Characterization of functional domains of equine infectious anemia virus Rev suggests a bipartite RNA-binding domain.

Equine infectious anemia virus (EIAV) Rev is an essential regulatory protein that facilitates expression of viral mRNAs encoding structural proteins and genomic RNA and regulates alternative splicing of the bicistronic tat/rev mRNA. EIAV Rev is characterized by a high rate of genetic variation in vivo, and changes in Rev genotype and phenotype have been shown to coincide with changes in clinical disease. To better understand how genetic variation alters Rev phenotype, we undertook deletion and mutational analyses to map functional domains and to identify specific motifs that are essential for EIAV Rev activity. All functional domains are contained within the second exon of EIAV Rev. The overall organization of domains within Rev exon 2 includes a nuclear export signal, a large central region required for RNA binding, a nonessential region, and a C-terminal region required for both nuclear localization and RNA binding. Subcellular localization of green fluorescent protein-Rev mutants indicated that basic residues within the KRRRK motif in the C-terminal region of Rev are necessary for targeting of Rev to the nucleus. Two separate regions of Rev were necessary for RNA binding: a central region encompassing residues 57 to 130 and a C-terminal region spanning residues 144 to 165. Within these regions were two distinct, short arginine-rich motifs essential for RNA binding, including an RRDRW motif in the central region and the KRRRK motif near the C terminus. These findings suggest that EIAV Rev utilizes a bipartite RNA-binding domain.

SU proteins from virulent and avirulent EIAV demonstrate distinct biological properties.

Biologic activity of equine infectious anemia virus (EIAV) surface (SU) glycoprotein was assayed in a mouse model. Recombinant SU from virulent EIAV17 (SU17), administered intraperitoneally to mouse pups, induced dose-dependent diarrheal responses similar to those reported for SIV SU (Virology 277 (2000) 250). SU17 caused fluid accumulation without histological lesions in mouse intestinal loops, induced chloride secretory currents in Ussing chambers and increased inositol 1,4,5 triphosphate (IP3) levels in HT29 cells. An SU17 peptide, SU17(299-330), provoked a dose-dependent diarrheal response akin to enterotoxic peptides from SIV. In contrast, SU from an avirulent EIAV strain failed to induce a dose response in mouse pups and produced lower levels of activity than SU17 in Ussing chambers and IP3 assays. These results demonstrate that a mouse pup model is useful to monitor EIAV SU biologic activity, showing clear differences between the activities of SU derived from virulent and avirulent viruses, and may provide a useful screen of EIAV virulence.

Late domain-dependent inhibition of equine infectious anemia virus budding.

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.