In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters

Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PI(4,5)P₂. These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYP(X)_nL motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system.     

In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 10⁵–10⁶ FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies.     

The small RING finger protein Z drives arenavirus budding: implications for antiviral strategies

By using a reverse genetics system that is based on the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we have identified the arenavirus small RING finger Z protein as the main driving force of virus budding. Both LCMV and Lassa fever virus (LFV) Z proteins exhibited self-budding activity, and both substituted efficiently for the late domain that is present in the Gag protein of Rous sarcoma virus. LCMV and LFV Z proteins contain proline-rich motifs that are characteristic of late domains. Mutations in the PPPY motif of LCMV Z severely impaired the formation of virus-like particles. LFV Z contains two different proline-rich motifs, PPPY and PTAP, which are separated by eight amino acids. Mutational analysis revealed that both motifs are required for efficient LFV Z-mediated budding. Both LCMV and LFV Z proteins recruited to the plasma membrane Tsg101, which is a component of the class E vacuolar protein sorting machinery that has been implicated in budding of HIV and Ebola virus. Targeting of Tsg101 by RNA interference caused a strong reduction in Z-mediated budding. These results indicate that Z is the arenavirus functional counterpart of the matrix proteins found in other negative strand enveloped RNA viruses. Moreover, members of the vacuolar protein sorting pathway appear to play an important role in arena-virus budding. These findings open possibilities for antiviral strategies to combat LFV and other hemorrhagic fever arenaviruses.     

Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function

Efficient budding of HIV-1 from the plasma membrane of infected cells requires the function of a 6-kDa protein known as p6. A highly conserved Pro-Thr-Ala-Pro (PTAP) motif (the "late" or "L" domain), is critical for the virus-budding activity of p6. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N terminus a domain highly related to ubiquitin-conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle assembly and release. We observed that this domain (referred to as TSG-5') potently inhibits virus production. Examination of cells coexpressing HIV-1 Gag and TSG-5' by electron microscopy reveals a defect in virus budding reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5' depends on an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5'. Furthermore, assembly and release of murine leukemia virus and Mason-Pfizer monkey virus are insensitive to TSG-5'. TSG-5' is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5' incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and indicate that TSG101 derivatives can act as potent and specific inhibitors of HIV-1 replication by blocking virus budding.     

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding

HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.Live-cell fluorescence microscopy of assembling HIV-1 virions has established the temporal sequence in which various viral and host molecules are recruited to the assembly site (1–6). The HIV-1 genome is recruited first to the plasma membrane by a subdetectable number of molecules of the structural protein, Gag (3), and a steady accumulation of Gag ensues for 6–10 min (1, 2, 4). After Gag recruitment is completed, members of the endosomal sorting complex required for transport III (ESCRT-III) complex and the ATPase vacuolar protein sorting-associated protein 4A (Vps4A) are recruited transiently, for just a few minutes, to the site of assembly (4, 6). The ESCRT machinery functions during membrane fission in processes such as the formation of multivesicular bodies, the terminal stages of cytokinesis (7), and the budding of enveloped viruses such as HIV-1 (8, 9). These processes all have inverted topologies compared with the topology of endocytic events at the plasma membrane. HIV-1 hijacks the ESCRT machinery by recruiting its members through specific amino acid sequences, called late domains, in the major structural protein Gag. Specifically, the PTAP motif recruits tumor susceptibility gene 101 (Tsg101) and the LXXLF motif recruits ALG-2 interacting protein X (Alix), with PTAP being the functionally more important motif (10, 11). Biochemical and genetic assays have defined specific molecular interactions between ESCRT proteins that are recruited by Gag (8, 12–14), but a fine temporal and spatial mapping of the recruitment of viral and host components relative to each other is lacking. Here, using live-cell multiple-color total internal reflection fluorescence microscopy (TIR-FM), we demonstrate that the ESCRT protein Tsg101 is corecruited with Gag and accumulates progressively, whereas charged multivesicular body protein 4b (Chmp4b) and Vps4A are recruited sequentially and transiently to Gag assembly sites. Moreover, because diffraction-limited microscopy cannot resolve spatial differences the size of an HIV-1 virion (∼100 nm) (15–17), we determined the relative spatial positions of Gag and of several members of the ESCRT machinery in nascent virions using two-color superresolution imaging.     

Ubiquitin-dependent virus particle budding without viral protein ubiquitination

An essential step in the release of an extracellular enveloped virus particle is a budding event that ultimately separates virion and host cell membranes. For many enveloped viruses, membrane fission requires the recruitment of the class E vacuolar protein sorting (VPS) machinery by short, virally encoded peptide sequences termed "late-budding" or "L" domains. Some L-domain peptide sequences (e.g., PSAP) bind directly to components of class E VPS machinery, whereas others (e.g., PPxY) access it indirectly by recruiting ubiquitin ligases. Additionally, ubiquitin itself is known to be generally important for the fission of virion from cellular membranes, and because ubiquitination of cellular transmembrane proteins can signal the recruitment of class E machinery, a popular model is that deposition of ubiquitin on viral structural proteins mediates class E machinery recruitment. To test this model, we took advantage of a retroviral Gag protein from the prototypic foamy virus (PFV) that is almost devoid of ubiquitin acceptors, and we engineered it to generate extracellular virus-like particles in the complete absence of other viral proteins. Notably, we found that particle budding, induced by a class E VPS machinery-binding L domain (PSAP), proceeded efficiently in the absence of ubiquitin acceptors in PFV Gag. Moreover, when particle release was engineered to be dependent on a viral PPXY motif, the requirement for a catalytically active ubiquitin ligase was maintained, irrespective of the presence or absence of ubiquitin acceptor sites in PFV Gag. Thus, in this model system, ubiquitin conjugation to transacting factors, not viral proteins, appears critical for ubiquitin-dependent enveloped viral particle release.     

Mutation of the Highly Conserved Ser-40 of the HIV-1 p6 Gag Protein to Phe Causes the Formation of a Hydrophobic Patch,Enhances Membrane Association,and Polyubiquitination of Gag

The HIV-1 p6 Gag protein contains two late assembly (l-) domains that recruit proteins of the endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane fission between the nascent virion and the cell membrane. It was recently demonstrated that mutation of the highly conserved Ser-40 to Phe (S40F) disturbs CA-SP1 processing, virus morphogenesis, and infectivity. It also causes the formation of filopodia-like structures, while virus release remains unaffected. Here, we show that the mutation S40F, but not the conservative mutation to Asp (S40D) or Asn (S40N), augments membrane association, K48-linked polyubiquitination, entry into the 26S proteasome, and, consequently, enhances MHC-I antigen presentation of Gag derived epitopes. Nuclear magnetic resonance (NMR) structure analyses revealed that the newly introduced Phe-40, together with Tyr-36, causes the formation of a hydrophobic patch at the C-terminal α-helix of p6, providing a molecular rationale for the enhanced membrane association of Gag observed in vitro and in HIV-1 expressing cells. The extended exposure of the S40F mutant to unidentified membrane-resident ubiquitin E3-ligases might trigger the polyubiquitination of Gag. The cumulative data support a previous model of a so far undefined property of p6, which, in addition to MA, acts as membrane targeting domain of Gag.     

Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells

The late assembly (L) domain of retrovirus Gag, required in the final steps of budding for efficient exit from the host cell, is thought to mediate its function through interaction with unknown cellular factors. Here, we report the identification of the Nedd4-like family of E3 ubiquitin protein ligases as proteins that specifically interact with the Rous sarcoma virus (RSV) L domain in vitro and in vivo. We screened a chicken embryo cDNA expression library by using a peptide derived from the RSV p2b sequence, isolating two unique partial cDNA clones. Neither clone interacted with a peptide containing mutations known to disrupt in vivo RSV L domain function or with human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) L domain-derived peptides. The WW domain region of one of the clones, late domain-interacting protein 1 (LDI-1), but not the C2 domain, bound RSV Gag and inhibited RSV Gag budding from human 293 cells in a dominant-negative manner, functionally implicating LDI-1 in RSV particle budding from cells. RSV Gag can be coimmune precipitated from cell extracts with an antisera directed at an exogenously expressed hemagglutinin (HA)-tagged LDI-1 or endogenous Nedd4 proteins. These findings mechanistically link the cellular ubiquitination pathway to retrovirus budding.     

Virus Hijacks Host Proteins and Machinery for Assembly and Budding,with HIV-1 as an Example

Viral assembly and budding are the final steps and key determinants of the virus life cycle and are regulated by virus–host interaction. Several viruses are known to use their late assembly (L) domains to hijack host machinery and cellular adaptors to be used for the requirement of virus replication. The L domains are highly conserved short sequences whose mutation or deletion may lead to the accumulation of immature virions at the plasma membrane. The L domains were firstly identified within retroviral Gag polyprotein and later detected in structural proteins of many other enveloped RNA viruses. Here, we used HIV-1 as an example to describe how the HIV-1 virus hijacks ESCRT membrane fission machinery to facilitate virion assembly and release. We also introduce galectin-3, a chimera type of the galectin family that is up-regulated by HIV-1 during infection and further used to promote HIV-1 assembly and budding via the stabilization of Alix–Gag interaction. It is worth further dissecting the details and finetuning the regulatory mechanism, as well as identifying novel candidates involved in this final step of replication cycle.     

Involvement of vacuolar protein sorting pathway in Ebola virus release independent of TSG101 interaction

Budding of Ebola virus (EBOV) particles from the plasma membrane of infected cells requires viral and host proteins. EBOV virus matrix protein VP40 recruits TSG101, an ESCRT-1 (host cell endosomal sorting complex required for transport-1) complex protein in the vacuolar protein sorting (vps) pathway, to the plasma membrane during budding. Involvement of other vps proteins in EBOV budding has not been established. Therefore, we used VP40 deletion analysis, virus-like particle-release assays, and confocal microscopy to investigate the potential role of ESCRT-1 proteins VPS4, VPS28, and VPS37B in EBOV budding. We found that VP40 could redirect each protein from endosomes to the cell surface independently of TSG101 interaction. A lack of VPS4 adenosine triphosphatase activity reduced budding by up to 80%. Inhibition of VPS4 gene expression by use of phosphorodiamidite morpholino antisense oligonucleotides protected mice from lethal EBOV infection. These data show that EBOV can use vps proteins independently of TSG101 for budding and reveal VPS4 as a potential target for filovirus therapeutics.     

Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly

The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.The endosomal sorting complexes required for transport (ESCRTs) are an ancient and conserved system for membrane scission (1, 2). ESCRT membrane remodeling activities are important in the budding of HIV-1 and other viruses from host cell membranes (3); cytokinesis (4); lysosomal transport (5); and more recently discovered functions that include membrane repair, exosome biogenesis, and nuclear envelope reformation (6). The ESCRTs are unique in that they promote membrane budding and sever membrane necks by working from the inner face of the bud (1, 2).The ESCRTs consist of the upstream complexes ESCRT-I, ESCRT-II, and ALIX, which recognize cargo, the ESCRT-III complex responsible for membrane scission, and the AAA+ ATPase VPS4, which releases and recycles ESCRT-III (1, 2). In this study, we focus on the human ESCRT-III subunit CHMP4B, which is considered a core component of the membrane scission machinery and is essential for HIV-1 budding (7). Its yeast counterpart is Snf7. CHMP4 can be recruited and activated through two different pathways in human cells. The first proceeds through ESCRT-I, ESCRT-II, and CHMP6, and the second through ALIX (3). The ESCRT-II– and CHMP6-dependent pathway functions downstream of ESCRT-I, which is in turn the essential link between HIV-1 Gag and the ESCRTs (3). Although there is uncertainty over whether ESCRT-II itself is essential in HIV-1 budding, the most recent virological data suggest that ESCRT-II is important for the efficient release of HIV-1 (8). Moreover, ESCRT-II and CHMP6 were required to bridge Gag and ESCRT-I to the rest of ESCRT-III in a reconstituted system (9).Most concepts of ESCRT recruitment to HIV-1 budding sites have focused on protein–protein interactions between the PTAP and YPXL late domain motifs of the Gag p6 domain and ESCRT-I and ALIX, respectively (3). ESCRT-I is recruited to HIV-1 budding sites simultaneously with Gag (10). However, ESCRT-III is recruited after a time lag and only to Gag that has already assembled on the plasma membrane (10, 11). In principle, either the oligomerization of Gag or its membrane association might trigger ESCRT-III recruitment to Gag-ESCRT-I assemblies. In one recent report, ESCRT-III assembly was visualized by superresolution light microscopy within the center of the Gag shell (12). This observation led to a model for virus scaffolding of ESCRT-III assembly, which downplayed the direct role of membrane shape. Another group, also using superresolution imaging, noted a displacement of the ESCRT-III localization closer to the plasma membrane than the mean position of Gag (13), consistent with ESCRT-III localization predominantly to the bud neck (3). The latter model implies that ESCRT-III could be a coincidence detector, responsive both to the presence of upstream interacting proteins and to membrane shape. Whereas an abundant literature describes the role of viral late domains and other protein interactions in ESCRT recruitment, almost no data are available on the role of membrane curvature in initiating ESCRT-III assembly.In this study, we set out to characterize the recruitment and assembly of purified ESCRT complexes on membranes of a defined geometry approximating that of an early stage HIV-1 budding site. At early stages, budding profiles with broad necks have been visualized in thin-section EM and in cryo-EM tomograms (14–17). During the process of their formation, HIV-1 budding intermediates are 50–100 nm deep and slightly over 100 nm wide. The well-developed methods for studying protein interactions with positively curvature membranes (18) cannot be applied to this type of geometry. Here, we used a focused ion beam to fabricate a 100-nm-deep invaginated template for negative curvature, which approximates the shape of a nascent HIV-1 bud. When coated with a supported lipid bilayer, we term this structure an invaginated supported lipid bilayer (invSLB). We went on to measure CHMP4B/Snf7 assembly in real time, which allowed us to dissect in vitro, and in real time, the role of membrane shape in the nucleation and growth of ESCRT polymers.     

Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein

Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.     

HIV capsid assembly

HIV Gag assembly is the first and most essential step in the formation of virus particles. Following protein synthesis, Gag relocates from ribosomes and forms a virus particle at the plasma membrane, using host factors and machinery. Early studies focused on mapping the regions within Gag required for assembly and identified three distinct domains (M, I, and L), although their precise locations within the three-dimensional structure of Gag awaited later study. In this review, I summarize the mapping results in the light of recent progress on Gag structures made by nuclear magnetic resonance and X-ray crystallography as well as further functional analysis. These data are largely consistent and provide sufficient information for an understanding of the interactions and functions of the assembly domains at a macromolecular level. Current studies have moved on to the identification of the host factors and machinery used in the process of Gag assembly. Cumulative data suggest that the dynamics of Gag assembly and transport are achieved not by simply using, but rather by taking control of, cellular machinery. Key area in the process include interactions with TSG101, L domain receptor which normally functions in the endosomal sorting pathway and with lipid rafts, a type of M domain receptor, which has been suggested to be the sites for effective concentration of Gag. The review provides a summary of these data and discusses the likely direction of future studies.     

The Foamy Virus Gag Proteins: What Makes Them Different?

Gag proteins play an important role in many stages of the retroviral replication cycle. They orchestrate viral assembly, interact with numerous host cell proteins, engage in regulation of viral gene expression, and provide the main driving force for virus intracellular trafficking and budding. Foamy Viruses (FV), also known as spumaviruses, display a number of unique features among retroviruses. Many of these features can be attributed to their Gag proteins. FV Gag proteins lack characteristic orthoretroviral domains like membrane-binding domains (M domains), the major homology region (MHR), and the hallmark Cys-His motifs. In contrast, they contain several distinct domains such as the essential Gag-Env interaction domain and the glycine and arginine rich boxes (GR boxes). Furthermore, FV Gag only undergoes limited maturation and follows an unusual pathway for nuclear translocation. This review summarizes the known FV Gag domains and motifs and their functions. In particular, it provides an overview of the unique structural and functional properties that distinguish FV Gag proteins from orthoretroviral Gag proteins.     

The Zinc Content of HIV-1 NCp7 Affects Its Selectivity for Packaging Signal and Affinity for Stem-Loop 3

The nucleocapsid (NC) protein of human immunodeficiency (HIV) is a small, highly basic protein containing two CCHC zinc-finger motifs, which is cleaved from the NC domain of the Gag polyprotein during virus maturation. We previously reported that recombinant HIV-1 Gag and NCp7 overexpressed in an E. coli host contains two and one zinc ions, respectively, and Gag exhibited much higher selectivity for packaging signal (Psi) and affinity for the stem-loop (SL)-3 of Psi than NCp7. In this study, we prepared NCp7 containing 0 (⁰NCp7), 1 (NCp7) or 2 (²NCp7) zinc ions, and compared their secondary structure, Psi-selectivity and SL3-affinity. Along with the decrease of the zinc content, less ordered conformations were detected. Compared to NCp7, ²NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas ⁰NCp7 exhibited a lower Psi-selectivity and SL3-affinity, similar to the H23&H44K double mutant of NCp7, indicating that the different RNA-binding property of Gag NC domain and the mature NCp7 may be resulted, at least partially, from their different zinc content. This study will be helpful to elucidate the critical roles that zinc played in the viral life cycle, and benefit further investigations of the functional switch from the NC domain of Gag to the mature NCp7.     

Molecular Epidemiology and Whole-Genome Analysis of Bovine Foamy Virus in Japan

Bovine foamy virus (BFV) is a member of the foamy virus family in cattle. Information on the epidemiology, transmission routes, and whole-genome sequences of BFV is still limited. To understand the characteristics of BFV, this study included a molecular survey in Japan and the determination of the whole-genome sequences of 30 BFV isolates. A total of 30 (3.4%, 30/884) cattle were infected with BFV according to PCR analysis. Cattle less than 48 months old were scarcely infected with this virus, and older animals had a significantly higher rate of infection. To reveal the possibility of vertical transmission, we additionally surveyed 77 pairs of dams and 3-month-old calves in a farm already confirmed to have BFV. We confirmed that one of the calves born from a dam with BFV was infected. Phylogenetic analyses revealed that a novel genotype was spread in Japan. In conclusion, the prevalence of BFV in Japan is relatively low and three genotypes, including a novel genotype, are spread in Japan.     

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.     

Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)_1–23, human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA_1–32, required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration.     

A role for ubiquitin ligase recruitment in retrovirus release

Retroviral Gag polyproteins have specific regions, commonly referred to as late assembly (L) domains, which are required for the efficient separation of assembled virions from the host cell. The L domain of HIV-1 is in the C-terminal p6(gag) domain and contains an essential P(T/S)AP core motif that is widely conserved among lentiviruses. In contrast, the L domains of oncoretroviruses such as Rous sarcoma virus (RSV) have a more N-terminal location and a PPxY core motif. In the present study, we used chimeric Gag constructs to probe for L domain activity, and observed that the unrelated L domains of RSV and HIV-1 both induced the appearance of Gag-ubiquitin conjugates in virus-like particles (VLP). Furthermore, a single-amino acid substitution that abolished the activity of the RSV L domain in VLP release also abrogated its ability to induce Gag ubiquitination. Particularly robust Gag ubiquitination and enhancement of VLP release were observed in the presence of the candidate L domain of Ebola virus, which contains overlapping P(T/S)AP and PPxY motifs. The release defect of a minimal Gag construct could also be corrected through the attachment of a peptide that serves as a physiological docking site for the ubiquitin ligase Nedd4. Furthermore, VLP formation by a full-length Gag polyprotein was sensitive to lactacystin, which depletes the levels of free ubiquitin through inhibition of the proteasome. Our findings suggest that the engagement of the ubiquitin conjugation machinery by L domains plays a crucial role in the release of a diverse group of enveloped viruses.     

Visualization of retrovirus budding with correlated light and electron microscopy

We have used correlated scanning EM (SEM) and multiphoton fluorescence microscopy to visualize budding of virus-like particles (VLPs) of Rous sarcoma virus (RSV) and HIV type 1 (HIV-1). When the Gag structural protein was expressed alone as a GFP fusion, most budding particles appeared morphologically aberrant, but normal assembly could be rescued by coexpression of untagged Gag protein. Imaging of live cells allowed budding to be seen in real time as the disappearance of fluorescent spots from the dorsal cell surface. The disappearance of very bright spots containing clusters of VLPs often occurred in a stepwise fashion. Even after imaging times >1 h, only a minority of the spots disappeared, suggesting that some might be budding-incompetent complexes. On individual cells, we enumerated both the fluorescent puncta and the budding structures visible by SEM and compared these numbers for WT Gag proteins and for Gag proteins that were blocked at the last step in budding by a late domain mutation. For the mutant HIV-1 and RSV proteins, almost all of the fluorescent spots corresponded to budding structures. For WT RSV, the dorsal side of cells showed 3-fold more fluorescent spots than budding structures, suggesting that formation of the polymerized Gag shell precedes bulging out of the membrane. For WT HIV-1, most fluorescent spots corresponded with budding structures, consistent with the slower budding rate of this virus. Combining these two types of microscopy will allow innovative approaches for elucidating the mechanism of retrovirus budding.