Transmission of Human T-Cell Leukemia Virus Type 1 to Mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10⁵ spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.     

Opposing Effects of a Tyrosine-Based Sorting Motif and a PDZ-Binding Motif Regulate Human T-Lymphotropic Virus Type 1 Envelope Trafficking

Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env) glycoprotein mediates binding of the virus to its receptor on the surface of target cells and subsequent fusion of virus and cell membranes. To better understand the mechanisms that control HTLV-1 Env trafficking and activity, we have examined two protein-protein interaction motifs in the cytoplasmic domain of Env. One is the sequence YSLI, which matches the consensus YXXΦ motifs that are known to interact with various adaptor protein complexes; the other is the sequence ESSL at the C terminus of Env, which matches the consensus PDZ-binding motif. We show here that mutations that destroy the YXXΦ motif increased Env expression on the cell surface and increased cell-cell fusion activity. In contrast, mutation of the PDZ-binding motif greatly diminished Env expression in cells, which could be restored to wild-type levels either by mutating the YXXΦ motif or by silencing AP2 and AP3, suggesting that interactions with PDZ proteins oppose an Env degradation pathway mediated by AP2 and AP3. Silencing of the PDZ protein hDlg1 did not affect Env expression, suggesting that hDlg1 is not a binding partner for Env. Substitution of the YSLI sequence in HTLV-1 Env with YXXΦ elements from other cell or virus membrane-spanning proteins resulted in alterations in Env accumulation in cells, incorporation into virions, and virion infectivity. Env variants containing YXXΦ motifs that are predicted to have high-affinity interaction with AP2 accumulated to lower steady-state levels. Interestingly, mutations that destroy the YXXΦ motif resulted in viruses that were not infectious by cell-free or cell-associated routes of infection. Unlike YXXΦ, the function of the PDZ-binding motif manifests itself only in the producer cells; AP2 silencing restored the incorporation of PDZ-deficient Env into virus-like particles (VLPs) and the infectivity of these VLPs to wild-type levels.Human T-lymphotropic virus type 1 (HTLV-1) envelope (Env), like most retroviral envelopes, is synthesized as a precursor protein in the endoplasmic reticulum, forms trimers, and is cleaved by a cellular furin-like protease as it transits through the trans-Golgi network on its way to the plasma membrane (7, 21, 31). Cleavage of the HTLV-1 Env precursor generates a 46-kDa surface subunit (SU, gp46) and a 21-kDa transmembrane protein (TM, gp21) (8, 43). SU contains the receptor-binding domain and is linked by a disulfide bond to TM, which anchors Env to the membrane and mediates fusion of virus and cell membranes after receptor engagement (11, 28, 40, 51). TM consists of extracellular, membrane-spanning, and cytoplasmic domains (31); the last contains motifs that direct Env trafficking, membrane targeting, and virion incorporation. HTLV-1 is poorly transmitted as cell-free virus, and there is good evidence supporting a model in which virions are transmitted in a polarized fashion between lymphocytes that are in close contact (22, 30). Unlike murine leukemia virus (MLV) and Mason-Pfizer monkey virus (MPMV) Envs, in which the cytoplasmic domain (CD) is cleaved by the virus-encoded protease to activate fusogenic activity (3, 6, 19, 42), the HTLV-1 Env cytoplasmic domain is not cleaved and HTLV-1 Env exists on the cell surface in a highly fusogenic state. In many respects, HTLV-1 Env resembles versions of MLV or MPMV Envs that lack C-terminal amino acids, e.g., with elevated cell-cell fusion activity and low virion infectivity. It is not exactly clear how HTLV-1 Env is controlled such that virus infection can proceed without cell-cell fusion, but it is probable that Env trafficking plays an important role. The cytoplasmic domain of HTLV-1 Env is relatively short and contains two important trafficking motifs: a YXXΦ motif (YSLI), which is involved in membrane protein trafficking and basolateral sorting in polarized epithelial cells (10), and a PDZ-binding motif (ESSL), which can interact with numerous PDZ proteins but is not found in other retroviral Envs (2).The tyrosine-based sorting motif (YXXΦ, where Y is tyrosine, X is any amino acid, and Φ is a bulky hydrophobic amino acid) determines the trafficking and turnover of many membrane-spanning proteins in the cell (5, 39) and is present in most retroviral Env proteins (7). The YXXΦ motif interacts with the μ subunit of the heterotetrameric adaptor protein complexes AP1, AP2, AP3, and AP4. Each adaptor complex is involved in a specific trafficking pathway: AP1 and AP4 deliver cargo from the trans-Golgi network to the plasma membrane (13, 33, 48), AP2 directs the endocytosis of proteins from the cell surface, and AP3 is involved in lysosomal sorting (5, 12, 24, 35). Each type of μ subunit interacts with a distinct but overlapping type of tyrosine-based motif; the tyrosine and the Φ residues are most critical, but affinity is determined in large part by the variable amino acids at positions +1 and +2 relative to tyrosine and also by surrounding amino acids (5, 37). Furthermore, interactions between AP2 and the YXXΦ motif may be regulated by phosphorylation of μ2 (38, 47), by localized changes in phosphoinositide concentration, or by interactions between AP2 and docking factors (47). Although most retroviral Env proteins contain YXXΦ-sorting motifs, the sequences of the motifs and their roles in Env trafficking and function appear to vary widely among different retroviruses. For example, mutation of the YXXΦ motif in MLV Env interferes with basolateral targeting of Env and diminishes viral pathogenesis in vivo but has little effect on Env accumulation at the plasma membrane (9, 16, 23, 25, 29). Mutations in the YXXΦ motif in MPMV Env are similar to those in MLV Evn and also were reported to affect Env incorporation into virions (45). Mutation of the YXXΦ motif in HTLV-1 Env was previously shown to decrease Env endocytosis, increase cell-cell fusion, increase Env incorporation into virions, abolish basolateral targeting, and decrease virus infectivity (1, 10).The most abundant protein-protein interaction domains in mammalian cells are the PDZ domains; more than 400 PDZ proteins are encoded in the human genome. PDZ domains are modular, recognize short C-terminal peptide motifs, and are often found in multiple copies or in combination with other protein interaction domains (36, 46, 50). PDZ proteins have the ability to form supramolecular scaffolds that coordinate signaling, synapse formation, cell polarity, and trafficking of interacting proteins (26, 44, 53). With respect to the last, it is important to note that PDZ proteins can delay the internalization of G protein-coupled receptors, ion channels, and membrane transporters (17, 41, 49, 52). Among retroviral Env proteins, only HTLV and simian T-lymphotropic virus (STLV) Envs contain putative PDZ-binding motifs. A yeast two-hybrid screen using the HTLV-1 Env cytoplasmic domain (CD) as bait identified the PDZ protein hDlg (human homolog of disc large protein) as a potential binding partner (2). In vitro pulldown experiments showed that a glutathione S-transferase (GST)-EnvCD fusion protein interacted with several PDZ proteins from cell lysates, one of which was hDlg. In one study, mutation of the PDZ-binding motif in HTLV-1 Env inhibited cell-cell fusion (2); in another study, hDlg small interfering RNA (siRNA) silencing caused a modest reduction in syncytium formation (54). Neither study examined how the PDZ-binding motif controls Env expression, membrane targeting, trafficking, or virus infectivity. Thus, it is still unclear which PDZ proteins interact with HTLV-1 Env in vivo and how those interactions affect Env trafficking and activity.In this paper, functional interactions between the YXXΦ motif and the PDZ-binding motif in the cytoplasmic domain of HTLV-1 Env were investigated by mutagenesis of Env and by siRNA silencing of potential cellular interacting proteins. The YXXΦ motif in HTLV-1 Env appears to interact primarily with AP2 and AP3, which regulate Env endocytosis and lysosomal degradation, respectively. Mutations that ablated the YXXΦ motif increased Env accumulation on the cell surface. The PDZ-binding motif at the C terminus of Env appears to delay Env turnover. Mutation of the PDZ-binding element diminished Env accumulation in cells to very low levels, indicating that loss of the PDZ-binding motif accelerates Env degradation. Expression of Env with a mutated PDZ-binding motif could be restored to normal levels by also mutating the YXXΦ motif or by silencing AP2 or AP3. The ability of the PDZ-binding motif to alter the activity of the YXXΦ motif depends on the particular sequence of the latter. The attenuating effect of the PDZ-binding motif on Env endocytosis could be overcome by substitution of the YSLI motif in HTLV-1 Env with YXXΦ elements from other cell or virus proteins that are predicted to have higher affinities for AP2 than the YSLI motif of HTLV-1 Env.     

Multimer Formation Is Not Essential for Nuclear Export of Human T-Cell Leukemia Virus Type 1 Rex trans-Activator Protein

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.     

Identification of a Domain within the Human T-Cell Leukemia Virus Type 2 Envelope Required for Syncytium Induction and Replication

In vitro infection by human T-cell leukemia virus type 1 and 2 (HTLV-1 and HTLV-2) can result in syncytium formation, facilitating viral entry. Using cell lines that were susceptible to HTLV-2-mediated syncytium formation but were nonfusogenic with HTLV-1, we constructed chimeric envelopes between HTLV-1 and -2 and assayed for the ability to induce syncytia in BJAB cells and HeLa cells. We have identified a fusion domain composed of the first 64 amino acids at the amino terminus of the HTLV-2 transmembrane protein, p21, the retention of which was required for syncytium induction. Construction of replication-competent HTLV genomic clones allowed us to correlate the ability of HTLV-2 to induce syncytia with the ability to replicate in BJAB cells. Differences in the ability to induce syncytia were not due to differences in the levels of total or cell membrane-associated envelope or in the formation of multimers. Therefore, we have localized a fusion domain within the amino terminus of the transmembrane protein of HTLV-2 envelope that is necessary for syncytium induction and viral replication.Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are type C retroviruses that have been associated with a variety of human malignancies. HTLV-1 is the etiological agent of adult T-cell leukemia as well as a degenerative neurological disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (28, 40, 58, 60, 83). Recent reports have also implicated HTLV-1 infection with arthropathy (42, 65), polymyosis (23, 37), and uveitis (48, 49, 51). HTLV-2 has been associated with a rare form of atypical hairy cell leukemia (62, 63, 68) as well as some cases of neuropathy (33, 39). It is estimated that between 10 million and 20 million individuals worldwide are infected with HTLV, with an overall risk of 5% of disease progression in infected individuals (14). HTLV is endemic in southern Japan, the Caribbean Basin, and Central and South America. In the United States, recent reports have identified a high proportion of HTLV, especially HTLV-2, infection in intravenous-drug abusers (44, 61, 64).Cell-to-cell contact is considered critical for the in vivo and in vitro transmission of HTLV-1 and HTLV-2, as infection by cell-free HTLV virus is inefficient in vitro and in vivo. By analogy with other enveloped viruses, HTLV infection of susceptible cells is likely mediated by the envelope glycoprotein. Antibodies against HTLV envelope are protective against infection in vivo (71, 80), and multiple epitopes that elicit neutralizing antibodies have been identified throughout the protein (31, 34, 56). Initially synthesized as a precursor protein, gp61, HTLV envelope is subsequently modified by glycosylation and cleaved into two subunits, gp46 and p21. The external surface glycoprotein, gp46, is anchored to the cell surface by noncovalent association with the transmembrane envelope glycoprotein, p21. Interaction of envelope with the as yet unidentified cellular receptor leads to cell-to-cell fusion and can result in syncytium formation.We were interested in identifying the molecular determinants of HTLV involved in syncytium formation and viral entry. Our laboratory has several cell lines that are permissive to HTLV-2- but not HTLV-1-mediated cell fusion. Therefore, we constructed recombinants between the HTLV-1 and -2 envelope genes and assayed for the loss of syncytium induction in BJAB cells and HeLa cells. Loss of a 64-amino-acid (aa) domain located at the amino terminus of the HTLV-2 transmembrane protein, p21, correlated with a loss in the ability of the envelope chimera to induce cell fusion. When the chimeric envelopes were expressed in the context of replication-competent genomic clones, there was a good correlation between syncytium induction and the ability to replicate in permissive cells. Present within the identified fusion domain is a hydrophobic region and a heptad repeat resembling a leucine zipper. We examined the contribution of the fusion domain to the structural integrity of the HTLV-2 envelope by using a vaccinia virus expression system. None of the recombinants affected the synthesis, transport, or oligomer formation of the HTLV glycoprotein complex.     

Palmitoylation and p8-Mediated Human T-Cell Leukemia Virus Type 1 Transmission

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.     

Multiple Functions for the Basic Amino Acids of the Human T-Cell Leukemia Virus Type 1 Matrix Protein in Viral Transmission

We studied the involvement of the human T-cell leukemia virus type 1 (HTLV-1) Gag matrix protein in the cell-to-cell transmission of the virus using missense mutations of the basic amino acids. These basic amino acids are clustered at the N terminus of the protein in other retroviruses and are responsible for targeting the Gag proteins to the plasma membrane. In the HTLV–bovine leukemia virus genus of retroviruses, the basic amino acids are distributed throughout the matrix protein sequence. The HTLV-1 matrix protein contains 11 such residues. A wild-type phenotype was obtained only for mutant viruses with mutations at one of two positions in the matrix protein. The phenotypes of the other nine mutant viruses showed that the basic amino acids are involved at various steps of the replication cycle, including some after membrane targeting. Most of these nine mutations allowed normal synthesis, transport, and cleavage of the Gag precursor, but particle release was greatly affected for seven of them. In addition, four mutated proteins with correct particle release and envelope glycoprotein incorporation did not however permit cell-to-cell transmission of HTLV-1. Thus, particle release, although required, is not sufficient for the cell-to-cell transmission of HTLV-1, and the basic residues of the matrix protein are involved in steps that occur after viral particle release.     

Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells

Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.     

Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system.     

Analysis of Human T-Cell Leukemia Virus Type 1 Particles by Using Cryo-Electron Tomography

The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.     

Efficient Expression and Rapid Purification of Human T-Cell Leukemia Virus Type 1 Protease

Human T-cell leukemia virus type 1 (HTLV-1) is an oncovirus that is clinically associated with adult T-cell leukemia. We report here the construction of a pET19-based expression clone containing HTLV-1 protease fused to a decahistidine-containing leader peptide. The recombinant protein is efficiently expressed in Escherichia coli, and the fusion protein can be easily purified by affinity chromatography. Active mature protease in yields in excess of 3 mg/liter of culture can then be obtained by a novel two-step refolding and autoprocessing procedure. The purified enzyme exhibited K_m and K_cat values of 0.3 mM and 0.143 sec⁻¹ at pH 5.3 and was inhibited by pepstatin A.     

The Humoral Immune Response to Human T-Cell Lymphotropic Virus Type 1 Envelope Glycoprotein gp46 Is Directed Primarily against Conformational Epitopes

Individuals infected with human T-cell lymphotropic virus type 1 (HTLV-1) develop a robust immune response to the surface envelope glycoprotein gp46 that is partially protective. The relative contribution of antibodies to conformation-dependent epitopes, including those mediating virus neutralization as part of the humoral immune response, is not well defined. We assess in this report the relationship between defined linear and conformational epitopes and the antibodies elicited to these domains. First, five monoclonal antibodies to linear epitopes within gp46 were evaluated for their ability to abrogate binding of three human monoclonal antibodies that inhibit HTLV-1-mediated syncytia formation and recognize conformational epitopes. Binding of antibodies to conformational epitopes was unaffected by antibodies to linear epitopes throughout the carboxy-terminal half and central domain of HTLV-1 gp46. Second, an enzyme-linked immunoadsorbent assay was developed and used to measure serum antibodies to native and denatured gp46 from HTLV-1-infected individuals. In sera from infected individuals, reactivity to denatured gp46 had an average of 15% of the reactivity observed to native gp46. Third, serum antibodies from 24 of 25 of HTLV-1-infected individuals inhibited binding of a neutralizing human monoclonal antibody, PRH-7A, to a conformational epitope on gp46 that is common to HTLV-1 and -2. Thus, antibodies to conformational epitopes comprise the majority of the immune response to HTLV-1 gp46, and the epitopes recognized by these antibodies do not appear to involve sequences in previously described immunodominant linear epitopes.     

Expanded Breadth of the T-Cell Response to Mosaic Human Immunodeficiency Virus Type 1 Envelope DNA Vaccination

An effective AIDS vaccine must control highly diverse circulating strains of human immunodeficiency virus type 1 (HIV-1). Among HIV-1 gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV-1 Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential T-cell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. One-, two-, and three-mosaic sets that increased theoretical epitope coverage were developed. The breadth and magnitude of T-cell immunity stimulated by these vaccines were compared to those for natural strain Envs; additional comparisons were performed on mutant Envs, including gp160 or gp145 with or without V regions and gp41 deletions. Among them, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs. Synthetic mosaic HIV-1 antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based HIV-1 vaccines.The development of AIDS vaccines has been advanced recently by demonstrations of increased survival and decreased viral load following vaccination with T-cell vaccines in nonhuman primate models (12, 19, 23, 26, 31, 37). Although such vaccine studies have implied that T cells may contribute to the control of viremia in the highly lethal simian immunodeficiency virus SIVmac251 challenge model, the applicability of these results in human studies remains uncertain. The major concern regarding the efficacy of human immunodeficiency virus (HIV) vaccines in humans is the extraordinary genetic diversity of the virus. The sequence similarity of HIV type 1 (HIV-1) envelope from diverse isolates within a clade can diverge by as much as 15%, and divergence between alternative clades may approach 30% (10). In addition, the diversity of the viral Gag gene product can approach similar levels, particularly in p17 and p15, which are much more diverse than p24 (6), although Gag does not have the extreme localized diversity seen in the highly variable regions of Env (6, 10). While the approach to viral diversity has been addressed in existing vaccines through the use of envelopes derived from representative viruses in the major clades, increasing knowledge about the genetic diversity of naturally occurring isolates has enabled alternative approaches that enhance population coverage of vaccine-elicited T-cell responses.Approaches under consideration include the use of central gene sequences based on ancestral, consensus, or center-of-the-tree genetic analyses (5, 10, 18, 31, 36). Such prototypes are derived by selection of the most common amino acids at each residue (10, 16, 17, 21, 25, 36), identifying the most recent common ancestor of diverging viruses in a vaccine target population (5, 10, 18, 36), or modeling the sequence at the center of the phylogenetic tree (29), respectively. Peptides based on any of these three centralized protein strategies enhanced the detection of T-cell responses in natural infection relative to the use of peptides based on natural strains; however, all three strategies behaved equivalently (7).The use of a single M group consensus/ancestral Env sequence has been shown to elicit T-cell responses with greater breadth of cross-reactivity than single natural strains in animal models (31, 36). Such central sequences do not exist in nature, and even phylogenetic ancestral reconstructions are just an approximate model of an ancestral state of the virus (8). Thus, central sequence strategies have provided evidence that various informatically derived gene products can elicit immune responses to T-cell epitopes found in diverse circulating strains, leading to the possibility of using computational strategies to design polyvalent vaccines which optimize T-cell coverage (6, 24). In this study, we have evaluated for the first time the ability of nonnatural mosaic Env immunogens (6) to elicit T-cell responses of increased cross-reactivity against epitopes represented in naturally circulating viruses in animals.Mosaic HIV-1 envelope genes were derived using an informatic approach, whereby in silico-generated recombinants of natural variants from the Los Alamos database M group Env alignment were created, scored, and selected in combination to optimize the coverage of 9-mers in the global database for a given vaccine cocktail size. While mosaic proteins are artificial constructs that do not occur in nature, they align well to natural proteins, and any short span found in mosaics will tend to be found repeatedly among natural strains (although some of the hypervariable loop regions of Env are so extremely variable that they are not repeated among circulating strains, and this necessitates bridging these regions with segments found in a single strain). In silico recombination breakpoints are constrained to create fusion points found in natural sequences. It is possible to provide increased breadth of coverage with a single mosaic, providing the maximum possible single-antigen diversity coverage for stretches of nine amino acids. Alternatively, multiple mosaics can increase the breadth of representation but have the drawback of requiring the synthesis of additional vectors for clinical use. Mosaics also preserve a natural Env-like sequence to retain normal antigen processing. Here, we have compared single-, double-, or triple-mosaic envelope antigen sets to naturally circulating strains or other derivatives for their ability to elicit immune responses of increased breadth. The data suggest that mosaic HIV-1 envelope sequences provide an approach that may be useful in the development of HIV vaccines that respond to T-cell epitopes represented in naturally circulating strains.