Quantitative Comparison of HTLV-1 and HIV-1 Cell-to-Cell Infection with New Replication Dependent Vectors

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.     

Trypsin-sensitive and -resistant components in human T-cell membranes required for syncytium formation by human T-cell lymphotropic virus type 1-bearing cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) envelope proteins play an important role in viral entry into target cells. In a syncytium formation assay consisting of a coculture of HTLV-1-bearing cells and target cells, mature gp46 and gp21 proteins each inhibited syncytium formation induced by HTLV-1-bearing cells. Experiments with 125I-labeled proteins showed that 125I-gp46 bound specifically with MOLT-4 target cells even in the presence of large amounts of gp21, whereas 125I-gp21 binding to target cells was completely blocked in the presence of large amounts of gp46. These observations suggest that HTLV-1 envelope proteins in syncytium formation interact with at least two components, which are located close to each other on the cell membrane. We isolated two components from MOLT-4 cell lysate, using Sepharose 4B columns coupled with peptides corresponding to amino acids 197 to 216 and 400 to 429, respectively, of the envelope protein. One is a trypsin digestion-sensitive component of approximately 34 to 35 kDa, which interacts specifically with gp46. The other is a nonprotein component, which interacts with gp21. This component was destroyed by sodium periodate oxidation and was partitioned into the methanol-chloroform phase. These observations suggest that these two components play an important role in HTLV-1 entry into target cells via membrane fusion.     

荧光蛋白标签对gD囊膜蛋白在BHK-21细胞亚细胞定位的影响

【目的】探究荧光蛋白标签对马疱疹病毒I型(Equine herpes virus type 1,EHV-1)gD囊膜蛋白亚细胞定位的影响。【方法】以EHV-1基因组为模板利用PCR扩增gD全基因,分别克隆至pAcGFP1-C1和p Ds Red2-N1质粒,构建p Ac-GFP-gD(GFP-gD)和p Ds-gD-Red(gD-Red)重组质粒;将GFP基因插入gD基因信号肽序列之后并克隆至PVAX-1质粒,构建PVAX-S-GFP-gD’(S-GFP-gD’)重组质粒;将Flag标签序列与gD囊膜蛋白N端序列融合后并克隆至p VAX-1表达载体,构建p VAX-Flag-gD(Flag-gD)重组质粒。将4种不同重组真核表达质粒分别转染BHK-21细胞,通过激光共聚焦显微镜对不同融合蛋白gD进行亚细胞定位。【结果】成功构建4种不同的融合蛋白gD真核表达载体;在BHK-21细胞单独表达时,不同融合蛋白gD绝大部分都定位于高尔基体,极少量定位于细胞核内。【结论】不同插入位点的荧光蛋白标签对gD囊膜蛋白亚细胞定位无明显影响,这对今后研究其它蛋白亚细胞定位提供参考。     

Characterization of envelope glycoprotein mutants for human T-cell leukemia virus type 1 infectivity and immortalization

The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4(+) lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax.

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

Differential requirements for activation of integrated and transiently transfected human T-cell leukemia virus type 1 long terminal repeat

Adult T-cell leukemia (ATL) cells contain integrated human T-cell leukemia virus type 1 (HTLV-1) proviruses. Although the exact sequence of events leading to the development of ATL remains incompletely resolved, expression of the integrated HTLV-1 long terminal repeat (LTR) is likely required at some point during the process of T-cell transformation. While much has been learned about the regulated expression of transiently transfected LTR reporter plasmids, an analysis of factors required for expression of chromosomally integrated HTLV-1 LTR has not been done. Here, we have constructed CHOK1 and HeLa cells that contain an integrated HTLV-1 LTR-luciferase gene. Using these cells, we have compared the requirements for activation of transiently transfected versus stably integrated HTLV-1 LTR. We observed different requirements for CREB, p300, and P/CAF in the expression of transiently transfected versus stably integrated HTLV-1 LTR. Furthermore, with dominant-negative mutants of CREB, p300, and P/CAF, we found that activation of integrated HTLV-1 LTR by an ambient stress signal, UV-C, proceeds through a path mechanistically distinct from that used by viral oncoprotein, Tax. Our findings point to additional complexities in the regulated expression of HTLV-1 proviruses compared with those hitherto revealed through transfection studies.     

The synthetic peptide P-197 inhibits human T-cell leukemia virus type 1 envelope-mediated syncytium formation by a mechanism that is independent of Hsc70

Entry of human T-cell leukemia virus type 1 (HTLV-1) into cells is mediated by the viral envelope glycoproteins gp46 and gp21. The gp46 surface glycoprotein binds to a poorly characterized cell surface receptor, thereby promoting the gp21-dependent fusion of the viral and cellular membranes. Interestingly, a synthetic peptide (P-197) simulating amino acids 197 to 216 of gp46 strongly inhibits envelope-dependent membrane fusion with Molt-4 target cells. It has been suggested that this peptide acts by competitively binding to Hsc70, a putative cellular receptor for HTLV-1. We now demonstrate that P-197 inhibits membrane fusion among diverse HTLV-1-permissive target cells. Importantly, most of these cells lack detectable levels of Hsc70, indicating that P-197 inhibits membrane fusion by a mechanism that is Hsc70 independent. We now suggest that competition for primary receptor binding is unlikely to account for the inhibitory activity of P-197. Understanding the mechanism by which P-197 functions may reveal concepts of general relevance to antiretroviral chemotherapy.     

Human T-lymphotropic virus-1 visualized at the virological synapse by electron tomography

Human T-lymphotropic virus 1 (HTLV-1) is transmitted directly between cells via an organized cell-cell contact called a virological synapse (VS). The VS has been studied by light microscopy, but the ultrastructure of the VS and the nature of the transmitted viral particle have remained unknown. Cell-free enveloped virions of HTLV-1 are undetectable in the serum of individuals infected with the human T-lymphotropic virus 1 (HTLV-1) and during in vitro culture of naturally infected lymphocytes. However, the viral envelope protein is required for infectivity of HTLV-1, suggesting that complete, enveloped HTLV-1 virions are transferred across the synapse. Here, we use electron tomography combined with immunostaining of viral protein to demonstrate the presence of enveloped HTLV-1 particles within the VS formed between naturally infected lymphocytes. We show in 3D that HTLV-1 particles can be detected in multiple synaptic clefts at different locations simultaneously within the same VS. The synaptic clefts are surrounded by the tightly apposed plasma membranes of the two cells. HTLV-1 virions can contact the recipient cell membrane before detaching from the infected cell. The results show that the HTLV-1 virological synapse that forms spontaneously between lymphocytes of HTLV-1 infected individuals allows direct cell-cell transmission of the virus by triggered, directional release of enveloped HTLV-1 particles into confined intercellular spaces.     

Neuropilin-1 is involved in human T-cell lymphotropic virus type 1 entry

Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted through a viral synapse and enters target cells via interaction with the glucose transporter GLUT1. Here, we show that Neuropilin-1 (NRP1), the receptor for semaphorin-3A and VEGF-A165 and a member of the immune synapse, is also a physical and functional partner of HTLV-1 envelope (Env) proteins. HTLV-1 Env and NRP1 complexes are formed in cotransfected cells, and endogenous NRP1 contributes to the binding of HTLV-1 Env to target cells. NRP1 overexpression increases HTLV-1 Env-dependent syncytium formation. Moreover, overexpression of NRP1 increases both HTLV-1 and HTLV-2 Env-dependent infection, whereas down-regulation of endogenous NRP1 has the opposite effect. Finally, overexpressed GLUT1, NRP1, and Env form ternary complexes in transfected cells, and endogenous NRP1 and GLUT1 colocalize in membrane junctions formed between uninfected and HTLV-1-infected T cells. These data show that NRP1 is involved in HTLV-1 and HTLV-2 entry, suggesting that the HTLV receptor has a multicomponent nature.     

Secretion of enzymatically active human renin from mammalian cells using an avian retroviral vector

Recombinant plasmid-based retroviral expression vectors were constructed using a modified spleen necrosis virus (SNV) containing the Herpes simplex virus thymidine kinase gene promoter controlling the expression of the Tn5 neomycin phosphotransferase II gene (NPTII gene). The human renin (HRn) gene (hrn) was inserted into the 5' end of the SNV sequences such that in concatemeric plasmid DNA its expression was controlled by the strong promoter in the SNV long terminal repeat (LTR). Dog cells transfected with the concatemeric plasmid DNA secreted a small amount of a HRn-like 43-kDa protein. After cotransfection of chicken cells with concatemeric plasmid DNA and proviral DNA of reticuloendotheliosis virus strain A, infectious stocks of viruses were recovered. Cells infected with the virus carrying the viral LTR-hrn gene oriented for expression secreted the 43-kDa HRn-like protein at about 100-fold higher levels than the cells transfected with the plasmid DNAs. Biological activity of secreted HRn was determined by measuring levels of angiotensin I generated by incubating culture media with either a porcine or human angiotensinogen substrate. Infected dog cells produce about 40 ng of enzymatically active HRn per 10(6) cells per 24 h. These data indicate that retroviral expression vectors provide a good system for obtaining the secretion of high levels of enzymatically active heterologous proteins from mammalian cells.     

Astrocyte-specific expression of human T-cell lymphotropic virus type 1 (HTLV-1) Tax: induction of tumor necrosis factor alpha and susceptibility to lysis by CD8+ HTLV-1-specific cytotoxic T cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with a chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP). Although the pathogenesis of this disease remains to be elucidated, the evidence suggests that immunopathological mechanisms are involved. Since HTLV-1 tax mRNA was colocalized with glial acidic fibrillary protein, a marker for astrocytes, we developed an in vitro model to assess whether HTLV-1 infection activates astrocytes to secrete cytokines or present viral immunodominant epitopes to virus-specific T cells. Two human astrocytic glioma cell lines, U251 and U373, were transfected with the 3' portion of the HTLV-1 genome and with the HTLV-1 tax gene under astrocyte-specific promoter control. In this study, we report that Tax-expressing astrocytic glioma transfectants activate the expression of tumor necrosis factor alpha mRNA in vitro. Furthermore, these Tax-expressing glioma transfectants can serve as immunological targets for HTLV-1-specific cytotoxic T lymphocytes (CTL). We propose that these events could contribute to the neuropathology of HAM/TSP, since infected astrocytes can become a source for inflammatory cytokines upon HTLV-1 infection and serve as targets for HTLV-1-specific CTL, resulting in parenchymal damage by direct lysis and/or cytokine release.     

Antibodies to the envelope glycoprotein of human T cell leukemia virus type 1 robustly activate cell-mediated cytotoxic responses and directly neutralize viral infectivity at multiple steps of the entry process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.     

Amino Acid Changes at Positions 173 and 187 in the Human T-Cell Leukemia Virus Type 1 Surface Glycoprotein Induce Specific Neutralizing Antibodies

The nucleotide sequence of human T-cell leukemia virus type 1 (HTLV-1) is highly conserved, most strains sharing at least 95% sequence identity. This sequence conservation is also found in the viral env gene, which codes for the two envelope glycoproteins that play a major role in the induction of a protective immune response against the virus. However, recent reports have indicated that some variations in env sequences may induce incomplete cross-reactivity between HTLV-1 strains. To identify the amino acid changes that might be involved in the antigenicity of neutralizable epitopes, we constructed expression vectors coding for the envelope glycoproteins of two HTLV-1 isolates (2060 and 2072) which induced human antibodies with different neutralization patterns. The amino acid sequences of the envelope glycoproteins differed at four positions. Vectors coding for chimeric or point-mutated envelope proteins were derived from 2060 and 2072 HTLV-1 env genes. Syncytium formation induced by the wild-type or mutated envelope proteins was inhibited by human sera with different neutralizing specificities. We thus identified two amino acid changes, I173-->V and A187-->T, that play an important role in the antigenicity of neutralizable epitopes located in this region of the surface envelope glycoprotein.     

Antibody reactivity to different regions of human T-cell leukemia virus type 1 gp61 in infected people.

The primary protein product of the human T-cell leukemia virus type 1 (HTLV-1) env gene, gp61, is cleaved to produce both the exterior (gp46) and the transmembrane (gp21) portions of the HTLV-1 envelope protein. To compare the reactivity with human antibodies of different regions of this gp61 protein, five plasmids (A, B, B1, C, and D) were constructed to express recombinant proteins (RPs) in Escherichia coli. RP-A, RP-B, RP-B1, and RP-C contain amino acid residues 26 to 165, 166 to 229, 166 to 201, and 229 to 308, respectively, of the exterior envelope protein gp46. Serum samples from HTLV-1-seropositive subjects were assayed for reactivity with these RPs by Western immunoblotting. The percentages of positive reactivity with each of the RPs were as follows: 18.9% (23 of 122) for RP-A, 89.6% (112 of 125) for RP-B, 70.2% (85 of 121) for RP-B1, and 92.9% (117 of 126) for RP-C. These results indicate that the C-terminal half of gp46 (RP-B plus RP-C) can detect 97.6% (123 of 126) of positive samples, while the N-terminal half of gp46 (RP-A) can only detect 18.9% of the HTLV-1-positive sera (P less than 0.005). Furthermore, RP-A, -B, and -C, which together span the entire length of gp46 except the first five amino acids at the N terminus and the last four amino acids at the C- terminus, detected 99.2% (125 of 126) of the HTLV-1-positive subjects. In contrast, RP-D, which contains the HTLV-1 transmembrane envelope protein gp21 minus the first amino acid at the N terminus, had a lower rate of antibody reactivity at 73.7% (84 of 114) (P less than 0.005). The difference in seropositive rates for RP-D between HTLV-1 carriers (55.6%) and adult T-cell leukemia patients (85.5%) is statistically significant (P less than 0.01). This study therefore indicates that the C-terminal half of gp46, especially the amino acid sequence from 200 to 308, contains the most reactive epitopes of the HTLV-1 gp61 envelope glycoprotein.     

Identification and mapping of functional domains on human T-cell lymphotropic virus type 1 envelope proteins by using synthetic peptides.

To identify the regions that are important in human T-cell leukemia virus type 1 (HTLV-1) envelope function, we synthesized 23 kinds of peptides covering the envelope proteins and examined the inhibitory effect of each peptide on syncytium formation induced by HTLV-1-bearing cells. Of the 23 synthetic peptides, 2, corresponding to amino acids 197 to 216 on gp46 and 400 to 429 on gp21, inhibited syncytium formation induced by HTLV-1-bearing cells but did not affect syncytium formation induced by human immunodeficiency virus type 1-producing cells. The peptide concentrations giving 50% inhibition of syncytium formation for gp46 197 to 216 and gp21 400 to 429 were 14.9 and 6.0 microM, respectively. A syncytium formation assay with overlapping synthetic peptides containing amino acids 175 to 236 and 391 to 448 of the envelope proteins showed that syncytium formation was inhibited by peptides that contained the amino acid sequences 197 to 205 (Asp-His-Ile-Leu-Glu-Pro-Ser-Ile-Pro) and 397 to 406 (Gln-Glu-Gln-Cys-Arg-Phe- Pro-Asn-Ile-Thr). These observations suggest that the two regions corresponding to amino acids 197 to 216 and 400 to 429 are involved] in HTLV-1 envelope function.