Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.

Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.

Evidence for inducible recruitment of Wiskott-Aldrich syndrome protein to T cell receptor-CD3 complex in Jurkat T cells.

BACKGROUND: The engagement of the T cell receptor (TCR)-CD3 complex induces the formation of multiple signalling complexes, which are required for actin cytoskeletal rearrangement. The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polymerization that is recruited to the TCR activation site. Since WASp is a binding partner of adaptor protein Nck, which is recruited directly to the TCR CD3? subunit upon TCR ligation, therefore we proposed that the direct recruitment of Nck to TCR-CD3 may also bring WASp directly to TCR-CD3. OBJECTIVE: The aim of this present study was to assess the distribution of WASp, in relation to Nck, to the TCR-CD3ε complex. METHODS: Jurkat T cells were stimulated with anti-TCR antibody and then the cell lysates were immunoprecipitated with anti-CD3 antibody before immunoblotting with antibodies specific to WASp, Nck1, Nck2, SLP-76 and CD3ε molecules. RESULTS: WASp was recruited to SLP-76 and also directly to the TCR-CD3 complex upon TCR triggering. The inducible recruitment of WASp to the TCR-CD3 complex is partially dependent of tyrosine phosphorylation. CONCLUSIONS: The present findings provide an alternative mechanism of WASp recruitment to the site of TCR activation that may be involved in recruitment of Nck.

Non-overlapping functions of Nck1 and Nck2 adaptor proteins in T cell activation.

BACKGROUND: Signalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes. Nck1 and Nck2 are two highly related adaptor proteins downstream of the TCR that each contains three SH3 and one SH2 domains. Their individual functions and the roles of their SH3 domains in human T cells remain mostly unknown. RESULTS: Using specific shRNA we down-regulated the expression of Nck1 or Nck2 to approximately 10% each in Jurkat T cells. We found that down-regulation of Nck1 impaired TCR-induced phosphorylation of the kinases Erk and MEK, activation of the AP-1 and NFAT transcription factors and subsequently, IL-2 and CD69 expression. In sharp contrast, down-regulation of Nck2 hardly impacts these activation read-outs. Thus, in contrast to Nck2, Nck1 is a positive regulator for TCR-induced stimulation of the Erk pathway. Mutation of the third SH3 domain of Nck1 showed that this domain was required for this activity. Further, TCR-induced NFAT activity was reduced in both Nck1 and Nck2 knock-down cells, showing that both isoforms are involved in NFAT activation. Lastly, we show that neither Nck isoform is upstream of p38 phosphorylation or Ca2+influx. CONCLUSIONS: In conclusion, Nck1 and Nck2 have non-redundant roles in human T cell activation in contrast to murine T cells.

Antiviral effects of dehydroascorbic acid.

IN THE PRESENT STUDY, DEHYDROASCORBIC ACID INHIBITED THE MULTIPLICATION OF VIRUSES OF THREE DIFFERENT FAMILIES: herpes simplex virus type 1 (HSV-1), influenza virus type A and poliovirus type 1. Although dehydroascorbic acid showed some cytotoxicity at higher concentrations, the observed antiviral activity was not the secondary result of the cytotoxic effect of the reagent, as the inhibition of virus multiplication was observed at reagent concentrations significantly lower than those resulting in cytotoxicity. Characterization of the mode of the antiviral action of dehydroascorbic acid against HSV-1 revealed that the addition of reagent at any time post infection inhibited the formation of progeny infectious virus in the infected cells, and a one-step growth curve showed that the addition of reagent allowed formation for an additional 2 h, but then almost completely suppressed it. These results indicate that the reagent inhibits HSV-1 multiplication after the completion of viral DNA replication, probably at the step of the envelopment of viral nucleocapsids at the Golgi apparatus of infected cells.

Vpx is critical for reverse transcription of the human immunodeficiency virus type 2 genome in macrophages.

The abilities of wild-type and vpx-defective human immunodeficiency virus type 2 (HIV-2) clones to synthesize viral DNA in human monocyte-derived macrophages (MDMs) and lymphocytic cells were comparatively and quantitatively evaluated. While the vpx-defective mutant directed the synthesis of viral DNA comparably to the wild-type virus and normally in lymphocytic cells, no appreciable viral DNA was detected in MDMs infected with the mutant. To substantiate this finding and to determine whether there is some specific region(s) in Vpx crucial for viral DNA synthesis in MDMs, we generated a series of site-specific point mutants of vpx and examined their phenotypes. The resultant five mutants, with no infectivity for MDMs, showed, without exception, the same defect as the vpx-defective mutant. Our results here clearly demonstrated that the entire Vpx protein is critical for reverse transcription of the HIV-2 genome in human MDMs.

Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study.

We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was far much higher than those of an APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. We finally monitored the incorporation of APOBEC3G into virions produced in A3G-C1. APOBEC3G was easily detected in progeny viral particles upon transfection of vif-minus proviral clone but not of wild type. These results indicated that our new A3G-C1 cell line is eminently useful for various studies on the interaction of human APOBEC3G and HIV-1 Vif.

Generation of HIV-1 derivatives that productively infect macaque monkey lymphoid cells.

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is caused in part by innate cellular factors such as apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) and TRIM5alpha, which restrict virus replication in monkey cells. Variant HIV-1 molecular clones containing both a 21-nucleotide simian immunodeficiency virus (SIV) Gag CA element, corresponding to the HIV-1 cyclophilin A-binding site, and the entire SIV vif gene were constructed. Long-term passage in a cynomolgus monkey lymphoid cell line resulted in the acquisition of two nonsynonymous changes in env, which conferred improved replication properties. A proviral molecular clone, derived from infected cells and designated NL-DT5R, was used to generate virus stocks capable of establishing spreading infections in the cynomolgus monkey T cell line and CD8-depleted peripheral blood mononuclear cells from five of five pig-tailed macaques and one of three rhesus monkeys. NL-DT5R, which genetically is >93% HIV-1, provides the opportunity, not possible with currently available SIV/HIV chimeric viruses, to analyze the function of multiple HIV-1 genes in a broad range of nonhuman primate species.

Effects of lysine to arginine mutations in HIV-1 Vif on its expression and viral infectivity.

We previously demonstrated that the expression in cells of human immunodeficiency virus type 1 (HIV-1) Vif is maintained at low level by proteasome-degradation. We examined the contribution of 16 lysines present in Vif (NL432 clone), which is composed of 192 amino acids (aa), to its expression within cells and to viral infectivity for non-permissive cells. To this end, various lysine-arginine mutations were introduced into wild-type (wt) Vif, and the mutational effects were monitored by transfection experiments. When all the lysines were changed to arginines, the mutant Vif was expressed in cells at much higher level than wt and was much more stable. Both N-terminal (aa nos. 34 and 36) and C-terminal (aa nos. 179 and 181) lysines were found to be almost sufficient for wt property. Different from this observation, one of the lysines at aa nos. 22 and 26 was demonstrated to be essential for the virus to grow in non-permissive cells. Our results showed that there is no clear co-relationship between the expression level of HIV-1 Vif and viral infectivity.

Morphological study on biologically distinct vpx/vpr mutants of HIV-2.

We have previously shown that human immunodeficiency virus type 2 (HIV-2) without functional vpx and vpr genes is severely defective for viral growth in lymphocytic cells, and suggested that the virions produced in the absence of Vpx and Vpr are critically damaged. To examine the nature of replication-defect for the vpx/vpr double mutant, we quantitatively and morphologically studied the virions produced in cells transfected or infected with wild type clone, single (vpx and vpr mutants) or the double mutant. While no significant difference in virion production was found for various virus clones in transfected cells, a major growth retardation in infected cells was readily observed for the vpx and vpx/vpr mutants. In particular, no viral growth was detected for the double mutant. By contrast to the very distinct growth characteristics of the three mutant clones, no appreciable difference in virion morphology was noted. These results indicated that Vpx and Vpr of HIV-2 may cooperatively contribute to virion infectivity without affecting virion morphogenesis.

Construction of gag-chimeric viruses between HIV-1 and SIVmac that are capable of productive multi-cycle infection.

Forty-nine recombinant viral clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus from the rhesus monkey (SIVmac), which carry chimeric gag (capsid/p2 region) genes in the background of the HIV-1 genome, were constructed to establish an HIV-1/monkey infection model system for human AIDS. Upon transfection, all the recombinants generated progeny virions at a level comparable to the parental HIV-1 clone and no major abnormalities were found in the virions, as examined by Western blot analysis. In infection experiments, 18 recombinants grew in human lymphocytic cells and six of these clones propagated as well as the parental virus, as monitored by virion associated-reverse transcriptase production. By contrast, none of the recombinants grew at a detectable level in monkey lymphocytic cells. The defective replication site(s) in human cells for non-infectious recombinants was mapped to the step before and/or during reverse transcription. Our results described here showed that HIV-1 type chimeric viruses between HIV-1 and SIVmac, which are capable of spreading productive infection, are readily constructed throughout the capsid/p2 region. In addition, it is suggested that there may be a viral determinant(s), other than Gag, responsible for the species-specific tropism of HIV-1 and which is associated with viral DNA synthesis.

Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins.

The three-dimensional (3-D) structure of human immunodeficiency virus type 2 (HIV-2) Vpr/Vpx was predicted by homology modeling based on the NMR structure of human immunodeficiency virus type 1 (HIV-1) Vpr. The three proteins similarly have three major amphipathic alpha-helices. In contrast to HIV-1 Vpr, Vpr/Vpx of HIV-2 have a long N-terminal loop and clustered prolines in the second half of the C-terminal loop. HIV-2 Vpx uniquely contains a long region between the second and third major helices, and bears several glycines in the first half of the C-terminal loop. Instead of the glycines, there is a group of hydrophilic amino acids and arginines in the corresponding regions of the two Vprs. To compare the cytopathogenic potentials of HIV-1 Vpr and HIV-2 Vpr/Vpx, we examined the production of luciferase as a marker of cell damage. We further analyzed the characteristics of cells transduced with vpr/vpx genes driven by an inducible promoter. The results obtained clearly show that structurally similar, but distinct, HIV Vpr/Vpx proteins are detrimental to target cells.

Unique characteristics of HIV-1 Vif expression.

We examined the steady-state expression in cells of four accessory proteins of human immunodeficiency virus type 1 (HIV-1). For this purpose, a series of single gene expression vectors for these viral proteins were constructed and were monitored for their production by transfection. Among them, the expression level of Vif was found to be lowest in both the absence and presence of APOBEC3G. In addition, we noticed the presence of its truncated form, which was not observed for the other accessory proteins. When a subgenomic vector was used for transfection, authentic and several small forms of Vif were produced. By mutational analysis, these forms were demonstrated to be mutant Vif proteins translated from M8, M16 and M29. When a full-length molecular clone was used, the smaller versions of Vif were hardly observed. Functional analysis of these mutant Vif proteins showed that they are incapable of modulating viral infectivity. The results described above, i.e. the low steady-state expression and the presence of truncated forms, represent the unique characteristics of HIV-1 Vif.