HIV-1 Tat蛋白对人类疱疹病毒8型复制的影响

用HindⅢ将HIV-1Tat101蛋白编码基因从pEV质粒中切出,BamHI、NotⅠ将绿色荧光蛋白(GFP)编码基因从表达质粒pcDNA3．1 ／GFP中切出,分别插入到质粒LZRSpBMN-Z中,构建成重组反转录病毒表达质粒LZRS—Tat101和LZRS—GFP。采用磷酸钙转染法将两重组质粒转染到含反转录病毒env,gal和pol编码基因的包装细胞Phoenix(φNX)中,嘌呤霉素筛选获得稳定细胞系。分别收集稳定细胞系分泌的病毒上清,并感染体外培养的原发性渗出性淋巴瘤(PEL)BC2BL-1细胞。收集LZRS—GFP重组病毒感染的BCBL-1细胞进行流式细胞计数,检测GFP表达水平。收集LZRS—Tat101重组病毒感染的BCBL-1细胞,提取蛋白作Western blot,检测Tat蛋白表达状况;取细胞总RNA作Northem blot和定量PCR,检查HHV-8次要衣壳蛋白ORF26 mRNA转录水平。重组LZRS—Tat101病毒进一步感染HL3T1细胞(HeLa细胞包含HIV-1-LTR／CAT报告基因),收集感染细胞提取蛋白,检测CAT活性,评价Tat生物学功能。PCR扩增HHV-8复制和转录激活蛋白Rta启动子区上游序列,并克隆至pGL-3载体中,构建Rta启动子 虫荧光素酶(Luciferase)报告基因重组质粒。此重组质粒进一步电转染预先感染了LZRS—Tat101病毒的BC-3细胞,TPA刺激后收集细胞,检测Luciferase活性。结果显示：①重组反转录病毒感染BCBL-1细胞,一次感染效率达56％;②重组LZRS—Tat101毒能够在其感染的BCBL-1细胞中表达Tat蛋白,且表达蛋白具有转录激活功能;③Tat蛋白不能有效上调HHV-8Rta启动子活性;④细胞内HIV-1Tat蛋白诱导HHV-8可溶性周期复制的能力较弱。提示,单纯HIV-1Tat蛋白并不能激活潜伏感染的HHV-8。     

Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.     

Modulation of Sp1 Phosphorylation by Human Immunodeficiency Virus Type 1 Tat

We previously reported (K. T. Jeang, R. Chun, N. H. Lin, A. Gatignol, C. G. Glabe, and H. Fan, J. Virol. 67:6224–6233, 1993) that human immunodeficiency virus type 1 (HIV-1) Tat and Sp1 form a protein-protein complex. Here, we have characterized the physical interaction and a functional consequence of Tat-Sp1 contact. Using in vitro protein chromatography, we mapped the region in Tat that contacts Sp1 to amino acids 30 to 55. We found that in cell-free reactions, Tat augmented double-stranded DNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation in a contact-dependent manner. Tat mutants that do not bind Sp1 failed to influence phosphorylation of the latter. In complementary experiments, we also found that Tat forms protein-protein contacts with DNA-PK. We confirmed that in HeLa and Jurkat cells, Tat expression indeed increased the intracellular amount of phosphorylated Sp1 in a manner consistent with the results of cell-free assays. Furthermore, using two phosphatase inhibitors and a kinase inhibitor, we demonstrated a modulation of reporter gene expression as a consequence of changes in Sp1 phosphorylation. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1 which is affected by Tat and DNA-PK.     

Vpu Increases Susceptibility of Human Immunodeficiency Virus Type 1-Infected Cells to Fas Killing

The importance of the Fas death pathway in human immunodeficiency virus (HIV) infection has been the subject of many studies. Missing from these studies is direct measurement of infected cell susceptibility to Fas-induced death. To address this question, we investigated whether T cells infected with HIV are more susceptible to Fas-induced death. We found that Fas cross-linking caused a decrease in the number of HIV-infected Jurkat T cells and CD4⁺ peripheral blood leukocytes (PBLs). We confirmed this finding by demonstrating that there were more apoptotic infected than uninfected cells after Fas ligation. The increase in sensitivity of HIV-infected cells to Fas killing mapped to vpu, while nef, vif, vpr, and second exon of tat did not appear to contribute. Furthermore, expression of Vpu in Jurkat T cells rendered them more susceptible to Fas-induced death. These results show that HIV-infected cells are more sensitive to Fas-induced death and that the Vpu protein of HIV contributes to this sensitivity. The increased sensitivity of HIV-infected cells to Fas-induced death might help explain why these cells have such a short in vivo half-life.     

Tat Protein Induces Human Immunodeficiency Virus Type 1 (HIV-1) Coreceptors and Promotes Infection with both Macrophage-Tropic and T-Lymphotropic HIV-1 Strains

Chemokine receptors CCR5 and CXCR4 are the primary fusion coreceptors utilized for CD4-mediated entry by macrophage (M)- and T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively. Here we demonstrate that HIV-1 Tat protein, a potent viral transactivator shown to be released as a soluble protein by infected cells, differentially induced CXCR4 and CCR5 expression in peripheral blood mononuclear cells. CCR3, a less frequently used coreceptor for certain M-tropic strains, was also induced. CXCR4 was induced on both lymphocytes and monocytes/macrophages, whereas CCR5 and CCR3 were induced on monocytes/macrophages but not on lymphocytes. The pattern of chemokine receptor induction by Tat was distinct from that by phytohemagglutinin. Moreover, Tat-induced CXCR4 and CCR5 expression was dose dependent. Monocytes/macrophages were more susceptible to Tat-mediated induction of CXCR4 and CCR5 than lymphocytes, and CCR5 was more readily induced than CXCR4. The concentrations of Tat effective in inducing CXCR4 and CCR5 expression were within the picomolar range and close to the range of extracellular Tat observed in sera from HIV-1-infected individuals. The induction of CCR5 and CXCR4 expression correlated with Tat-enhanced infectivity of M- and T-tropic viruses, respectively. Taken together, our results define a novel role for Tat in HIV-1 pathogenesis that promotes the infectivity of both M- and T-tropic HIV-1 strains in primary human leukocytes, notably in monocytes/macrophages.     

Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors.     

Binding of Human Immunodeficiency Virus Type 1 gp120 to CXCR4 Induces Mitochondrial Transmembrane Depolarization and Cytochrome c-Mediated Apoptosis Independently of Fas Signaling

Apoptosis of CD4(+) T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochrome c from the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4(+) T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4(+) T-cell depletion in AIDS.     

人免疫缺陷病毒1型TAT蛋白点突变对其反式激活作用的影响

不同免疫缺陷病毒（ＨＩＶ－１,ＨＩＶ－２和ＳＩＶ）的Ｔａｔ均有３个高度保守的结构域：Ｃｙｓ富集域、核心域和碱性氨基酸富集域,用ＰＣＲ定点突变法在ＨＩＶ－１Ｔａｔ蛋白的这些区域引入单氨基酸或多氨基酸突变;构建了以ＨＩＶ－１ＬＴＲ－１５８到＋８Ｏ区域为启动子,含有不同突变点的突变Ｔａｔ基因表达质粒;以荧光酶基因为报告基因,瞬时共转染Ｊｕｒｋａｔ细胞：分析不同氨基酸突变对Ｔａｔ的反式激活作用的影响。结果发现,突变后的Ｔａｔ蛋白的活性均极大地降低或丧失,表明这些序列内的氨基酸的确定性对Ｔａｔ的活性至关重要。     

Amplification of the Inflammatory Cellular Redox State by Human Immunodeficiency Virus Type 1-Immunosuppressive Tat and gp160 Proteins

In the course of our studies on oxidative stress as a component of pathological processes in humans, we showed that microintrusion into cells with microcapillary and ultramicroelectrochemical detection could mimic many types of mechanical intrusion leading to an instant (0.1 s) and high (some femtomoles) burst release of H₂O₂. Specific inhibitors of NADPH enzymes seem to support the assumption that this enzyme is one of the main targets of our experiments. Also, human immunodeficiency virus type 1 (HIV-1) gp160 inhibits the cooperative response of uninfected T cells as well as Tat protein release by infected cells does. In this study, we analyzed in real time, lymphocyte per lymphocyte, the T-cell response following activation in relation to the redox state. We showed that the immunosuppressive effects of HIV-1 Tat and gp160 proteins and oxidative stress are correlated, since the native but not the inactivated Tat and gp160 proteins inhibit the cellular immune response and enhance oxidative stress. These results are consistent with a role of the membrane NADPH oxidase in the cellular response to immune activation.     

The Transmembrane Domain of BST-2 Determines Its Sensitivity to Down-Modulation by Human Immunodeficiency Virus Type 1 Vpu

Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin) restricts the production of a number of enveloped viruses by blocking virus release from the cell surface. This antiviral activity is counteracted by such viral factors as Vpu of human immunodeficiency virus type 1 (HIV-1). Here, we report that Vpu antagonizes human BST-2 but not BST-2 derived from African green monkeys. The determinants of susceptibility to Vpu map to the transmembrane domain of BST-2. In accordance with this, expression of human BST-2 containing a modified transmembrane domain effectively blocks the replication of wild-type Vpu-expressing HIV-1 in CD4⁺ T cells. Furthermore, these BST-2 variants, as opposed to wild-type human BST-2, are refractory to Vpu-mediated down-regulation as a result of an attenuated interaction with Vpu. In view of the work by others pointing to a key role of the transmembrane domain of Vpu in promoting virus release, our data suggest that a direct interaction through the transmembrane domain of each of these two proteins is a prerequisite for Vpu to down-modulate BST-2.Human immunodeficiency virus type 1 (HIV-1) encodes four accessory proteins, Vif, Vpr, Vpu, and Nef. Although they are dispensable for HIV-1 replication in certain transformed cell lines, these accessory proteins play important roles in HIV-1 pathogenesis by modulating host immunity and overcoming antagonism by cellular factors (10). For example, Vif counteracts APOBEC3G by recruiting the cullin 5-elongin B/C ubiquitin ligase complex and sending polyubiquitinated APOBEC3G to proteasomes for degradation (29). In the absence of Vif, newly synthesized APOBEC3G is incorporated into virus particles and hampers the production of infectious proviral DNA in the new round of infection (4, 10, 23). In addition to its role in down-modulating the cell surface expression of CD4 in infected T cells (11), Vpu stimulates HIV-1 production in cells such as HeLa cells (26). The mechanism behind this latter activity of Vpu was unknown until it was recently discovered that bone marrow stromal cell antigen 2 (BST-2, also known as tetherin, CD317, or HM1.24) blocks the release of HIV-1 and that this inhibitory effect is antagonized by viral Vpu (16, 25).BST-2 harbors an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol anchor that together create an unusual topology with both termini of BST-2 inserted into the plasma membrane (8, 18). This unique topology of BST-2 may underlie the mechanism for the retention of progeny virus particles at the cell surface (16). An indirect mechanism behind this tethering effect has not been ruled out, especially in view of the difficulty of detecting BST-2 protein in purified HIV-1 particles (14). In addition to HIV-1, a number of enveloped viruses are subject to inhibition by BST-2, including simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, Mason-Pfizer monkey virus, and Lassa virus, as well as Ebola and Marburg viruses (5, 6, 16, 19, 25). This suggests that BST-2 has a broad antiviral effect spectrum.The bst-2 gene has in its promoter the IRF-1/2 and ISGF3 response elements and thus belongs to the interferon-stimulated gene family (17). In line with its ability to impair the release of enveloped viruses, BST-2 has been demonstrated to be the effector in human embryonic kidney (HEK293T) cells that leads to the interferon-induced block of Vpu deletion-containing HIV-1 production (15). However, the African green monkey kidney cell line COS-7 responds to interferon treatment with a different outcome in that the production of both Vpu deletion-containing and Vpu-expressing HIV-1 is inhibited (15). This indicates that interferon induces a block to HIV-1 in COS-7 cells that cannot be overcome by Vpu. A conceivable candidate that creates this block is BST-2 in COS-7 cells (hereafter named agmBST-2). In this study, we provide evidence that depletion of endogenous BST-2 in COS-7 cells greatly alleviates interferon-induced inhibition of HIV-1 production. The refractoriness of agmBST-2 to Vpu results from a weak association of these two proteins and a resistance of agmBST-2 to Vpu-mediated down-regulation.     

Coreceptor Utilization by Human Immunodeficiency Virus Type 1 Is Not a Primary Determinant of Neutralization Sensitivity

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.1 regardless of the coreceptor utilized. This study suggests that coreceptor utilization is not a primary determinant of differential neutralization sensitivity in PI and TCLA viruses.Although CD4 had long been recognized as the cellular receptor to which the human immunodeficiency virus type 1 (HIV) envelope protein binds (9, 21, 22), it had also been recognized that expression of CD4 alone is insufficient to render nonhuman cells susceptible to HIV infection (4, 5, 22). Similarly, different HIV isolates display different abilities to infect CD4-positive human macrophages, T lymphocytes, and established T-cell lines (31, 32, 35), suggesting that additional molecules may be responsible for cell tropism specificity. During the past year, cellular molecules that act in conjunction with CD4 have been identified as required cofactors for HIV envelope protein-mediated binding and entry (1, 6, 10–12, 14). These HIV coreceptors are members of the superfamily of seven-transmembrane segment G-protein-coupled receptors and act primarily as cellular receptors for chemokines.The discovery of cellular coreceptors for HIV has provided new perspectives for understanding these early events in HIV infection (see review in reference 2). Thus, phenotypically distinct isolates of HIV utilize as coreceptors different chemokine receptor molecules. Although all primary isolates of HIV infect primary T lymphocytes, some also infect cells of the macrophage lineage (31, 32). These monocyteropic isolates utilize the CCR5 chemokine receptor, whose natural ligands include the chemokines RANTES, MIP-1α, and MIP-1β (1, 6, 10–12). Monocytropic isolates do not induce syncytia in primary lymphocyte culture and do not infect established T-cell lines (31). During the late course of HIV infection, syncytium-inducing (SI) primary viruses often arise from the population of monocytropic viruses (31, 32). These SI primary isolates no longer infect macrophages, and they utilize both CCR5 and another chemokine receptor, CXCR4 (7, 33, 38). CXCR4, whose natural chemokine ligand is SDF-1 (3, 27), was originally identified by Feng et al. as the cofactor used by laboratory-adapted viruses (14). In fact, the common laboratory viruses (IIIb/LAI, LAV, and RF) are unable to utilize CCR5 coreceptor (1, 6, 10–12), presumably reflecting the lack of CCR5 expression in most established T-cell lines (1, 13). Although some primary isolates utilize additional chemokine receptor molecules, notably CCR3 and CCR2b (6, 11, 18), the relationship between these coreceptors and viral phenotypes is less clear. The ability to utilize CCR5 coreceptor, however, is unique to primary-isolate (PI) viruses.Paralleling these differences in coreceptor utilization and cell tropism are differences in sensitivity to virus neutralization. Although laboratory-adapted isolates of HIV can be potently neutralized by sera elicited by recombinant gp120 (rgp120) protein, primary isolates are largely refractory to neutralization by rgp120 vaccine sera (23, 37). Similarly, PI viruses are significantly more resistant than T-cell line-adapted (TCLA) viruses to neutralization by gp120-directed monoclonal antibodies (MAbs) (25, 37) and to inhibition by soluble forms of CD4 (8). We and others have demonstrated that neutralization sensitivity develops concomitantly with adaptation of primary isolates to persistent growth in established T-cell lines (24, 37). By studying pedigreed PI and TCLA viruses (168P and 168C, respectively), we have shown that adaptation renders the TCLA virus sensitive not only to rgp120 vaccine sera and CD4 immunoadhesin but also to MAbs directed to the V3 loop of gp120 (37). However, the basis for this increase in neutralization sensitivity remains unclear.In this report, we explore the relationship between neutralization sensitivity and coreceptor utilization, especially with regard to changes that accompany adaptation. We examined neutralization sensitivity of the well-characterized SI primary isolate 168P under experimental conditions where infection can be directed via either the CXCR4 or the CCR5 pathway. The pedigreed TCLA derivative 168C utilizes only CXCR4 and was sensitive to neutralization by the panel of V3-directed MAbs used in these assays. However, the primary isolate 168P remained refractory to neutralization regardless of coreceptor pathway taken. Our findings suggest that envelope protein structure, and not coreceptor utilization, is the primary determinant of differential neutralization sensitivity in PI and TCLA viruses.

Coreceptor utilization by pedigreed PI and TCLA viruses.

Cross-sectional surveys of coreceptor use have shown that primary SI isolates generally utilize CXCR4 and CCR5 coreceptors, whereas unrelated laboratory-adapted isolates utilize only CXCR4 (1, 6, 7, 10–12, 14, 33, 38). We wished to confirm this trend in a longitudinal study of adaptation. We previously described the adaptation of the SI primary isolate 168P to persistent growth in the FDA/H9 T-cell line and the concomitant development of neutralization sensitivity in the resulting TCLA virus 168C (37). In the present study, the ability of these pedigreed viruses to utilize specific coreceptors was tested by infection of U87 human glioma cell lines expressing CD4 (U87-CD4) and the specific coreceptor (19).For this assay, virus stocks were prepared from cell culture supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) (168P) or FDA/H9 cells (168C) and standardized to yield a submaximal number of foci of infection on U87-CD4-CXCR4 cells (approximately 100 to 200 foci/96-well microplate culture). To confirm coreceptor specificity, in some assays CCR5 chemokines (each at 500 ng/ml) were added to cells 1 h prior to infection. After 2 days of incubation, cell monolayers were fixed with methanol-acetone and immunochemically stained with HIV immunoglobulin (HIVIG) (29), anti-human ABC kit (Biomeda Corp.), and diaminobenzidine substrate.Figure ​Figure11 confirms the ability of the SI 168P virus to utilize both CXCR4 and CCR5 and the subsequent loss of this latter specificity in the 168C TCLA virus. Infection was dependent on coreceptor expression, and both PI and TCLA viruses could also utilize CCR3 (data not presented). Open in a separate windowFIG. 1Coreceptor utilization by pedigreed PI and TCLA 168 viruses. U87-CD4 cell lines expressing CXCR4 (▪) or CCR5 () were used to define the ability of 168P and 168C viruses to utilize the respective coreceptor. CCR5 utilization was further tested by the addition to U87-CD4-CCR5 cells of CCR5-specific chemokines (RANTES, MIP-1α, and MIP-1β; R&D Systems) (□). For details, see text. ∗, no foci were observed.In keeping with the determined coreceptor specificity, infection could be blocked by addition of coreceptor-specific ligands. Thus, 168P virus infection of CCR5-expressing cells was blocked by the CCR5-specific ligands RANTES, MIP-1α, and MIP-1β (1, 6, 10–12) (Fig. ​(Fig.1).1). Similarly, infection of CXCR4-expressing U87-CD4 cells by either virus could be blocked by the CXCR4-specific chemokine ligand SDF-1 (3, 27) (data not presented).

Coreceptor pathway and neutralization sensitivity.

In previous work, we demonstrated that the PI 168P virus is refractory to neutralization by HIV MN gp120 vaccine sera and by several well-characterized V3-directed murine MAbs which strongly neutralize infectivity of the TCLA 168C virus (37). In the present study, we extended the panel of MAbs to include two V3-directed human MAbs, 257-D and 268-D (17). These well-characterized human MAbs recognize core epitopes at the crown of the V3 loop of gp120 (KRIHI and HIGPGR, respectively), linear sequences known to be present in both 168P and 168C envelope proteins (37). These epitope predictions were confirmed by gp120 capture enzyme-linked immunosorbent assay (ELISA) (26) which demonstrated equal binding to envelope protein in detergent-solubilized 168P and 168C virions (data not presented). Sensitivity to neutralization by these human MAbs was determined in a standard assay using PHA-activated PBLs (37). MAbs 257-D and 268-D were found to potently neutralize 168C but fail to neutralize 168P (Fig. ​(Fig.2).2). This pattern of neutralization sensitivity is similar to that previously described for the V3-directed murine MAb 50.1 (30, 36, 37). Open in a separate windowFIG. 2Neutralization sensitivity of 168 viruses in PBL culture. Virus neutralization assays in PHA-stimulated PBL culture were performed as previously described (37). 168P (○, •) and 168C (□, ▪) virus stocks were standardized to yield submaximal extents of virus spread during the 5-day infection. CCR5-specific chemokines (•, ▪) were added as described for Fig. ​Fig.1.1. The V3-directed MAbs are indicated. p24 antigen was determined by p24 antigen capture ELISA (SAIC Frederick) and was normalized to infected cell control values (168P, 190 ng/ml [170 ng/ml with chemokines]; 168C, 36 ng/ml [33 ng/ml with chemokines]).To examine whether sensitivity to neutralization was affected by the coreceptor pathway utilized in infection of PBLs, we used inhibitory concentrations of CCR5-specific chemokine ligands RANTES, MIP-1α, and MIP-1β in order to restrict infection to the CXCR4 pathway. Addition of these chemokines to the PBL cultures did not affect virus growth, nor did it affect sensitivity to neutralization by the V3-directed human MAbs (Fig. ​(Fig.2).2). To the extent that CCR5 blockade was complete, these results suggest that the simple availability of the CCR5 pathway is not a factor in the resistance of PI viruses to neutralization.To strengthen this conclusion, we examined neutralization sensitivity in human U87-CD4 cell lines expressing only CXCR4 or CCR5. Using this method, we confirmed that the SI 168P virus remained refractory to neutralization by human MAbs 257-D and 268-D as well as by the murine MAb 50.1, regardless of whether infection occurred via CXCR4 or CCR5 (Fig. ​(Fig.3).3). These results suggest that availability of the CCR5 pathway is not a primary determinant for the resistance of PI viruses to neutralization. The TCLA 168C virus utilized CXCR4 only and was sensitive to neutralization. Open in a separate windowFIG. 3Neutralization sensitivity of 168 viruses in U87-CD4 cell lines expressing CCR5 or CXCR4 coreceptor. 168P (○, •) and 168C (▪) viruses were used to infect U87-CD4 cell lines expressing CXCR4 (•, ▪) or CCR5 (○) as described for Fig. ​Fig.1.1. The V3-directed MAbs were incubated with virus for 1 h prior to infection.

Molecularly cloned PI and TCLA envelope genes.

To understand better the changes that accompany adaptation and those that determine coreceptor utilization and neutralization sensitivity, we molecularly cloned the envelope genes of the 168P and 168C viruses. High-fidelity XL PCR (rTth and Vent DNA polymerases; PE Applied Biosystems) and primers envA and envN (15) were used to amplify a 3.1-kb region of proviral DNA encoding the rev and envelope genes. PCR products were isolated by unidirectional T/A cloning in the eucaryotic expression vector pCR3.1-Uni (Invitrogen). Expression in pCR3.1-Uni is driven by the cytomegalovirus immediate-early promoter. Multiple clones were isolated from each virus, and transient transfection studies in COS-7 cells confirmed the surface expression and fusion competence of all clones tested (data not presented).DNA sequence analysis demonstrated that all 168C molecular clones analyzed encoded the three adaptation-associated amino acid changes previously identified by PCR sequencing of the 168C virus population (V2, I166R; C2, I282N; and V3, G318R) (37). Two molecular clones of each 168P and 168C envelope were subjected to complete DNA sequence analysis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF035532","term_id":"2827997","term_text":"AF035532"}}AF035532 to {"type":"entrez-nucleotide","attrs":{"text":"AF035534","term_id":"2828001","term_text":"AF035534"}}AF035534). Molecular clones 168C23 and 168C60 were identical throughout the envelope gene. Molecular clones 168P5 and 168P23 differed from each other and from the previously determined sequence at four to five positions distinct from those associated with adaptation. These scattered changes within the primary virus quasispecies are considered inconsequential at the present level of analysis; the significance of the three adaptation-associated changes is under separate investigation.Functional analysis of these molecularly cloned envelope genes was performed by incorporation of the molecularly cloned envelope protein into pseudotyped HIV virions. We used an envelope-defective provirus derived from the molecularly cloned NL4-3 provirus (kindly provided by I. S. Y. Chen, University of California, Los Angeles). The pNLthyΔBgl provirus (28) contains a BglII-BglII deletion within the envelope gene and a substitution of the viral nef gene with a cDNA encoding the murine Thy1.2 cell surface protein. The simian virus 40 ori was subsequently introduced into the plasmid to generate pSVNLthyΔBgl (27a). Cotransfection of COS-7 cells (16, 20) with pSVNLthyΔBgl provirus and the envelope expression plasmid resulted in the production of pseudotyped HIV virions. Culture supernatants were harvested 3 days posttransfection, filtered, and used to infect U87-CD4 cell lines expressing coreceptor. Cells infected by virions bearing the complementing envelope protein were identified by immunostaining for murine Thy1.2 or HIV proteins.As anticipated, the molecularly cloned envelope proteins recapitulated the coreceptor specificity of the parental virus population (see the legend to Fig. ​Fig.4).4). Pseudotyped virions containing 168C60 were able to infect only U87-CD4 cells expressing CXCR4, while virions containing 168P23 envelope were able to infect U87-CD4 cells expressing either CCR5 or CXCR4. Thus, the viral envelope protein appears to be the major, if not sole, determinant of viral coreceptor use. These findings also indicate that dual coreceptor use is a direct property of the envelope protein complex and not a result of a mixture of distinct envelope proteins in the SI virus population. This conclusion is corroborated by the failure of CCR5-specific chemokine ligands to diminish 168P virus infection in PBL culture (Fig. ​(Fig.22).Open in a separate windowFIG. 4Neutralization sensitivity of pseudotyped virions in U87-CD4 cell lines expressing CCR5 or CXCR4 coreceptor. Pseudotyped virions were derived by cotransfection of COS-7 cells with pSVNLthyΔBgl provirus and plasmid expressing 168P23 (○, •) or 168C60 (▪) envelope protein. Virion preparations were incubated with U87-CD4 cell lines expressing CXCR4 (•, ▪) or CCR5 (○) as described for Fig. ​Fig.1;1; V3-directed MAbs were added as indicated. The number of foci was normalized to control values (60 to 100 foci/well for U87-CD4-CXCR4 cells; 10 foci/well for U87-CD4-CCR5 cells). ∗, no foci were observed.Finally, we wished to determine the neutralization sensitivity of pseudotyped virions containing the molecularly cloned 168P23 and 168C60 envelope proteins and to confirm that coreceptor pathway is not a primary determinant of neutralization sensitivity. We found that infection of U87-CD4-CXCR4 cells by pseudotyped virions containing 168C60 envelope protein was sensitive to neutralization by MAbs 257-D, 268-D, and 50.1 at concentrations comparable to those determined in assays using 168C virus (Fig. ​(Fig.4).4). Pseudotyped virions containing 168P23 envelope protein remained refractory to neutralization by all three V3-directed MAbs, regardless of the coreceptor expressed by the U87-CD4 cell line. In summary, we examined the relationship between coreceptor utilization and sensitivity to neutralization by V3-directed MAbs. The observed dichotomy in the sensitivity to neutralization of PI and TCLA viruses had suggested a discrete difference between these viruses, and we tested one hypothesis: that PI viruses are refractory to neutralization as a result of their unique ability to utilize the CCR5 coreceptor. We examined neutralization sensitivity of a well-characterized SI primary isolate under experimental conditions wherein the virus was forced to utilize either CCR5 or CXCR4 for infection. We showed that coreceptor pathway is not a direct determinant of neutralization sensitivity. The primary virus envelope protein remained refractory to neutralization by V3-directed MAbs regardless of the coreceptor pathway utilized. Similarly, coreceptor utilization did not affect neutralization sensitivity by soluble CD4 (34) or HIVIG (data not presented).In discarding the otherwise attractive hypothesis that PI viruses escape neutralization through their unique ability to utilize CCR5, we are left to consider the as yet undefined structural differences between the envelope protein complex of PI and TCLA viruses. Several studies have suggested that critical determinants in the envelope protein of PI viruses are less accessible than those of TCLA viruses and that it is this differential access that determines neutralization sensitivity (reviewed in reference 25). By contrast, our studies have indicated similar binding of V3-directed MAbs to PBLs infected with neutralization-resistant isolate 168P or neutralization-sensitive isolate 168C (37). Thus, the basis for the differential neutralization sensitivity of PI and TCLA viruses remains unresolved.Our present studies also do not address whether changes in coreceptor utilization and/or neutralization sensitivity are necessarily linked as a consequence of adaptation. The analysis of independently derived PI and TCLA viruses may allow further separation of these viral phenotypes. Subsequent dissection of the amino acid changes that distinguish pedigreed PI and TCLA envelope proteins will help to define the structural bases underlying the changes that accompany adaptation.     

Common Themes of Antibody Maturation to Simian Immunodeficiency Virus, Simian-Human Immunodeficiency Virus, and Human Immunodeficiency Virus Type 1 Infections

Characterization of virus-specific immune responses to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) is important to understanding the early virus-host interactions that may determine the course of virus infection and disease. Using a comprehensive panel of serological assays, we have previously demonstrated a complex and lengthy maturation of virus-specific antibody responses elicited by attenuated strains of SIV that was closely associated with the development of protective immunity. In the present study, we expand these analyses to address several questions regarding the nature of the virus-specific antibody responses to pathogenic SIV, SIV/HIV-1 (SHIV), and HIV-1 infections. The results demonstrate for the first time a common theme of antibody maturation to SIV, SHIV, and HIV-1 infections that is characterized by ongoing changes in antibody titer, conformational dependence, and antibody avidity during the first 6 to 10 months following virus infection. We demonstrate that this gradual evolution of virus-specific antibody responses is independent of the levels of virus replication and the pathogenicity of the infection viral strain. While the serological assays used in these studies were useful in discriminating between protective and nonprotective antibody responses during evaluation of vaccine efficacy with attenuated SIV, these same assays do not distinguish the clinical outcome of infection in pathogenic SIV, SHIV, or HIV-1 infections. These results likely reflect differences in the immune mechanisms involved in mediating protection from virus challenge compared to those that control an established viral infection, and they suggest that additional characteristics of both humoral and cellular responses evolve during this early immune maturation.     

Expression of CCR5 Increases during Monocyte Differentiation and Directly Mediates Macrophage Susceptibility to Infection by Human Immunodeficiency Virus Type 1

The stage of differentiation and the lineage of CD4⁺ cells profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). While CD4⁺ T lymphocytes in patients are readily susceptible to HIV-1 infection, peripheral blood monocytes are relatively resistant during acute or early infection, even though monocytes also express CD4 and viral strains with macrophage (M)-tropic phenotypes predominate. CCR5, the main coreceptor for M-tropic viruses, clearly contributes to the ability of CD4⁺ T cells to be infected. To determine whether low levels of CCR5 expression account for the block in infection of monocytes, we examined primary monocyte lineage cells during differentiation. Culturing of blood monocytes for 5 days led to an increase in the mean number of CCR5-positive cells from <20% of monocytes to >80% of monocyte-derived macrophages (MDM). Levels of CCR5 expression per monocyte were generally lower than those on MDM, perhaps below a minimum threshold level necessary for efficient infection. Productive infection may be restricted to the small subset of monocytes that express relatively high levels of CCR5. Steady-state CCR5 mRNA levels also increased four- to fivefold during MDM differentiation. Infection of MDM by M-tropic HIV-1_JRFL resulted in >10-fold-higher levels of p24, and MDM harbored >30-fold more HIV-1 DNA copies than monocytes. In the presence of the CCR5-specific monoclonal antibody (MAb) 2D7, virus production and cellular levels of HIV-1 DNA were decreased by >80% in MDM, indicating a block in viral entry. There was a direct association between levels of CCR5 and differentiation of monocytes to macrophages. Levels of CCR5 were related to monocyte resistance and macrophage susceptibility to infection because infection by the M-tropic strain HIV-1_JRFL could be blocked by MAb 2D7. These results provide direct evidence that CCR5 functions as a coreceptor for HIV-1 infection of primary macrophages.     

Apoptosis Induction by the Binding of the Carboxyl Terminus of Human Immunodeficiency Virus Type 1 gp160 to Calmodulin

The role of calmodulin (CaM) in apoptosis induced by gp160 of human immunodeficiency virus type 1 was investigated with cells undergoing single-cell killing. These cells were found to express, under the control of an inducible promoter, wild-type gp160 or mutant gp160 devoid of various lengths of the carboxyl terminus. Immunoprecipitation accompanied by immunoblotting revealed binding of CaM to wild-type gp160 but not to mutant gp160 bearing a carboxyl terminus with a deletion spanning more than five amino acid residues. A significant coenzyme activity was detected in the CaM bound to gp160 even in the presence of a Ca²⁺ chelater, EGTA. The cells forming this gp160-CaM complex exhibited an elevated intracellular Ca²⁺ level followed by DNA fragmentation, which is a hallmark of apoptosis, and finally cell killing, while the cells not forming this complex did not show any significant elevation in Ca²⁺ level or DNA fragmentation. These results thus indicated that CaM plays a key role in gp160-induced apoptosis.