The C-terminal domain of glyceraldehyde 3-phosphate dehydrogenase plays an important role in suppression of tRNALys3 packaging into human immunodeficiency virus type-1 particles.

Human immunodeficiency virus type-1 (HIV-1) requires the packaging of human tRNALys3 as a primer for effective viral reverse transcription. Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) suppresses the packaging efficiency of tRNALys3. Although the binding of GAPDH to Pr55 gag is important for the suppression mechanism, it remains unclear which domain of GAPDH is responsible for the interaction with Pr55 gag . In this study, we show that Asp256, Lys260, Lys263 and Glu267 of GAPDH are important for the suppression of tRNALys3 packaging. Yeast two-hybrid analysis demonstrated that the C-terminal domain of GAPDH (151-335) interacts with both the matrix region (MA; 1-132) and capsid N-terminal domain (CA-NTD; 133-282). The D256R, K263E or E267R mutation of GAPDH led to the loss of the ability to bind to wild-type (WT) MA, and the D256R/K260E double mutation of GAPDH resulted in the loss of detectable binding activity to WT CA-NTD. In contrast, R58E, Q59A or Q63A of MA, and E76R or R82E of CA-NTD abrogated the interaction with the C-terminal domain of GAPDH. Multiple-substituted GAPDH mutant (D256R/K260E/K263E/E267R) retained the oligomeric formation with WT GAPDH in HIV-1 producing cells, but the incorporation level of the hetero-oligomer was decreased in viral particles. Furthermore, the viruses produced from cells expressing the D256R/K260E/K263E/E267R mutant restored tRNALys3 packaging efficiency because the mutant exerted a dominant negative effect by preventing WT GAPDH from binding to MA and CA-NTD and improved the reverse transcription. These findings indicate that the amino acids Asp256, Lys260, Lys263 and Glu267 of GAPDH is essential for the mechanism of tRNALys3-packaging suppression and the D256R/K260E/K263E/E267R mutant of GAPDH acts in a dominant negative manner to suppress tRNALys3 packaging.

ATP generation in a host cell in early-phase infection is increased by upregulation of cytochrome c oxidase activity via the p2 peptide from human immunodeficiency virus type 1 Gag.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) must take advantage of its own proteins with two or more functions to successfully replicate. Although many attempts have been made to determine the function of viral proteins encoded in the HIV-1 genome, the role of the p2 peptide, a spacer between the capsid and the nucleocapsid in HIV-1 Gag in early-phase HIV infection still remains unclarified. RESULTS: In this study, we show that the p2 peptide enhances HIV-1 acute infection by increasing intracellular ATP production via the activation of mitochondrial cytochrome c oxidase (MT-CO) involved in the respiratory chain. We found that cell-permeable p2-peptide-treated cells were more effectively infected by HIV-1 than control cells. To characterize the effect of the p2 peptide on HIV-1 replication in MAGIC-5 cells, various HIV-1 cDNA products were measured by quantitative real-time PCR. The levels of the late (R/gag), 2-LTR circular (2-LTR), and integrated (Alu) forms of viral cDNAs increased in the presence of the p2 peptide. Interestingly, yeast two-hybrid analysis revealed a novel interaction between the p2 peptide and the mitochondrial intermembrane space domain (N(214)-F(235)) of MT-CO subunit I (MT-CO1). Mutational analysis indicated that Gln(6) in the p2 peptide is important for the interaction with MT-CO1. The p2 peptide activated MT-CO1 in vitro in a concentration-dependent manner, and fluorescence-microscopy analysis demonstrated that the p2 peptide had a significant effect on mitochondrial targeting. Furthermore, the analysis of HIV-1 lacking a functional p2 peptide demonstrated the inhibition of intracellular ATP production in MT-4 cells and monocyte-derived macrophages (MDMs) and a decrease in reverse transcription efficiency following infection of MT-4 cells and MDMs. CONCLUSIONS: These findings provide evidence that the p2 peptide is a viral positive allosteric modulator of MT-CO and the increased intracellular ATP production after HIV infection in a p2-peptide-dependent manner is essential for efficient reverse transcription in early-phase HIV-1 infection.

N-Myristoyltransferase 1 enhances human immunodeficiency virus replication through regulation of viral RNA expression level.

N-myristoyltransferase (NMT) catalyzes protein N-myristoylation. It has been suggested that the isozyme NMT1 enhances the replication of human immunodeficiency virus type-1 (HIV-1). However, the details of the mechanism by which NMT1 does so remain unclear. In this study, we investigated NMT1-binding proteins by co-immunoprecipitation and mass spectrometry. As a result, several RNA-binding proteins including ribosomal proteins, NMT isozymes, and hnRNP A2/B1 were observed to bind to NMT1, as mediated mainly by RNA. Interestingly, only hRNP A2/B1 was found to associate with NMT1 without mediation by RNA. It was also suggested that hnRNP A2/B1 contributes to the formation of complexes of high molecular weights involving NMT1. Knockdown of hnRNP A2/B1 resulted in the enhancement of viral replication with an increase in the expression level of viral RNA in HIV-1-producing cells. On the other hand, knockdown of NMT1 resulted in the attenuation of viral replication with the decrease in the expression level of viral RNA in HIV-1-producing cells. Additionally, overexpression of NMT1 induced the enhancement of viral replication with the increase in the expression level of the viral RNA. These findings suggest that both NMT1 and hnRNP A2/B1 take part in the regulation of HIV-1 RNA expression through their mutual opposite effects on the viral RNA expression in HIV-1-producing cells.

Clearly different mechanisms of enhancement of short-lived Nef-mediated viral infectivity between SIV and HIV-1.

BACKGROUND: One of the major functions of Nef is in the enhancement of the infectivity of the human and simian immunodeficiency viruses (HIV and SIV, respectively). However, the detailed mechanism of the enhancement of viral infectivity by Nef remains unclear. Additionally, studies of mechanisms by which Nef enhances the infectivity of SIV are not as intensive as those of HIV-1. METHODS: We generated short-lived Nef constructed by fusing Nef to a proteasome-mediated protein degradation sequence to characterize the Nef role in viral infectivity. RESULTS: The apparent expression level of the short-lived Nef was found to be extremely lower than that of the wild-type Nef. Moreover, the expression level of the short-lived Nef increased with the treatment with a proteasome inhibitor. The infectivity of HIV-1 with the short-lived Nef was significantly lower than that with the wild-type Nef. On the other hand, the short-lived Nef enhanced the infectivity of SIVmac239, an ability observed to be interestingly equivalent to that of the wild-type Nef. The short-lived Nef was not detected in SIVmac239, but the wild-type Nef was, suggesting that the incorporation of Nef into SIVmac239 is not important for the enhancement of SIVmac239 infectivity. CONCLUSIONS: Altogether, the findings suggest that the mechanisms of infectivity enhancement by Nef are different between HIV-1 and SIVmac239. Lastly, we propose the following hypothesis: even when the expression level of a protein is extremely low, the protein may still be sufficiently functional.

Identification of essential cis element in 5'UTR of Nef mRNA for Nef translation.

Nef is one of the accessory proteins of the human immunodeficiency virus type 1 (HIV-1). Nef is translated from multiple-spliced mRNAs transcribed from the viral genome, whose mRNAs have a relatively long 5' untranslated region (5'UTR). Here, we identified a cis element in the 5'UTR of Nef mRNA essential for efficient Nef translation, which was named the Nef-translation essential region (NER). Mutants with a deleted NER in the 5'UTR of the HIV-1 NL4-3 strain showed an almost undetectable Nef expression owing to a low Nef translation efficiency. The NER of the NL4-3 strain was predicted to form putative stem loops. Although the 5'UTR showed significant but relatively low internal ribosome entry site (IRES) activity, the mechanism of 5'cap-dependent translation mainly contributed to the Nef translation from its Nef mRNA. Altogether, it was clarified that not only the 5' cap but also the NER in the 5'UTR is an essential cis element for efficient Nef translation, which is not a typical 5'-cap-dependent mechanism, and that there must be an as yet unknown mechanism using the NER for efficient Nef translation.

Phosphorylation of human immunodeficiency virus type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2.

We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser(16)-Pro(17) motif. However, the specific kinase responsible for Ser(16) phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser(16). The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser(16) is phosphorylated after the initiation of Pr55(Gag) processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser(16) residue in the Ser(16)-Pro(17) motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser(16) phosphorylation level inside virions, and virus partially defective in Ser(16) phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser(16) phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating.

Bovine alpha-2-HS-glycoprotein functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and SIVmac239 envelope glycoprotein.

The presence of anti-CCR5 and anti-HIV-1 envelope glycoprotein (ENV) gp41 antibodies (Abs) at sites of HIV-1 exposure was effective in preventing its transmission to HIV-1-exposed seronegative (ESN) subjects. Here, we design an immunogen that can induce Abs against CCR5 and SIVmac239 ENV simultaneously and show that bovine alpha-2-HS-glycoprotein (bAHSG) functions as a booster antigen for efficiently stimulating humoral immune responses to CCR5 and ENV. Initially, we generated a rhesus CCR5-derived cyclopeptide (cDDR5) conjugated with a recombinant trimeric SIVmac239 Env. When inguinally administered to rhesus macaques, the immunogen simultaneously induced both anti-CCR5 and anti-ENV Abs in sera, and the purified serum IgG fraction exerted an inhibitory effect on SIVmac239 infection in vitro. When further boosted with bAHSG, the responses of both Abs were significantly enhanced. To examine the cross-reactivity of bAHSG, it was administered to naïve cynomolgus macaques. The results showed a statistically significant increase in IgG response against cynomolgus CCR5 and SIVmac239 ENV, and the induction of neutralizing activity against SIVmac239. These findings suggest that bAHSG is useful for immune strategies aimed at generating Abs against CCR5 and ENV simultaneously to confer HIV-protective immunity.

Glyceraldehyde 3-phosphate dehydrogenase negatively regulates human immunodeficiency virus type 1 infection.

BACKGROUND: Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. Although the identification efficiency of host proteins is improved by mass spectrometry, how those host proteins affect HIV-1 replication has not yet been fully clarified. RESULTS: In this study, we show that virion-associated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) does not allosterically inactivate HIV-1 reverse transcriptase (RT) but decreases the efficiency of reverse transcription reactions by decreasing the packaging efficiency of lysyl-tRNA synthetase (LysRS) and tRNA(Lys3) into HIV-1 virions. Two-dimensional (2D) gel electrophoresis demonstrated that some isozymes of GAPDH with different isoelectric points were expressed in HIV-1-producing CEM/LAV-1 cells, and a proportion of GAPDH was selectively incorporated into the virions. Suppression of GAPDH expression by RNA interference in CEM/LAV-1 cells resulted in decreased GAPDH packaging inside the virions, and the GAPDH-packaging-defective virus maintained at least control levels of viral production but increased the infectivity. Quantitative analysis of reverse transcription products indicated that the levels of early cDNA products of the GAPDH-packaging-defective virus were higher than those of the control virus owing to the higher packaging efficiencies of LysRS and tRNA(Lys3) into the virions rather than the GAPDH-dependent negative allosteric modulation for RT. Furthermore, immunoprecipitation assay using an anti-GAPDH antibody showed that GAPDH directly interacted with Pr55(gag) and p160(gag)-pol and the overexpression of LysRS in HIV-1-producing cells resulted in a decrease in the efficiency of GAPDH packaging in HIV particles. In contrast, the viruses produced from cells expressing a high level of GAPDH showed decreased infectivity in TZM-bl cells and reverse transcription efficiency in TZM-bl cells and peripheral blood mononuclear cells (PBMCs). CONCLUSIONS: These findings indicate that GAPDH negatively regulates HIV-1 infection and provide insights into a novel function of GAPDH in the HIV-1 life cycle and a new host defense mechanism against HIV-1 infection.

Induction of extremely low protein expression level by fusion of C-terminal region of Nef.

Nef is one of the accessory proteins of human immunodeficiency viruses. Here, we noted that the relative expression level of Nef(NL4-3) is much lower than that of NefJR-CSF in HEK293 cells. By evaluating the expression level using a Nef mutant, it was indicated that amino acids 129-206 of Nef(NL4-3), that is, the C-terminal region named NLAA129-206, could contain the region responsible for the induction of the low protein expression level. In addition, the expression levels of the enhanced green fluorescent protein and Renilla luciferase became extremely low with the fusion of NLAA129-206. Interestingly, the NLAA129-206-corresponding sequences of other Nef variants with relatively high expression levels also induced the extremely low protein expression level by fusion. These results suggest that the C-terminal region of Nef can generally induce an extremely low protein expression level. Here, we propose that the C-terminal region of Nef could become an excellent tool for the induction of an extremely low expression level of arbitrary proteins by attachment as fusion proteins.

Suppression of human immunodeficiency virus type-1 production by coexpression of catalytic-region-deleted N-myristoyltransferase mutants.

N-Myristoyltransferase (NMT) isozymes, i.e., NMT1 and NMT2, are essential host factors for the AIDS-causing human immunodeficiency virus type-1 (HIV-1), by which the viral proteins Pr55(gag) and Nef are N-myristoylated. N-Myristoylation is important for the membrane targeting of modified proteins. Since it is predicted that approximately 0.5% of all proteins in the human genome are N-myristoylated, selective inhibition of closely HIV-1-associated NMT isozymes is thought to be important for the improvement of specificity in the anti-HIV-1 strategy with the inhibition of NMT function. NMT isozymes contain two characteristic structures, the N-terminal region and the catalytic region. Here, it was shown that the N-terminal region of each NMT isozyme is required for isozyme-specific binding to the ribosome. The specific binding of each isozyme to the ribosome was associated with HIV-1 production, in which NMT1 and NMT2 in the ribosome were suggested to be mainly related to Pr55(gag) and Nef, respectively. These results indicate that the N-terminal region that mediates binding to the ribosome can become a target for NMT-isozyme-specific inhibition, which could block HIV-1 production.

Treatment of breast cancer cells with proteasome inhibitor lactacystin increases the level of sensitivity to cell death induced by human immunodeficiency virus type 1.

Upon binding to CD4, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 undergoes conformational changes that facilitate subsequent interactions with the chemokine coreceptor CXCR4 on the T cells. Our previous study showed that HIV-1 induces breast cancer cell death through gp120-CXCR4 interaction without CD4-induced conformational change of gp120. To characterize the structural properties of CXCR4 on breast cancer cells, the structural differences in CXCR4 between breast cancer cell lines and T cells were investigated. Immunoblots of whole cell lysates from breast cancer cell and T cell lines demonstrated that the predominant forms of CXCR4 on the breast cancer cell lines and T cell lines were three species (45, 61, 100 kDa) and one species (45 kDa), respectively. Cell surface biotin labeling revealed that the 100-kDa polyubiquitinated form of CXCR4 is specifically expressed on the surface of breast cancer cell line DU4475 but not T cell line Molt4#8. The treatment of breast cancer cell lines MDA-MB231 and DU4475 with proteasome inhibitor lactacystin leads to increased surface expression of the 100-kDa polyubiquitinated form of CXCR4 and increases the level of sensitivity to cell death induced by HIV-1. These data suggest that the 100-kDa polyubiquitinated form of CXCR4 plays an important role as a trigger for gp120-induced breast cancer cell death.

Uncoating of human immunodeficiency virus type 1 requires prolyl isomerase Pin1.

The process by which the human immunodeficiency virus type 1 (HIV-1) conical core dissociates is called uncoating, but not much is known about this process. Here, we show that the uncoating process requires the interaction of the capsid (CA) protein with the peptidyl-prolyl isomerase Pin1 that specifically recognizes the phosphorylated serine/threonine residue followed by proline. We found that the HIV-1 core is composed of some isoforms of the CA protein with different isoelectric points, and one isoform is preferentially phosphorylated in the Ser(16)-Pro(17) motif. The mutant virus S16A/P17A shows a severely attenuated HIV-1 replication and an impaired reverse transcription. The S16A/P17A change increased the amount of particulate CA cores in the cytosol of target cells and correlated with the restriction of HIV-1 infection. Glutathione S-transferase pulldown assays demonstrated a direct interaction between Pin1 and the HIV-1 core via the Ser(16)-Pro(17) motif. Suppression of Pin1 expression by RNA interference in a target cell results in an attenuated HIV-1 replication and increases the amount of particulate CA cores in the cytosol of target cells. Furthermore, heat-inactivated, inhibitor-treated, or W34A/K63A Pin1 causes an attenuated in vitro uncoating of the HIV-1 core. The Pin1-dependent uncoating is inhibited by antisera raised against a CA peptide phosphorylated at Ser(16) or treatment of the HIV-1 core with alkaline phosphatase. These findings provide insights into this obscure uncoating process in the HIV-1 life cycle and a new cellular target for HIV-1 drug development.

Targeted delivery of immunogen to primate m cells with tetragalloyl lysine dendrimer.

Effective uptake of Ags by specialized M cells of gut-associated lymphoid tissues is an important step in inducing efficient immune responses after oral vaccination. Although stable nontoxic small molecule mimetics of lectins, such as synthetic multivalent polygalloyl derivatives, may have potential in murine M cell targeting, it remains unclear whether synthetic multivalent polygalloyl derivatives effectively target nonhuman and human M cells. In this study, we evaluated the ability of a tetragalloyl derivative, the tetragalloyl-D-lysine dendrimer (TGDK), to target M cells in both in vivo nonhuman primate and in vitro human M-like cell culture models. TGDK was efficiently transported from the lumen of the intestinal tract into rhesus Peyer's patches by M cells and then accumulated in germinal centers. Oral administration of rhesus CCR5-derived cyclopeptide conjugated with TGDK in rhesus macaque resulted in a statistically significant increase in stool IgA response against rhesus CCR5-derived cyclopeptide and induced a neutralizing activity against SIV infection. Furthermore, TGDK was specifically bound to human M-like cells and efficiently transcytosed from the apical side to the basolateral side in the M-like cell model. Thus, the TGDK-mediated vaccine delivery system represents a potential approach for enabling M cell-targeted mucosal vaccines in primates.

Development of cell-expressed and virion-incorporated CCR5-targeted vaccine.

Our previous study demonstrated that the immunization with a cycloimmunogen derived from extracellular loop-2 (ECL-2) of CCR5 (cDDR5) attenuated acute phase of CCR5-tropic simian-human immunodeficiency virus (SHIV)(SF162P3) replication in vivo. Although the study showed that the antisera raised against cDDR5 reacted with cell-expressed CCR5, we have not yet demonstrated whether the antisera can react with virion-incorporated CCR5. Here, we show that rhesus cDDR5 (rcDDR5)-specific antibodies react with not only cell-expressed but also virion-incorporated simian CCR5s (siCCR5s), but may predominantly exert their inhibitory effects on simian immunodeficiency virus (SIV) infection by the binding of cell-expressed rather than virion-incorporated CCR5s. These results suggest that the virion-incorporated CCR5 may contribute to the reactivation of the anti-rcDDR5 antibody-producing B-cells by SIV particles after rcDDR5 immunization, although the binding of anti-rcDDR5 antibody to virion-incorporated CCR5 results in a partial inhibitory effect on SIV infection.

HIV-1 production is specifically associated with human NMT1 long form in human NMT isozymes.

The N-myristoylation of the N-terminal of human immunodeficiency virus type-1 (HIV-1) Pr55(gag) by human N-myristoyltransferase (hNMT) is a prerequisite modification for HIV-1 production. hNMT consists of multiple isozymes encoded by hNMT1 and hNMT2. The hNMT1 isozyme consists of long, medium, and short forms. Here, we investigated which isozyme is crucial for HIV-1 production. Human embryonic kidney (HEK) 293 cells transfected with infectious HIV-1 vectors were used as models of HIV-1-infected cells in this study. The significant reduction in HIV-1 production and the failure of the specific localization of Pr55(gag) in a detergent-resistant membrane fraction were dependent on the knockdown of the different forms of the hNMT1 isozyme but not of the hNMT2 isozyme. Additionally, the coexpression of an inactive mutant hNMT1 isozyme, namely the hNMT1 long form (hNMT1(L)), but not that of other hNMT mutants resulted in a significant reduction in HIV-1 production. These results strongly suggest that HIV-1 production is specifically associated with hNMT1, particularly hNMT1(L), but not with hNMT2 in vivo, contributing to the understanding of a step in HIV-1 replication.

Human immunodeficiency virus-induced apoptosis of human breast cancer cells via CXCR4 is mediated by the viral envelope protein but does not require CD4.

HIV-1 infection results in an increased risk of malignancy as well as immune suppression. However, analyses of cancer incidence in chronically immunosuppressed transplant recipients and HIV-infected person have demonstrated an unexpected low incidence of certain types of cancer, such as breast cancers, and the mechanism behind this remains unclarified. In this study, we show that most breast cancer cell lines express CXCR4 but are not susceptible to HIV-1 infection. The apoptosis of breast cancer cells is induced by HIV-1 in a viral-dose- and time-dependent manner without productive infection. The apoptosis is induced by R5X4 and X4 HIV-1 but not by R5 HIV-1, and is inhibited by an anti-CXCR4 antibody, an anti-gp120 antibody, AMD3100, or pertussis toxin. The apoptosis is mediated via CXCR4 in breast cancer cells that exhibit conformational heterogeneity in comparison with CXCR4 in T-cells. Furthermore, the gp120 mutant (E370R) with a low CD4 binding ability can specifically induce apoptosis in breast cancer cells but not in T-cells. Taken together, these results indicate that HIV-1 and gp120 can induce breast cancer cell apoptosis through gp120-CXCR4 interaction without a CD4-induced conformational change of gp120, and may lead to a novel HIV-1-based therapy for breast cancer.

Nonhuman primate intestinal villous M-like cells: an effective poliovirus entry site.

Humans and some Old World monkeys, chimpanzees, and cynomolgus macaques, are susceptible to oral poliovirus (PV) infection. Interestingly, rhesus macaques, although sensitive to injected PV, are not susceptible to gut infection. Not much is known about the initial event of gut infection by PV in rhesus macaques so far. Here, we show that PV can efficiently enter the lamina propria (LP) by penetrating across intestinal villous M-like cells in rhesus macaques. We found by immunofluorescence analysis that PV effectively invades LP rather than germinal centers (GCs) in rhesus macaques despite expressing PV receptor CD155 on cells within GCs and LP. Furthermore, energy dispersive X-ray spectroscopy demonstrated that gold-labeled PV is spatiotemporally internalized into villous M-like cells and engulfed by macrophage-like cells in LP. These results suggest that rhesus macaques may be resistant to productive gut PV infection owing to a defective translocation of PV to GCs.

Immunoreactive cycloimmunogen design based on conformational epitopes derived from human immunodeficiency virus type 1 coreceptors: cyclic dodecapeptides mimic undecapeptidyl arches of extracellular loop-2 in chemokine receptor and inhibit human immunodeficiency virus type 1 infection.

Human immunodeficiency virus type 1 (HIV-1) requires a chemokine receptor (CCR5 or CXCR4) as a coreceptor not only for initiate viral entry but also protecting highly conserved neutralization epitopes from the attack of neutralizing antibodies. Over the past decade, many studies have provided new insights into the HIV entry mechanism and have focused on developing an effective vaccine strategy. However, to date, no vaccine that can provide protection from HIV-1 infection has been developed. One reason for the disappointing results has been the inability of current vaccine candidates to elicit a broadly reactive immunity to viral proteins such as the envelope (env) protein. Here, we propose that chemokine receptors are attractive targets of vaccine development because their structures are highly conserved and that our synthetic cycloimmunogens can mimic conformational-specific epitopes of undecapeptidyl arches (UPAs: R(168)-C(178) in CCR5, N(176)-C(186) in CXCR4) and be useful for HIV-1 novel vaccine development.

A transgenic rat with the human ATTR V30M: a novel tool for analyses of ATTR metabolisms.

Amyloidogenic transthyretin (ATTR) is the pathogenic protein of familial amyloidotic polyneuropathy (FAP). To establish a tool for analyses of ATTR metabolisms including after liver transplantations, we developed a transgenic rat model expressing human ATTR V30M and confirmed expressions of human ATTR V30M in various tissues. Mass spectrometry for purified TTR revealed that rat intrinsic TTR and human ATTR V30M formed tetramers. Congo red staining and immunohistochemistry revealed that nonfibrillar deposits of human ATTR V30M, but not amyloid deposits, were detected in the gastrointestinal tracts of the transgenic rats. At 24h after liver transplantation, serum human ATTR V30M levels in transgenic rats that received livers from normal rats became lower than detectable levels. These results thus suggest that this transgenic rat may be a useful animal model which analyzes the metabolism of human ATTR V30M including liver transplantation studies.

Analysis of amyloid fibrils in the cheetah (Acinonyx jubatus).

Recently, a high prevalence of amyloid A (AA) amyloidosis has been documented among captive cheetahs worldwide. Biochemical analysis of amyloid fibrils extracted from the liver of a Japanese captive cheetah unequivocally showed that protein AA was the main fibril constituent. Further characterization of the AA fibril components by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis revealed three main protein AA bands with approximate molecular weights of 8, 10 and 12 kDa. Mass spectrometry analysis of the 12-kDa component observed in SDS-PAGE and Western blotting confirmed the molecular weight of a 12,381-Da peak. Our finding of a 12-kDa protein AA component provides evidence that the cheetah SAA sequence is longer than the previously reported 90 amino acid residues (approximately 10 kDa), and hence SAA is part of the amyloid fibril.