Functional epitopes on porcine endogenous retrovirus envelope protein interacting with neutralizing antibody combining sites

Porcine cell and organ transplantation provides promise for maintaining normal physiological conditions in patients with end-stage organ failure. The approach however poses serious risk of transmitting pig pathogens to humans. Among many potential pathogens, porcine endogenous retroviruses (PERV) are of particular concern due to their ubiquitous nature in pigs and capability of infecting human cells. Major antigenic determinants and receptor binding domains on PERV remain unclear until now. Two monoclonal antibodies (mAb), named 8E10 and 7C4 capable of neutralizing PERV infection in HEK293 cells are isolated at an IC(50) of 3.0 and 2.7 microg/ml, respectively, in this work. Epitope location for mAb 8E10 was mapped to amino acids 427-434, residing at the C-terminal region of the gp70 component of type A PERV Env protein. The mAb 8E10 bound directly to the PERV indicating that the epitope is exposed on the virion surface. The mAb 7C4 epitope was assigned to the region comprising amino acids 517-537 on the p15E component of PERV. In contrast to mAb 8E10, the 7C4 mAb bound native PERV inefficiently suggesting that its epitope is accessible only after the virus interacts with its receptor. Finally, both mAbs variable regions were cloned and nucleotide sequence determined. All together, these results reveal that both mAbs 8E10 and 7C4 effectively neutralize PERV infection and may be used as a mean to prevent PERV infection in patients receiving xenotransplantation.     

Distribution of human endogenous retrovirus type W receptor in normal human villous placenta

BACKGROUND: The fusion of trophoblast cells into the villous syncytiotrophoblast is crucial for appropriate placental function and fetal development. Fusion occurs following the interaction of syncytin-1, an envelope protein of the endogenous retrovirus HERV-W, and the RD114/mammalian type D retrovirus receptor (RDR/ASCT2) on adjacent cell membranes. This process must be tightly regulated in order to maintain the proliferative pool of cytotrophoblast cells as well as the function of the syncytia. AIM: We sought to investigate whether syncytial fusion of placental cytotrophoblast cells may be regulated via modulation of RDR/ASCT2 expression. METHODS: Expression of RDR/ASCT2 in term and first trimester villous placenta was assessed along with a number of molecular markers using immunofluorescent staining. In a complementary approach, Western blotting was used to investigate RDR/ASCT2 expression in a panel of choriocarcinoma cell lines before and after stimulation of fusion. RESULTS: Villous placental RDR/ASCT2 expression was found to be restricted to the cytotrophoblast compartment, being largely absent in the syncytiotrophoblast. Local variations in RDR/ASCT2 expression were not associated with the proliferative status of cytotrophoblast cells. RDR/ASCT2 expression was also shown to be down-regulated in BeWo choriocarcinoma cells after stimulation of syncytial fusion. CONCLUSION: This first report of the localisation and distribution of RDR/ASCT2 in human placental villi suggests that the fusion of placental trophoblast cells is not regulated by local or temporal variations of RDR/ASCT2 expression in villous cytotrophoblast cells.     

HERV-K Gag RNA and Protein Levels Are Elevated in Malignant Regions of the Prostate in Males with Prostate Cancer

Heightened expression of human endogenous retrovirus (HERV) sequences has been associated with a range of malignancies, including prostate cancer, suggesting that they may serve as useful diagnostic or prognostic cancer biomarkers. We analysed the expression of HERV-K (Gag and Env/Np9 regions), HERV-E 4.1 (Pol and Env regions), HERV-H (Pol) and HERV-W (Gag) sequences in prostate cancer cells lines and normal prostate epithelial cells using qRT-PCR. HERV expression was also analysed in matched malignant and benign prostate tissue samples from men with prostate cancer (n = 27, median age 65.2 years (range 47–70)) and compared to prostate cancer-free male controls (n = 11). Prostate cancer epithelial cell lines exhibited a signature of HERV RNA overexpression, with all HERVs analysed, except HERV-E Pol, showing heightened expression in at least two, but more commonly all, cell lines analysed. Analysis of primary prostate material indicated increased expression of HERV-E Pol but decreased expression of HERV-E Env in both malignant and benign regions of the prostate in men with prostate cancer as compared to those without. Expression of HERV-K Gag was significantly higher in malignant regions of the prostate in men with prostate cancer as compared to matched benign regions and prostate cancer-free men (p < 0.001 for both), with 85.2% of prostate cancers donors showing malignancy-associated upregulation of HERV-K Gag RNA. HERV-K Gag protein was detected in 12/18 (66.7%) malignant tissues using immunohistochemistry, but only 1/18 (5.6%) benign tissue sections. Heightened expression of HERV-K Gag RNA and protein appears to be a sensitive and specific biomarker of prostate malignancy in this cohort of men with prostate carcinoma, supporting its potential utility as a non-invasive, adjunct clinical biomarker.     

Retroviral transduction of acute myeloid leukaemia-derived dendritic cells with OX40 ligand augments their antigen presenting activity

Recent studies have shown that human myeloid leukaemia cells can differentiate into dendritic cell (DC)-like cells (leukaemia-DCs) when cultured with a combination of cytokines. In the present study, we examined whether the transduction of leukaemia-DCs with OX40 ligand (OX40L), a member of the tumour necrosis factor (TNF) family, resulted in augmentation of their antigen presenting activity. Bicistronic retroviral vectors expressing both human OX40L and enhanced green fluorescent protein (EGFP) or EGFP alone were generated and used for transduction. Fresh leukaemic cells from five patients with acute myeloid leukaemia (AML) were isolated and retrovirally transduced with OX40L during the culture with a combination of cytokines from stem cell factor, fms-like tyrosine kinase (Flt)-3 ligand, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and TNF-alpha. After 7 d, the majority of cells showed DC-like morphology, and expressed higher levels of CD80, CD86 and HLA-DR than fresh leukaemic cells. The transduction efficiency was 8.5-27.2%. Leukaemia-DCs transduced with OX40L elicited higher proliferative response of allogeneic CD4+ T cells than fresh leukaemic cells, non-transduced, or mock-transduced leukaemia-DCs. Co-culture of allogeneic CD4+ T cells with OX40L-transduced leukaemia-DCs was superior in the generation of interferon (IFN)-gamma producing CD4+ T cells and in production of IFN-gamma. Furthermore, OX40L-transduced leukaemia-DCs could elicit significant proliferative response of human leucocyte antigen-matched T cells from the donor in allogeneic stem cell transplantation. These results indicate that retroviral transduction of leukaemia-DCs with OX40L augments their antigen presenting cell activity and thus renders them more suitable for tumour vaccines or ex vivo stimulation of leukaemia-specific T cells.     

Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.The assembly and budding of retroviruses involve a series of regulated steps, driven primarily by the viral Gag protein (reviewed in refs. 1 and 2). In the case of HIV-1, assembly and budding occur predominantly at the plasma membrane. The HIV-1 Gag protein is expressed as a 55-kDa polyprotein, comprising four major domains and two spacer peptides (SPs). The major domains are matrix (MA), capsid (CA), nucleocapsid (NC), and p6; the spacer peptides are known as SP1 and SP2 and are located between CA-NC and NC-p6, respectively. Assembly and budding from the host cell are driven by the full-length Gag protein; concomitant with, or shortly after budding, viral particles undergo maturation, wherein the viral protease (PR) cleaves Gag in an ordered cascade to release the mature proteins (3).In addition to Gag, the other major structural component of retroviral particles is the envelope glycoprotein (Env). HIV-1 Env is synthesized as a 160-kDa precursor that traffics to the plasma membrane via the Golgi apparatus, where it is processed to form the surface glycoprotein gp120 and the transmembrane glycoprotein gp41 (reviewed in ref. 4). The processed Env glycoproteins remain noncovalently associated as a heterodimer; the Env spike is a homotrimer of these dimers (5, 6). On the surface of the viral particle, gp120 binds the viral receptor CD4 and the chemokine coreceptors CXCR4 or CCR5. Binding of gp120 to receptor and coreceptor triggers structural changes in gp41 that lead to fusion of the viral and target cell membranes. The fusion activity of gp41 is conferred by the ecto- and transmembrane domains of the protein (7). A third domain, the cytoplasmic tail (CT), is dispensable for fusion but plays important roles in Env trafficking and cell signaling. Like most lentiviruses, HIV-1 encodes an Env bearing a very long CT composed of ∼150 amino acids. In contrast, most other retroviruses encode Env CTs that are ∼25–35 amino acids in length (4). The reasons for the greater length of lentivirus Env CTs are not fully understood. For HIV-1, the CT is required for Env incorporation into particles in physiologically relevant cell types, such as peripheral blood mononuclear cells, monocyte-derived macrophages, and most T-cell lines (8, 9). Recent work has shown that the CT mediates an interaction with Rab11 family-interacting protein 1c (FIP1c), which in turn interacts with Rab14 and appears to direct trafficking of HIV-1 Env to sites of assembly; it is likely that interactions with FIP1c contribute to the observed requirement for the CT in Env incorporation (10, 11). It is, however, unclear why lentiviral CTs are so large, because far smaller Env CTs also contain essential functional trafficking and signaling motifs (12).A consequence of the large lentiviral CT is the potential for, or inevitability of, interactions with the MA domain of Gag during particle assembly. The Gag protein forms a hexameric lattice, driven primarily by CA–CA interactions. Whereas the structure of CA in mature particles has been studied extensively, with many high-resolution structures now available (13–16), and recently progress has been made in determining the structure of the immature Gag lattice (17), the organization of MA in particles has proven difficult to address directly, with no long-range order discernable for the MA shell. The structure of HIV-1 MA has been solved in vitro, using both NMR and crystallography approaches (18, 19). The structure of the monomer is very similar using either approach; however, crystallography suggests a trimeric arrangement for both HIV-1 and simian immunodeficiency virus (SIV) MA proteins (19, 20), whereas NMR reveals only structures for the monomer, with no evidence for higher-order interactions (18). A third approach visualized 2D lattices of MA or MA-CA, using myristylated proteins on a synthetic lipid bilayer with a composition intended to mimic that of the plasma membrane at sites of assembly (21). Under these conditions, MA was seen to arrange as hexamers of trimers, although the low resolution of this approach precluded more-detailed structural analysis. A hexamer-of-trimers arrangement for MA would be compatible with the most recently suggested model for the immature CA lattice, which proposes a hexamer-of-trimers arrangement for the CA amino-terminal domain (CA-NTD), which lies immediately below MA (17).The available structures of MA can be used to place the data acquired through molecular and genetic approaches into context. Mutations have been identified in MA that prevent the incorporation of Env into particles (22–26). The majority of these mutations map to the tips of the MA trimer (27), a region of the protein that lies around the central aperture of the hexamer of trimers. These findings implicate this central aperture as the site of Env incorporation in the particle. This idea is further supported by the observation that, in cell lines permissive for packaging of CT-truncated Env, removal of the CT relieves the inhibition of Env incorporation imposed by the MA mutations (22, 25, 28). The ability to rescue Env incorporation by removing the CT suggests that steric hindrance of Env incorporation may be a key mechanism for the loss of Env incorporation imposed by mutations in MA. This view is consistent with our recent data showing that a mutation at the trimer interface was able to rescue the Env incorporation defects imposed by several MA mutations and a deletion in the Env CT (24). These data suggest that the MA trimer interface regulates the ability to rescue mutants that are defective for Env incorporation.In this study, we sought to develop a system that would allow us to directly determine whether MA forms trimers in virions and, if so, whether MA trimerization plays a role in Env incorporation. By using a combination of biochemical, genetic, and virological approaches, we demonstrate the presence of MA trimers in replication-competent HIV-1 particles, and show a strong correlation between loss of MA trimerization and impaired Env incorporation. These MA trimerization-defective mutants could be rescued by removal of the long Env CT, demonstrating that the requirement for MA trimerization in Env incorporation is linked to the presence of the long gp41 CT. This report both demonstrates the existence of MA trimers in infectious HIV-1 particles and establishes the importance of this structure for Env incorporation.     

Mash1 efficiently reprograms rat astrocytes into neurons简

To date, it remains poorly understood whether astrocytes can be easily reprogrammed into neurons. Mashl and Brn2 have been previously shown to cooperate to reprogram fibroblasts into neurons. In this study, we examined astrocytes from 2-month-old Sprague-Dawley rats, and found that Brn2 was expressed, but Mashl was not detectable. Thus, we hypothesized that Mashl alone could be used to reprogram astrocytes into neurons. We transfected a recombinant MSCV-MASH1 plasmid into astrocytes for 72 hours, and saw that all cells expressed Mashl. One week later, we observed the changes in morphology of astrocytes, which showed typical neuro- nal characteristics. Moreover, β-tubulin expression levels were significantly higher in astrocytes expressing Mashl than in control cells. These results indicate that Mashl alone can reprogram astrocytes into neurons.     

Inhibition of porcine endogenous retrovirus in PK15 cell line by efficient multitargeting RNA interference

To effectively suppress porcine endogenous retroviruses (PERV)s, RNAi technique was utilized. RNAi is the up‐to‐date skill for gene knockdown which simultaneously multitargets both gag and pol genes critical for replication of PERVs. Previously, two of the most effective siRNAs (gag2, pol2) were found to reduce the expression of PERVs. Concurrent treatment of these two siRNAs (gag2+pol2) showed knockdown efficiency of up to 88% compared to negative control. However, despite the high initial knockdown efficiency 48 h after transfection caused by siRNA, it may only be a transient effect of suppressing PERVs. The multitargeting vector was designed, containing both gag and pol genes and making use of POL II miR Expression Vector, which allowed for persistent and multiple targeting. This is the latest shRNA system technique expressing and targeting like miRNA. Through antibiotics resistance characteristics utilizing this vector, miRNA‐transfected PK15 cells (gag2‐pol2) were selected during 10 days. An 88.1% reduction in the level of mRNA expression was found. In addition, we performed RT‐activity analysis and fluorescence in situ hybridization assay, and it demonstrated the highest knockdown efficiency in multitargeting (gag2+pol2) miRNA group. Therefore, according to the results above, gene knockdown system (siRNA and shRNA) through multitargeting strategy could effectively inhibit PERVs.     

Pathology and Pathogenesis of Ovine Pulmonary Adenocarcinoma

Ovine pulmonary adenocarcinoma (OPA), also known as jaagsiekte, is a transmissible lung tumour of sheep caused by jaagsiekte sheep retrovirus (JSRV). JSRV induces neoplastic transformation of alveolar and bronchiolar secretory epithelial cells and the resulting tumours can grow to occupy a significant portion of the lung. Tumour growth is frequently accompanied by the overproduction of fluid in the lung, which further compromises normal respiration. The period between infection and the appearance of clinical signs may be several months or years and many JSRV-infected sheep do not exhibit clinical signs at all during their lifespan. This allows the spread of OPA into new flocks through contact with infected but apparently normal animals. OPA was first described in the early 19th century; however, it has still not been possible to devise effective methods for controlling its spread and it remains an important problem in most countries where sheep are farmed. This is due in part to the absence of an immunological response to JSRV in infected animals, which has hindered the development of serological diagnostic tests and vaccines. In addition to its veterinary importance, OPA is regarded as a potential large animal model for human lung adenocarcinoma and this has stimulated research into the pathogenesis of the ovine disease. This work has produced some significant results, including the finding that one of the JSRV structural proteins is directly involved in oncogenesis. The recent advances in understanding JSRV and the pathogenesis of OPA should lead to novel strategies for diagnosis and control of this disease and for its exploitation as a comparative model for human lung cancer.     

Structure and assembly of immature HIV

The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to ≈17-Å resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein–protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.     

人硫氧还蛋白基因克隆及其重组逆转录病毒载体的构建和鉴定

目的 克隆人硫氧还蛋白（hTRX）基因,并构建含有该目的 基因的重组逆转录病毒载体。方法 利用RT-PCR法,以PHA活化的人外周血单个核细胞总RNA为模板,扩增hTRX编码蛋白的cDNA基因,并亚克隆至逆转录病毒载体pSIV-1中进行PCR、双酶切和测序鉴定。结果 将所得的序列与GenBank（BC003377）报道的序列比较,其酶活性中心（Trp-Cys-Gly-Pro-Cys）与已知序列一致,第132、136、170、264位碱基与已知序列不同,其中第136、170位相应密码子编码的氨基酸发生了变化（Phe→Leu,Ile→Thr）,成功获得了pSIV-1-hTRX重组逆转录病毒载体。结论 hTRX基因的克隆及其重组逆转录病毒载体pSIV-1-hTRX的构建,为进一步探讨hTRX的生物学活性和应用奠定了基础。