Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements

The Rex proteins of types I and II human T-cell leukemia viruses (HTLV-I, HTLV-II) are required for expression of the viral structural gene products, gag and env and, thus, are essential for the replication of these pathogenic retroviruses. The action of Rex is sequence specific, requiring the presence of a cis-acting Rex response element located in the 3' long terminal repeat. This element corresponds to a predicted RNA secondary structure and functions in an orientation-dependent but position-independent manner. Rex acts through this response element to stimulate the nuclear export of the unspliced or singly spliced viral mRNA species encoding the virion structural proteins that are normally excluded from the cytoplasm. Although the Rex proteins of HTLV-I and HTLV-II can also function via the related Rev response element present in the env gene of the type I human immunodeficiency virus (HIV-1), the analogous HIV-1 Rev protein is unable to act on the HTLV-I Rex response element. This nonreciprocal pattern of genetic complementation by Rex and Rev suggests that these viral trans-regulators may interact directly with their RNA response elements.     

A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev

HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait". DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev- cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics.     

Mutational analysis of PTPRT phosphatase domains in common human cancers

A recent report revealed that the protein-tyrosine phosphatase, receptor-type, T (PTPRT) gene is somatically mutated in several types of human cancer, suggesting that the mutated PTPRT gene is a tumor suppressor gene in human cancers. However, because previously the mutational search has focused primarily on colon cancers, data on PTPRT mutations in other types of human cancer have largely been lacking. Here, we performed a mutational analysis of the PTPRT phosphatase domain by polymerase chain reaction-based single-strand conformation polymorphism (PCR-SSCP) assay in 345 cases of common human cancers, including colon carcinomas, hepatocellular carcinomas, acute leukemias, gastric carcinomas, breast carcinomas and non-small cell lung cancers. We detected PTPRT phosphatase domain mutations in 1 of 105 colon carcinomas (1%) and 1 of 48 gastric carcinomas (2%), but none in acute leukemias, hepatocellular carcinomas, breast carcinomas and non-small cell lung cancers. The PTPRT mutation detected in the colon carcinoma was a missense mutation and the mutation in the gastric carcinomas was a splice-site mutation. Contrary to the previous report on the frequent PTPTR phosphatase domain mutations in colon cancers, this study demonstrated that the somatic mutation of the PTPRT phosphatase domain rarely occurred in common human cancers. The data suggested that alterations of the PTPRT-mediated signaling pathway by PTPRT phosphatase domain mutation may not play a critical role in the development of common human cancers.     

Functional Variability of Rev Response Element in HIV-1 Primary Isolates

We have previously studied sequence heterogeneity of HIV-1 Rev response element (RRE), and showed uneven variations in different stem–loops of both primary sequence and secondary structure. Here we studied the functional variation of RRE clones from a set of 10 primary isolates, and demonstrated a variation in the function of these RRE clones on the expression of Gag proteins from a truncated HIV-1 genome. The difference in Gag level was, in part, if not exclusively, resulted from the differential efficiency of RNA transport and enhancing of translation. These data suggested that variation of HIV-1 RRE may play a role in regulation of viral replication rate in HIV-1 primary isolates.     

Histographic recording of human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev and nuclear factors

Summary.  HeLa cells and HeLa cells expressing the HIV-1 regulatory protein Rev were immunostained for Rev and pre-mRNA processing factors and examined histographically by confocal laser scanning microscopy. Following short pulse-labelling with bromouridine tri-phosphate nascent RNA gave a granular nucleoplasmic staining increasing somewhat towards the periphery as did also the heterogeneous ribonucleoproteins (hnRNPs) A1 and particularly C1/C2, a distribution pattern which has not been described. The sm-antigen of the small ribonucleoprotein particle (snRNP) proteins U1, U2, U4/U6 and U5 stained the nucleoplasm diffusely in addition to speckles which co-localised with speckles of the non-snRNP splicing factor SC-35. Brominated RNA and the hnRNPs A1 and C1/C2 were to varying degrees excluded from the speckles. Rev concentrated in the nucleolus and often as a perinucleolar ring/zone. Rev also stained the nucleoplasm and cytoplasm without co-localising with the above-mentioned proteins or brominated RNA and was not enriched or excluded in SC-35 speckles. The nucleolar proteins B23 and C23, like Rev, gave primarily a perinucleolar ring and stained the nucleoplasm but did not otherwise co-localise with Rev or with nuclear proteins. Histographic recording of immunofluorescence images proved to be a valuable tool in the study of localisation of HIV-1 Rev and cellular components and of possible co-localisations. A parallel comparison of the subcellular patterns of pre-mRNA processing factors versus major nucleolar antigens is new and suggests that the factors are not strictly separated in the nucleoplasm. Accepted September 12, 1997 Received April 28, 1997     

Alterations in HIV-1 Rev transport in response to cell stress

Movement of HIV-1 Rev between the nucleus and cytoplasm is essential to its function. While normally nuclear, the protein can be induced to accumulate in the cytoplasm upon inhibition of RNA polymerase I/II. Nuclear accumulation of Rev in the presence of these inhibitors was found to be rescued upon addition of leptomycin B, an inhibitor of Rev nuclear export. This finding, in conjunction with kinetic data on nuclear import, indicates that the effect of the RNA polymerase inhibitors is due to an inversion of the rates of nuclear import versus export possibly achieved by increasing the rate of Rev nuclear export. We also examined whether changes in Rev localization could be due to a stress response. While neither ultraviolet radiation nor heat shock affected Rev subcellular localization, both oxidative and osmotic shocks induce changes in Rev localization comparable to that observed with the RNA polymerase inhibitors. The ability of certain serine/threonine kinase inhibitors, including CKI/II inhibitors, to cause cytoplasmic accumulation of Rev suggested that the alteration in Rev distribution could be due to changes in Rev or CRM1 phosphorylation. However, no change in extent of phosphorylation of either protein is observed upon treatment of cells with any of the agents tested, indicating involvement of another cellular factor.     

The Alzheimer's disease-associated gamma-secretase complex: functional domains in the presenilin 1 protein

Alzheimer's disease is neuropathologically characterized by the presence of neurofibrillary tangles and amyloid plaques in the brain. Amyloid plaques are extracellular deposits primarily composed of the amyloid beta-peptide, which is derived from the amyloid beta-precursor protein (APP) by sequential cleavages at the beta-secretase and gamma-secretase sites. gamma-Secretase cleavage is performed by a high molecular weight protein complex containing presenilin (PS), nicastrin, Aph-1 and Pen-2. The gamma-secretase complex is an unusual transmembrane aspartyl protease that cleaves APP within the transmembrane domain. In addition to APP, a large number of other single membrane-spanning proteins have been shown to be cleaved within their transmembrane domains by the gamma-secretase complex in a process referred to as regulated intramembrane proteolysis. Here we review recent research leading to the identification and understanding of the gamma-secretase complex components with emphasis on PS, which harbors the catalytic site. In addition, we summarize our own work focused on identifying and studying domains in PS1 that are critical for mediating gamma-secretase activity. Biochemical understanding of the gamma-secretase complex is important from a basic biological and physiological point of view, and could help in the development of small molecules that modulate gamma-secretase processing in an APP-specific manner.     

Mutational patterns in the frameshift-regulating site of HIV-1 selected by protease inhibitors

Sustained suppression of viral replication in HIV-1 infected patients is especially hampered by the emergence of HIV-1 drug resistance. The mechanisms of drug resistance mainly involve mutations directly altering the interaction of viral enzymes and inhibitors. However, protease inhibitors do not only select for mutations in the protease but also for mutations in the precursor Gag and Pol proteins. In this study, we analysed the frameshift-regulating site of HIV-1 subtype B isolates, which also encodes for Gag and Pol proteins, classified as either treatment-naïve (TN) or protease inhibitor resistant (PI-R). HIV-1 Gag cleavage site mutations (G435E, K436N, I437V, L449F/V) especially correlated with protease inhibitor resistance mutations, but also Pol cleavage site mutations (D05G, D05S) could be assigned to specific protease resistance profiles. Additionally, two Gag non-cleavage site mutations (S440F, H441P) were observed more often in HIV-1 isolates carrying protease resistance mutations. However, in dual luciferase assays, the frameshift efficiencies of specific clones did not reveal any effect from these mutations. Nevertheless, two patterns of mutations modestly increased the frameshift rates in vitro, but were not specifically accumulating in PI-resistant HIV-1 isolates. In summary, HIV-1 Gag cleavage site mutations were dominantly selected in PI-resistant HIV-1 isolates but also Pol cleavage site mutations influenced resistance profiles in the protease. Additionally, Gag non-cleavage site mutations accumulated in PI-resistant HIV-1 isolates, but were not related to an increased frameshift efficiency.     

以Rev依赖性凋亡增强HIV-1gp160的抗原性

目的　检测Rev(HIV的调控基因 )依赖性肿瘤坏死因子受体 (TNFR 1)和gp16 0双重表达质粒pDM12 8 TNFR 1(pT12 8)的凋亡诱导功能。方法　采用基因枪转导及流式细胞仪检测新质粒的表达功能。结果　新结构具有特异的选择性表达作用。当Rev存在时 ,能间接表达TNFR 1,明显诱导HeLa细胞凋亡 ,使绿荧光细胞百分率非常显著地低于阴性对照 (P <0 .0 1)。等质量转染时 ,TNFR 1表达量少于Hup6 0TNFR 1的pDC30 2 (pT6 0 ) ,故间接表达不及单纯pT6 0的直接表达 ,绿荧光细胞百分率显著高于pT6 0转染组 (P <0 .0 1)。培养 40h ,才有明显杀伤功能并接近单纯pT6 0 ,差异无显著性 (P>0 .0 1)。单纯pT12 8不能直接表达TNFR 1,绿荧光细胞百分率非常显著地高于单纯pT6 0转染组 (P<0 .0 1) ,接近阴性对照 ,培养 40h时差异无显著性 (P >0 .0 1)。当AD8或pMD +pRnv存在并表达Rev时 ,pT12 8均能表达TNFR 1,杀伤HeLa细胞 ,绿荧光细胞百分率非常显著地低于阴性对照 (P <0 .0 1)。pT12 8与pRev或AD8转染人正常的角质生成细胞时 ,能表达TNFR 1,诱导细胞凋亡。培养 72h后 ,阴性对照和单纯pT12 8组的绿荧光角质生成细胞数皆显著地超过pT12 8+pRev和pT12 8+pAD8组 (P <0 .0 1)。结论　pT12 8可调控的凋亡诱导保证了HIVDNA疫苗足量的抗原表     

Functional variation of HIV-1 Rev Response Element in a longitudinally studied cohort

We showed previously that HIV-1 Rev Response Element (RRE) contains a certain degree of structural variation, and in a set of limited samples, RRE from HIV-1 natural isolates were found to have functional variability. The significance of the RRE heterogeneity is addressed further by analyzing the functional variation of RREs in a longitudinal cohort. While the RRE activity at early time points was not a good predictor of disease outcome, the RRE activity at late time points was correlated with rates of CD4+ count decline. These data suggest that RRE heterogeneity may be important in viral pathogenesis and disease progression.     

Mutational analysis of the feline immunodeficiency virus matrix protein

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.     

High throughput functional analysis of HIV-1 env genes without cloning

Functional human immunodeficiency virus type 1 (HIV-1) env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3 Delta env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple env genes from HIV-1 infected individuals.