Structural Model of the Rev Regulatory Protein from Equine Infectious Anemia Virus

Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to generate and evaluate a structural model of the Rev protein. The modeled EIAV Rev (ERev) structure includes a total of 6 helices, four of which form an anti-parallel four-helix bundle. The first helix contains the leucine-rich nuclear export signal (NES). An arginine-rich RNA binding motif, RRDRW, is located in a solvent-exposed loop region. An ERLE motif required for Rev activity is predicted to be buried in the core of modeled structure where it plays an essential role in stabilization of the Rev fold. This structural model is supported by existing genetic and functional data as well as by targeted mutagenesis of residues predicted to be essential for overall structural integrity. Our predicted structure should increase understanding of structure-function relationships in Rev and may provide a basis for the design of new therapies for lentiviral diseases.     

Biological Characterization of Rev Variation in Equine Infectious Anemia Virus

Sequence analysis identified significant variation in the second exon of equine infectious anemia virus (EIAV) rev. Functional analysis indicated that limited amino acid variation in Rev significantly altered the export activity of the protein but did not affect Rev-dependent alternative splicing. EIAV Rev can mediate export through two independent cis-acting Rev-responsive elements (RREs), and differences among Rev variants were more pronounced when both RREs were present. Variation in Rev may be an important mechanism for regulation of virus replication in vivo and may contribute to changes in clinical disease.     

Differential Requirements for Alternative Splicing and Nuclear Export Functions of Equine Infectious Anemia Virus Rev Protein

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. None of the nuclear export signal (NES) mutants are defective for alternative splicing. Furthermore, the NES of ERev is similar in composition but distinct in spacing from other leucine-rich NESs. Basic residues at the C terminus of ERev are involved in nuclear localization, and disruption of the C-terminal residues affects both functions of ERev. ERev forms multimers, and no mutation disrupts this activity. In two mutants with substitutions of charged residues in the middle of ERev, RNA export is affected. One of these mutants is also defective for ERev-mediated alternative splicing but is identical to wild-type ERev in its localization, shuttling, and multimerization. Together, these results demonstrate that the two functions of ERev both require nuclear import and at least one other common activity, but RNA export can be separated from alternative splicing based on its requirement for a functional NES.     

Apolipoprotein E but Not B Is Required for the Formation of Infectious Hepatitis C Virus Particles

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.Hepatitis C virus (HCV) is the leading cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (8, 40). HCV coinfection with human immunodeficiency virus (HIV) is also common, occurring overall in 25 to 30% of HIV-positive persons (1). Individuals with chronic HCV infection are at high risk for the development of cirrhosis and hepatocellular carcinoma. A pegylated interferon and ribavirin combination is the standard therapy to treat hepatitis C but suffers from limited efficacy (<50% antiviral response among patients infected with the dominant genotype 1 HCV) and severe side effects (18, 27). More efficacious and safer antiviral drugs for effective treatment of hepatitis C are urgently needed. A thorough understanding of the HCV life cycle will likely provide novel targets for antiviral drug discovery and development to control HCV infection.HCV is an enveloped RNA virus containing a single-stranded, positive-sense RNA genome and is classified as a Hepacivirus in the Flaviviridae family (11, 33). The viral RNA genome carries a single open reading frame flanked by untranslated regions (UTRs) at both the 5′ and 3′ ends. The 5′ and 3′ UTRs contain cis-acting RNA elements important for the initiation of HCV polyprotein translation and viral RNA replication (24). Upon translation, the HCV polyprotein precursor is proteolytically processed by cellular peptidases and viral proteases into at least 10 different viral proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Studies with subgenomic HCV RNAs demonstrated that the NS3 to NS5B proteins, in association with intracellular membranes and cellular proteins, are essential and sufficient for HCV RNA replication in the cell (5, 14, 25). The newly synthesized HCV proteins and RNA genome are assembled to form progeny HCV particles by undetermined mechanisms.Our earlier work found that infectious HCV particles are highly enriched in apolipoprotein E (apoE), which is a major determinant of HCV infectivity and production in cell culture (10). ApoE-specific monoclonal antibodies (MAbs) effectively neutralized HCV infectivity, in a dose-dependent manner. The knockdown of apoE expression by specific small interfering RNA (siRNA) remarkably suppressed HCV production, suggesting that apoE is also important for the formation of infectious particles and/or egression (10). However, studies by others suggested that HCV assembly and production are dependent on microsomal transfer protein (MTP) and apolipoprotein B (apoB), both of which are essential components required for the assembly and secretion of very-low-density lipoproteins (VLDLs) (19, 21). In those studies, both apoB-specific siRNAs and MTP inhibitors were found to suppress HCV production (19, 21). It was speculated that HCV shares the same assembly and secretion pathway with VLDLs.To define the roles of apoB and apoE in the formation of HCV particles and egression, we developed a single-cycle HCV growth assay. Using this assay system, we have demonstrated that apoE but not apoB is required for the infectivity and formation of infectious HCV particles. First of all, apoB-specific MAb and polyclonal antibodies did not affect HCV infection. Additionally, apoE-specific siRNA potently inhibited the formation of infectious HCV particles, whereas HCV production was unaffected by the siRNA-mediated knockdown of apoB expression. Furthermore, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked apoB secretion but did not significantly affect HCV production prior to the blockage of apoE expression/secretion. At higher concentrations, however, both MTP inhibitors blocked apoE secretion and consequently suppressed the formation of infectious HCV particles. To further understand the role of apoE in HCV assembly, we carried out coimmunoprecipitation (co-IP) experiments and found that apoE-specific MAb pulled down NS5A but not other HCV proteins from lysed HCV particles, suggesting a specific interaction between apoE and NS5A during the formation of infectious HCV particles. Collectively, our findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.     

Inhibition of Human Immunodeficiency Virus Rev and Human T-Cell Leukemia Virus Rex Function, but Not Mason-Pfizer Monkey Virus Constitutive Transport Element Activity, by a Mutant Human Nucleoporin Targeted to Crm1

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran · GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed ΔCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, ΔCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, ΔCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.     

马传染性贫血病毒驴白细胞弱毒疫苗gag基因的序列测定及分析

以马传染性贫血病毒（ＥＬＡＶ）驴白细胞弱毒疫苗株（ＤＬＡ）病毒基因组ＲＮＡ为材料,用ＲＴ－ＰＣＲ方法扩增出ＥＩＡＶ ｇａｇ基因,以平端针其克隆到质粒载体ｐＵＣ１９中,由于疫苗不是克隆株,因此通过５次单独克隆与测序,推导出ＥＩＡＶ－ＤＬＡ ｇａｇ基因的优势序列。ｇａｇ基因全长１４５８个碱基,编码一４８６个氨基酸残基的前体蛋白。与美国ＥＩＡＶ Ｗｙｏｍｉｎｇ１３６９株比较,核苷酸同源性为８０％,氨基酸     

Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication

We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.     

中国株EIAV S2基因逆向突变感染性克隆的构建及体外感染性评价

以中国株EIAV的驴强毒株EIAVDV115与疫苗株EIAVFDDV15的S2基因变化规律为依据,利用反向遗传技术对EIAV弱毒疫苗株全基因组感染性克隆pFDDV3-8的S2基因区进行逆向突变,构建了含有强毒株EIAVDV115S2基因区内4个稳定点突变的克隆质粒pFDDVS2r1-3-4-5,将该质粒转染FDD后进行体外继代盲传,经RT-PCR、逆转录酶活性、间接免疫荧光等检测后,显示经EIAV克隆质粒pFDDVS2r1-3-4-5转染的FDD盲传3代后,可在转染的FDD细胞培养物的上清液中检测到EIAV逆转录酶活性;该细胞培养物经RT-PCR和间接免疫荧光检测均呈EIAV阳性;在电镜下可观察到典型EIAV粒子。证明已成功拯救经EIAVS2基因逆向突变后的衍生病毒vpFDDVS2r1-3-4-5。比较vpFDDVS2r1-3-4-5衍生病毒与亲本克隆衍生病毒的复制动力学,表明前者的复制比后者略滞后。以上结果提示,对疫苗株S2基因的突变未明显影响EIAV的体外复制。     

马传贫驴白细胞弱毒疫苗株酸性蛋白p9基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码p9蛋白的基因,并在大肠杆菌中进行了表达.所表达的蛋白是一种可溶性的融合蛋白,其氨基端带有6个组氨酸的标签,因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化.在间接酶联免疫吸附试验(ELISA)和免疫印迹试验中,重组的酸性蛋白p9可与马传贫阳性血清样品发生反应,而与健康马血清无任何反应.这表明该重组蛋白具有良好的抗原性和特异性,可用于马传贫弱毒疫苗株在体内外复制及在接种马体内免疫应答的研究.     

Seroprevalence of Human Immunodeficiency Virus,Hepatitis B Virus,Hepatitis C Virus,and Treponema pallidum Infections among Blood Donors on Bioko Island,Equatorial Guinea

Background

Regular screening of transfusion-transmissible infections (TTIs), such as human immunodeficiency virus (HIV), hepatitis B and hepatitis C virus (HBV and HCV, respectively), and Treponema pallidum, in blood donors is essential to guaranteeing clinical transfusion safety. This study aimed to determine the seroprevalence of four TTIs among blood donors on Bioko Island, Equatorial Guinea (EG).

Methods

A retrospective survey of blood donors from January 2011 to April 2013 was conducted to assess the presence of HIV, HBV, HCV and T. pallidum. The medical records were analyzed to verify the seroprevalence of these TTIs among blood donations stratified by gender, age and geographical region.

Results

Of the total 2937 consecutive blood donors, 1098 (37.39%) had a minimum of one TTI and 185 (6.29%) harbored co-infections. The general seroprevalence of HIV, HBV, HCV and T. pallidum were 7.83%, 10.01%, 3.71% and 21.51%, respectively. The most frequent TTI co-infections were HBV-T. pallidum 60 (2.04%) and HIV-T. pallidum 46 (1.57%). The seroprevalence of HIV, HBV, HCV and T. pallidum were highest among blood donors 38 to 47 years, 18 to 27 years and ≥ 48 years age, respectively (P<0.05). The seroprevalence of TTIs varied according to the population from which the blood was collected on Bioko Island.

Conclusions

Our results firstly provide a comprehensive overview of TTIs among blood donors on Bioko Island. Strict screening of blood donors and improved hematological examinations using standard operating procedures are recommended.     

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.Recent estimates predict that the prevalence of hepatitis C virus (HCV) infection is approximately 2.2% worldwide, equivalent to about 130 million persons (22). The virus typically establishes a chronic infection that frequently leads to serious liver disease (1), and current models indicate that both morbidity and mortality as a consequence of HCV infection will continue to rise for about the next 20 years (10, 11, 29).HCV is the only assigned species of the Hepacivirus genus within the family Flaviviridae. The virus can be classified into six genetic groups or clades (numbered 1 to 6) and then further separated into subtypes (e.g., 1a, 1b, 2a, 2b, etc.) (53, 55). HCV has a single-stranded, positive-sense RNA genome that is approximately 9.6 kb in length (reviewed in reference 46). Genomic RNA carries a single open reading frame flanked by 5′ and 3′ nontranslated regions, which are important for both replication and translation (19, 20, 34, 47, 56). Viral RNA is translated by the host ribosomal machinery, and the resultant polyprotein is co- and posttranslationally cleaved to generate the mature viral proteins. The structural proteins (core, E1, and E2) and a small hydrophobic polypeptide called p7 are produced by the cellular proteases signal peptidase and signal peptide peptidase (28, 45, 54). Two virus-encoded proteases, the NS2-3 autoprotease and the NS3 serine protease (5, 13, 26), are responsible for maturation of the nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). With the exception of NS2, the NS proteins are necessary for genome replication (8, 40) and form replication complexes (RCs), which are located at the endoplasmic reticulum (ER) membrane (14, 24, 52, 57, 59). The functions of all viral constituents of RCs have not been characterized in detail. It is known that NS5B is the RNA-dependent RNA polymerase (6), while NS3 possesses helicase and nucleoside triphosphatase activities in addition to acting as a protease (32, 58). However, the precise roles of the other proteins remain to be firmly established.Expression of NS4B, one of the replicase proteins, generates rearrangements at the ER membrane that have been termed the “membranous web” (14, 24) and “membrane-associated foci” (MAFs) (25). Detection of viral RNA at such foci suggests that NS4B is involved in creating the sites where genome synthesis occurs (18, 24, 59). It is predicted that NS4B has an amphipathic α-helix within its N-terminal region, which is followed by four transmembrane domains (TMDs) in the central portion of the protein (17, 42). As a result, the majority of NS4B is likely to be tightly anchored to membranes, and experimental evidence indicates that it has characteristics consistent with an integral membrane protein (27). It is thought that after membrane association, NS4B rearranges membranes into a network, thereby generating foci which act as a “scaffold” to facilitate RNA replication. The mechanisms engaged in formation of foci are not known but include the notion that the NS4B N terminus can translocate into the ER lumen, resulting in rearrangement of cellular membranes (41, 42). Alternatively, palmitoylation, a lipid modification, might facilitate polymerization of NS4B, in turn promoting formation of RCs on the ER membrane (68).Apart from inducing membranous changes required for replication, NS4B may perform other tasks in HCV RNA synthesis. For example, studies of cell culture adaptive mutations in subgenomic replicons (SGRs) have identified amino acid changes that can stimulate RNA production (39), suggesting that NS4B may exert a regulatory role in determining replication efficiency. In support of a regulatory function, replacement of NS4B sequences in an SGR from strain H77 (a genotype 1a strain) with those from strain Con-1 (a genotype 1b strain) gave higher levels of replication than for a wild-type (wt) strain H77 SGR (7). The corresponding replacement of strain Con-1 NS4B sequences with those from strain H77 reduced the replication efficiency of a Con-1 SGR (7). Moreover, interactions of NS4B with the RC can affect the behavior of other replicase proteins. For example, NS4B is needed for hyperphosphorylation of NS5A (35, 48) and restricts its intracellular movement (30).To try to gain greater insight into the functional organization of the components that constitute RCs, trans-complementation assays using defective and helper SGRs have been established (2, 64). Such studies reveal that the only protein capable of trans-complementation is NS5A, while active replication cannot be restored for replicons harboring deleterious mutations in NS3, NS4B, and NS5B. These data led to the conclusion that functional NS5A may be able to exchange between RCs (2), whereas, by inference, such exchange would not be possible for other HCV replicase proteins. In transient-replication assays, complementation by NS5A also relied on its expression as part of a polyprotein (minimally NS3-NS5A), and production of the protein alone failed to restore replication for an inactive SGR (2). However, in a separate study, stable expression of wt NS5A was capable of complementing a defective replicon (64). Thus, different assay systems can give dissimilar results for complementation by NS5A.In this study, we have created a series of mutations in the NS4B gene of HCV strain JFH1 (31) to explore the function of the protein in the HCV life cycle. We focused our attention on the C-terminal portion of NS4B, downstream from the predicted TMD regions, since it is relatively well conserved and is predicted to lie on the cytosolic side of the ER membrane (15, 42). Our analysis examines the impact of mutations on replication efficiency and the intracellular characteristics of the mutants compared to the behavior of the wt protein. In addition, we have utilized this series of mutants to reassess trans-complementation of NS4B in replication assays. Finally, we also analyze the impact of mutations which do not affect replication on the production of infectious virus to determine whether NS4B plays a role in virus assembly and release.