Wolbachia reduces the transmission potential of Dengue-infected Aedes aegypti

BACKGROUND: Dengue viruses (DENV) are the causative agents of dengue, the world's most prevalent arthropod-borne disease with around 40% of the world's population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia's efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito's ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites. METHODOLOGY/PRINCIPAL FINDINGS: We used a non-destructive assay to repeatedly quantify DENV in saliva from wMel-infected and Wolbachia-free wild-type control mosquitoes following the consumption of a DENV-infected blood meal. We show that wMel lengthens the EIP, reduces the frequency at which the virus is expectorated and decreases the dengue copy number in mosquito saliva as compared to wild-type mosquitoes. These observations can at least be partially explained by an overall reduction in saliva produced by wMel mosquitoes. More generally, we found that the concentration of DENV in a blood meal is a determinant of the length of EIP, saliva virus titer and mosquito survival. CONCLUSIONS/SIGNIFICANCE: The saliva-based traits reported here offer more disease-relevant measures of Wolbachia's effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.

Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardmentwith the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

Oncolytic adenoviruses for treatment of ovarian cancer

Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.

Genetically Engineered Oncolytic Adenoviruses for the Treatment of Kidney and Breast Cancer

Metastatic kidney and breast cancer are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment are oncolytic adenoviruses, which have demonstrated excellent safety in many clinical trials. However, antitumor efficacy needs to be improved in order to make oncolytic viruses a viable treatment alternative. To be able to follow oncolytic virus replication in vivo, we set up a non-invasive imaging system based on coinjection of a replication deficient luciferase expressing virus and a replication competent virus. The system was validated in vitro and in vivo and used in other projects of the thesis. In another study we showed that capsid modifications on adenoviruses result in enhanced gene transfer and increased oncolytic effect on renal cancer cells in vitro. Moreover, capsid modified oncolytic adenoviruses demonstrated significantly improved antitumor efficacy in murine kidney cancer models. To transcriptionally target kidney cancer tissue we evaluated two hypoxia response elements for their usability as tissue specific promoters using a novel dual luciferase imaging system. Based on the results of the promoter evaluation and the studies on capsid modifications, we constructed a transcriptionally and transductionally targeted oncolytic adenovirus armed with an antiangiogenic transgene for enhanced renal cell cancer specificity and improved antitumor efficacy. This virus exhibited kidney cancer specific replication and significantly improved antitumor effect in a murine model of intraperitoneal disseminated renal cell cancer. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in breast cancer relapse and the formation of metastasis. Therefore, we examined if capsid modified oncolytic adenoviruses are able to kill these cells proposed to be breast cancer initiating. Efficient oncolytic effect and significant antitumor efficacy on tumors established with breast cancer initiating cells was observed, suggesting that oncolytic adenoviruses might be able to prevent breast cancer relapse and could be used in the treatment of metastatic disease. In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of metastatic kidney and breast cancer.

Approaches for improving the safety and efficacy of adenoviral gene therapy

Cancer is a devastating disease with poor prognosis and no curative treatment, when widely metastatic. Conventional therapies, such as chemotherapy and radiotherapy, have efficacy but are not curative and systemic toxicity can be considerable. Almost all cancers are caused due to changes in the genetic material of the transformed cells. Cancer gene therapy has emerged as a new treatment option, and past decades brought new insights in developing new therapeutic drugs for curing cancer. Oncolytic viruses constitute a novel therapeutic approach given their capacity to replicate in and kill specifically tumor cells as well as reaching tumor distant metastasis. Adenoviral gene therapy has been suggested to cause liver toxicity. This study shows that new developed adenoviruses, in particular Ad5/19p-HIT, can be redirected towards kidney while adenovirus uptake by liver is minimal. Moreover, low liver transduction resulted in a favorable tumor to liver ratio of virus load. Further, we established a new immunocompetent animal model Syrian hamsters. Wild type adenovirus 5 was found to replicate in Hap-T1 hamster tumors and normal tissues. There are no antiviral drugs available to inhibit adenovirus replication. In our study, chlorpromazine and cidofovir efficiently abrogated virus replication in vitro and showed significant reduction in vivo in tumors and liver. Once safety concerns were addressed together with the new given antiviral treatment options, we further improved oncolytic adenoviruses for better tumor penetration, local amplification and host system modulation. Further, we created Ad5/3-9HIF-Δ24-VEGFR-1-Ig, oncolytic adenovirus for improved infectivity and antiangiogenic effect for treatment of renal cancer. This virus exhibited increased anti-tumor effect and specific replication in kidney cancer cells. The key player for good efficacy of oncolytic virotherapy is the host immune response. Thus, we engineered a triple targeted adenovirus Ad5/3-hTERT-E1A-hCD40L, which would lead to tumor elimination due to tumor-specific oncolysis and apoptosis together with an anti-tumor immune response prompted by the immunomodulatory molecule. In conclusion, the results presented in this thesis constitute advances in our understanding of oncolytic virotherapy by successful tumor targeting, antiviral treatment options as a safety switch in case of replication associated side-effects, and modulation of the host immune system towards tumor elimination.  

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

The Structure and Functions of Hantavirus Nucleocapsid Protein

Hantaviruses, members of the genus Hantavirus in the Bunyaviridae family, are enveloped single-stranded RNA viruses with tri-segmented genome of negative polarity. In humans, hantaviruses cause two diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which vary in severity depending on the causative agent. Each hantavirus is carried by a specific rodent host and is transmitted to humans through excreta of infected rodents. The genome of hantaviruses encodes four structural proteins: the nucleocapsid protein (N), the glycoproteins (Gn and Gc), and the polymerase (L) and also the nonstructural protein (NSs). This thesis deals with the functional characterization of hantavirus N protein with regard to its structure. Structural studies of the N protein have progressed slowly and the crystal structure of the whole protein is still not available, therefore biochemical assays coupled with bioinformatical modeling proved essential for studying N protein structure and functions. Presumably, during RNA encapsidation, the N protein first forms intermediate trimers and then oligomers. First, we investigated the role of N-terminal domain in the N protein oligomerization. The results suggested that the N-terminal region of the N protein forms a coiled-coil, in which two antiparallel alpha helices interact via their hydrophobic seams. Hydrophobic residues L4, I11, L18, L25 and V32 in the first helix and L44, V51, L58 and L65 in the second helix were crucial for stabilizing the structure. The results were consistent with the head-to-head, tail-to-tail model for hantavirus N protein trimerization. We demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein. We also added new details to the head-to-head, tail-to-tail model of trimerization by suggesting that the initial step is based on interaction(s) between intact intra-molecular coiled-coils of the monomers. We further analyzed the importance of charged aa residues located within the coiled-coil for the N protein oligomerization. To predict the interacting surfaces of the monomers we used an upgraded in silico model of the coiled-coil domain that was docked into a trimer. Next the predicted target residues were mutated. The results obtained using the mammalian two-hybrid assay suggested that conserved charged aa residues within the coiled-coil make a substantial contribution to the N protein oligomerization. This contribution probably involves the formation of interacting surfaces of the N monomers and also stabilization of the coiled-coil via intramolecular ionic bridging. We proposed that the tips of the coiled-coils are the first to come into direct contact and thus initiate tight packing of the three monomers into a compact structure. This was in agreement with the previous results showing that an increase in ionic strength abolished the interaction between N protein molecules. We also showed that residues having the strongest effect on the N protein oligomerization are not scattered randomly throughout the coiled-coil 3D model structure, but form clusters. Next we found evidence for the hantaviral N protein interaction with the cytoplasmic tail of the glycoprotein Gn. In order to study this interaction we used the GST pull-down assay in combination with mutagenesis technique. The results demonstrated that intact, properly folded zinc fingers of the Gn protein cytoplasmic tail as well as the middle domain of the N protein (that includes aa residues 80 248 and supposedly carries the RNA-binding domain) are essential for the interaction. Since hantaviruses do not have a matrix protein that mediates the packaging of the viral RNA in other negatve stranded viruses (NSRV), hantaviral RNPs should be involved in a direct interaction with the intraviral domains of the envelope-embedded glycoproteins. By showing the N-Gn interaction we provided the evidence for one of the crucial steps in the virus replication at which RNPs are directed to the site of the virus assembly. Finally we started analysis of the N protein RNA-binding region, which is supposedly located in the middle domain of the N protein molecule. We developed a model for the initial step of RNA-binding by the hantaviral N protein. We hypothesized that the hantaviral N protein possesses two secondary structure elements that initiate the RNA encapsidation. The results suggest that amino acid residues (172-176) presumably act as a hook to catch vRNA and that the positively charged interaction surface (aa residues 144-160) enhances the initial N-RNA interacation. In conclusion, we elucidated new functions of hantavirus N protein. Using in silico modeling we predicted the domain structure of the protein and using experimental techniques showed that each domain is responsible for executing certain function(s). We showed that intact N terminal coiled-coil domain is crucial for oligomerization and charged residues located on its surface form a interaction surface for the N monomers. The middle domain is essential for interaction with the cytoplasmic tail of the Gn protein and RNA binding.

Oncolytic Adenoviruses with Radiation Therapy for Treatment of Prostate Cancer

Prostate cancer is the most common cancer in males. Although many patients with localized disease can be cured with surgery and radiotherapy, advanced disease and especially castration resistant metastatic disease remains incurable, with a median life expectancy of less than 18 months. Oncolytic adenoviruses (Ads) are a new promising treatment against cancer due to their innate capacity to kill cancer cells. Viral replication in tumor cells leads to oncolysis and production of a multiplicity of new virions that are capable of further destroying cancerous tissue. Oncolytic Ads can be modified for tumor targeted infection and replication and be armed with therapeutic transgenes to maximize the oncolytic effect. Worldwide, clinical trials with oncolytic Ads have demonstrated good safety while the antitumor efficacy remains to be improved. Importantly, the best responses have been reported when oncolytic adenoviruses have been combined with standard cancer treatments, such as chemotherapy and radiation. Further, a challenge in many virotherapy approaches has been the monitoring of virus replication in vivo. Reporter genes have been extensively used as transgenes to evaluate the biodistribution of the virus and activity of specific promoters. However, these techniques are often limited to preclinical evaluation and not amenable to human use. The aim of the thesis was to find and develop new oncolytic Ads with maximum efficacy against metastatic, castration resistant prostate cancer and study them in vitro and in vivo combined to different forms of radiation therapy. Using combination therapy, we were aiming for better antitumor efficacy with reduced side effects. Capsid modified Ads for enhanced transduction were studied. Serotype 3 targeted chimera, Ad5/3, was found to have enhanced infectivity for prostate cancer and was used for developing new viruses for the study. Correlation between Ad-encoded marker peptide secretion and simultaneous viral replication was evaluated and the effects of radiotherapy on viral replication were studied in detail. We found that the repair of double strand breaks caused by ionizing radiation was inhibited by adenoviral proteins and led to autophagic cell death. Both subcutaneous models and intrapulmonary tumor models mimicking metastatic, aggressive disease were used in vivo. Virus efficacy was evaluated by intratumoral injections. Also, intravenous administration was evaluated to study the effectiveness in metastatic disease. Oncolytic adenovirus treatment led to significant tumor growth control and increased the survival rate of the mice. These results were further improved when oncolytic Ads were combined with radiation therapy. Oncolytic Ads expressing human sodium/iodide transporter (hNIS) as a transgene were evaluated for their oncolytic potency and for the functionality of hNIS in vitro and in vivo. Monitoring of viral replication was also assessed using different imaging modalities relative to clinical use. SPECT imaging of tumor-bearing mice was evaluated and combined with simultaneous CT-scanning to obtain important anatomical information on biodistribution, also in a three-dimensional form. It was shown that hNIS-expressing adenoviruses could harbour a bi-functional transgene allowing for localization and imaging of viral replication. Targeted radiotherapy was applied by systemic radioiodide administration and resulted in iodide accumulation into Ad-infected tumor. The combination treatment showed significantly enhanced antitumor efficacy in mice bearing prostate cancer tumors. In summary, the results presented above aim to provide new treatment modalities for castration resistant prostate cancer. Molecular insights were provided for better understanding of the benefits of combined radiation therapy and oncolytic adenoviruses, which will hopefully facilitate the translation of the approach into clinical use for humans.

Functions of Alphavirus Macrodomain-containing protein nsP3

Alphaviruses are positive strand RNA viruses that replicate in association with cellular membranes. The viral RNA replication complex consists of four non-structural proteins nsP1-nsP4 which are essential for viral replication. The functions of nsP1, nsP2 and nsP4 are well established, but the roles of nsP3 are mainly unknown. In this work I have clarified some of the functions of nsP3 in order to better understand the importance of this protein in virus replication. Semliki Forest virus (SFV) has been mostly used as a model alphavirus during this work, but some experiments have also been conducted with Sindbis and Chikungunya viruses. NsP3 is composed of three different protein domains. The N-terminus of nsP3 contains an evolutionarily conserved macrodomain, the central part of nsP3 contains a domain that is only found in alphaviruses, and the C-terminus of the protein is hypervariable and predicted to be unstructured. In this work I have analyzed the functions of nsP3 macrodomain, and shown that viral macrodomains bind poly(ADP-ribose) and that they do not resemble cellular macrodomains in their properties. Furthermore, I have shown that some macrodomains, including viral macrodomains of SFV and hepatitis E virus, also bind poly(A). Mutations in the ligand binding pocket of SFV macrodomain hamper virus replication but do not confer lethality, indicating that macrodomain function is beneficial but not mandatory for virus replication. The hypervariable C-terminus of nsP3 is heavily phosphorylated and is enriched in proline residues. In this work it is shown that this region harbors an SH3 domain binding motif (Sh3BM) PxRxPR through which cellular amphiphysin is recruited to viral replication sites and to nsP3 containing cytoplasmic aggregate structures. The function of Sh3BM was destroyed by a single point mutation, which led to impaired viral RNA replication in HeLa cells, pointing out the functional importance of amphiphysin recruitment by the Sh3BM. In addition, evidence is provided tho show that the endosomal localization of alphavirus replication is mediated by nsP3 and that the phosphorylation of hypervariable region might be important for the endosomal targeting. Together these findings demonstrate that nsP3 contains multiple important host interaction motifs and domains, which facilitate successful viral propagation in host cells.

Hantavirus infection: Insights into entry, assembly and pathogenesis

Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells. (C) 2004 Published by Elsevier B.V.

Anti-AIDS agents 81. Design, synthesis, and structure-activity relationship study of betulinic acid and moronic acid derivatives as potent HIV maturation inhibitors.

In our continuing study of triterpene derivatives as potent anti-HIV agents, different C-3 conformationally restricted betulinic acid (BA, 1) derivatives were designed and synthesized in order to explore the conformational space of the C-3 pharmacophore. 3-O-Monomethylsuccinyl-betulinic acid (MSB) analogues were also designed to better understand the contribution of the C-3' dimethyl group of bevirimat (2), the first-in-class HIV maturation inhibitor, which is currently in phase IIb clinical trials. In addition, another triterpene skeleton, moronic acid (MA, 3), was also employed to study the influence of the backbone and the C-3 modification toward the anti-HIV activity of this compound class. This study enabled us to better understand the structure-activity relationships (SAR) of triterpene-derived anti-HIV agents and led to the design and synthesis of compound 12 (EC(50): 0.0006 microM), which displayed slightly better activity than 2 as a HIV-1 maturation inhibitor.

Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation.

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Plasmacytoid Dendritic Cells Are Productively Infected and Activated through TLR-7 Early after Arenavirus Infection.

The antiviral response is largely mediated by dendritic cells (DCs), including conventional (c) DCs that function as antigen-presenting cells, and plasmacytoid (p) DCs that produce type I interferons, making them an attractive target for viruses. We find that the Old World arenaviruses lymphocytic choriomeningitis virus clone 13 (LCMV Cl13) and Lassa virus bind pDCs to a greater extent than cDCs. Consistently, LCMV Cl13 targets pDCs early after in vivo infection of its natural murine host and establishes a productive and robust replication cycle. pDCs coproduce type I interferons and proinflammatory cytokines, with the former being induced in both infected and uninfected pDCs, demonstrating a dissociation from intrinsic virus replication. TLR7 globally mediates pDC responses, limits pDC viral load, and promotes rapid innate and adaptive immune cell activation. These early events likely help dictate the outcome of infections with arenaviruses and other DC-replicating viruses and shed light on potential therapeutic targets.