Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression.

The nonlytic suppression of human immunodeficiency virus (HIV) production from infected CD4+ T cells by CD8+ lymphocytes from HIV-infected individuals is one of the most potent host-mediated antiviral activities observed in vitro. We demonstrate that the pleiotropic cytokine interleukin 2 (IL-2), but not IL-12, is a potent inducer of the CD8+ HIV suppressor phenomenon. IL-2 induces HIV expression in peripheral blood or lymph node mononuclear cells from HIV-infected individuals in the absence of CD8+ T cells. However, IL-2 induces CD8+ T cells to suppress HIV expression when added back to these cultures, and this effect dramatically supersedes the ability to IL-2 to induce HIV expression. Five to 25 times fewer CD8+ cells were required to obtain comparable levels of inhibition of viral production if they were activated in the presence of IL-2 as compared with IL-12 or no exogenous cytokine. Furthermore, IL-2 appeared either to induce a qualitative increase in HIV suppressor cell activity or to increase the relative frequency of suppressor cells in the activated (CD25+) CD8+ populations. Analyses of proviral levels in peripheral blood mononuclear cells suggest that CD8+ T cell-mediated lysis of in vivo infected cells is not induced by IL-2. These results have implications for our understanding of the effects of impaired IL-2 production during HIV disease as well as the overall effects of IL-2-based immunotherapy on HIV replication in vivo.

Association of a M(r) 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2 alpha and premature shutoff of protein synthesis after infection with gamma 134.5- mutants of herpes simplex virus 1.

The protein encoded by the gamma 134.5 gene of herpes simplex virus precludes premature shutoff of protein synthesis in human cells triggered by stress associated with onset of viral DNA synthesis. The carboxyl terminus of the protein is essential for this function. This report indicates that the shutoff of protein synthesis is not due to mRNA degration because mRNA from wild-type or gamma 134.5- virus-infected cells directs protein synthesis. Analyses of the posttranslational modifications of translation initiation factor eIF-2 showed the following: (i) eIF-2 alpha was selectively phosphorylated by a kinase present in ribosome-enriched fraction of cells infected with gamma 134.5- virus. (ii) Endogenous eIF-2 alpha was totally phosphorylated in cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene but was not phosphorylated in mock-infected or wild-type virus-infected cells. (iii) Immune precipitates of the PKR kinase that is responsible for regulation of protein synthesis of some cells by phosphorylation of eIF-2 alpha yielded several phosphorylated polypeptides. Of particular significance were two observations. First, phosphorylation of PKR kinase was elevated in all infected cells relative to the levels in mock-infected cells. Second, the precipitates from lysates of cells infected with gamma 134.5- virus or a virus lacking the 3' coding domain of the gamma 134.5 gene contained an additional labeled phosphoprotein of M(r) 90,000 (p90). This phosphoprotein was present in only trace amounts in the immunoprecipitate from cells infected with wild-type virus or mutants lacking a portion of the 5' domain of gamma 134.5. We conclude that in the absence of gamma 134.5 protein, PKR kinase complexes with the p90 phosphoprotein and shuts off protein synthesis by phosphorylation of the alpha subunit of translation initiation factor eIF-2.

Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

We and other groups have recently reported the potentiation by ribonucleotide reductase inhibitors such as hydroxyurea of the anti-human immunodeficiency virus type 1 (HIV-1) activity of purine and pyrimidine 2',3'-dideoxynucleosides in both resting and phytohemagglutinin-stimulated peripheral blood mononuclear cells. Little agreement prevails, however, as to the mechanism of the synergistic effects described. We report here that in phytohemagglutinin-stimulated peripheral blood mononuclear cells, two mechanisms exist for the potentiation of the anti-HIV-1 activity by low-dose hydroxyurea of the purine-based dideoxynucleoside 2',3'-dideoxyinosine and the pyrimidine-based dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine. For 2',3'-dideoxyinosine, the enhancement arises from a specific depletion of dATP by hydroxyurea, resulting in a favorable shift of the 2',3'-dideoxyadenosine 5'-triphosphate/dATP ratio. For the pyrimidine dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, the more modest anti-HIV enhancement results from hydroxyurea-induced increases of pyrimidine kinase activities in the salvage pathway and, hence, increased 5'-phosphorylation of these drugs, while depletion of the corresponding deoxynucleoside 5'-triphosphates (dTTP and dCTP) plays no significant role.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Inhibitory effect of 9-(2-phosphonylmethoxyethyl)adenine on visna virus infection in lambs: a model for in vivo testing of candidate anti-human immunodeficiency virus drugs.

The acyclic nucleoside phosphonate analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was recently found to be effective as an inhibitor of visna virus replication and cytopathic effect in sheep choroid plexus cultures. To study whether PMEA also affects visna virus infection in sheep, two groups of four lambs each were inoculated intracerebrally with 10(6.3) TCID50 of visna virus strain KV1772 and treated subcutaneously three times a week with PMEA at 10 and 25 mg/kg, respectively. The treatment was begun on the day of virus inoculation and continued for 6 weeks. A group of four lambs were infected in the same way but were not treated. The lambs were bled weekly or biweekly and the leukocytes were tested for virus. At 7 weeks after infection, the animals were sacrificed, and cerebrospinal fluid (CSF) and samples of tissue from various areas of the brain and from lungs, spleen, and lymph nodes were collected for isolation of virus and for histopathologic examination. The PMEA treatment had a striking effect on visna virus infection, which was similar for both doses of the drug. Thus, the frequency of virus isolations was much lower in PMEA-treated than in untreated lambs. The difference was particularly pronounced in the blood, CSF, and brain tissue. Furthermore, CSF cell counts were much lower and inflammatory lesions in the brain were much less severe in the treated lambs than in the untreated controls. The results indicate that PMEA inhibits the propagation and spread of visna virus in infected lambs and prevents brain lesions, at least during early infection. The drug caused no noticeable side effects during the 6 weeks of treatment.

Análise de ligação do gene de resistência Zym-2 com marcadores microssatélites e reação de acessos de meloeiro ao Zucchini yellow mosaic virus (ZYMV)

As viroses causam perdas significativas na cultura do melão. Dentre essas, o vírus do mosaico amarelo da abobrinha-de-moita (Zucchini yellow mosaic virus- ZYMV) possui grande importância para a cultura e é encontrado em todos os locais de plantio de cucurbitáceas. O controle desse vírus através da resistência genética é a forma mais eficiente de manejo. O acesso PI414723 é a única fonte de resistência de meloeiro ao ZYMV. Essa resistência é oligogênica e supostamente condicionada por três genes dominantes: Zym-1, Zym-2 e Zym-3. A localização cromossômica do gene Zym-1 já foi confirmada no grupo de ligação 2, próximo ao marcador CMAG36. Entretanto, a localização de Zym-2 ainda carece de confirmação experimental, muito embora existam evidências de sua localização no grupo de ligação 10 (LGX). Sendo assim, um dos objetivos do presente trabalho foi confirmar a localização do gene Zym-2 através de análises de ligação com marcadores microssatélites (SSRs). Para tanto, foi utilizada uma população F2 derivada do cruzamento PI414723 x \'Védrantais\'. As plantas foram inoculadas mecanicamente com o isolado RN6-F, patótipo 0, duas vezes em um intervalo de 24 h. A confirmação da infecção e a quantificação dos títulos virais nas plantas F2 foram realizadas através do teste PTA-ELISA. O DNA genômico das plantas foi extraído da primeira folha verdadeira e utilizado nas reações de PCR com primers específicos para SSRs selecionados pertencentes ao LGX. Observou-se uma distribuição assimétrica de classes de absorbância e maior frequência de indivíduos F2 na classe com menor valor (0,1 a 0,2), sugerindo a existência de um gene de efeito maior. O teste chi-quadrado mostrou que todos os marcadores segregaram na frequência esperada (1:2:1), exceto o marcador CMCT134b. A ligação do Zym-2 aos marcadores foi confirmada por meio de regressão linear simples. Dos marcadores analisados, a regressão linear foi significativa para MU6549 e CMBR55, com p-valores de 0,011 e 0,0054, respectivamente. As análises de ligação mostraram que as ordens e as distâncias entre os marcadores condizem com os mapas presentes na literatura. Um segundo objetivo do estudo foi o de avaliar a reação ao ZYMV de 42 acessos de meloeiro oriundos da região Nordeste do Brasil, com o intuito de explorar novas fontes de resistência. Foram realizados dois experimentos utilizando a mesma metodologia citada anteriormente. O título viral médio entre os acessos variou de 0,123 a 0,621 no experimento 1 e de 0,019 a 0,368 no experimento 2. Alguns acessos apresentaram consistentemente baixos títulos virais, próximos aos do acesso resistente PI414723 e dos controles negativos (plantas não inoculadas da cultivar \'Védrantais\'). Portanto, estes acessos mostram-se como potenciais fontes de resistência ao vírus para o emprego em programas de melhoramento.

Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins

Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8 hr or 18-24 hr etoposide treatment (80 muM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p < 0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Determination of organ tropism of Newcastle disease virus (strain I-2) by virus isolation and reverse transcription-polymerase chain reaction

The vaccines 1-2 and V4 are avirulent strains of Newcastle disease virus. Organ tropism of strain V4 has been determined and the virus has a predilection for the digestive tract. Tropism of strain 1-2 has not yet been determined. The objective of this study was to determine the distribution of strain 1-2 in various body organs and fluids following vaccination in comparison with V4. Four-week-old chickens were vaccinated by eye drop separately with these two avirulent strains. Virus isolation and the reverse transcription-polymerase chain reaction technique were employed to detect 1-2 and V4 viruses in various tissues and body fluids for 7 days following vaccination. Tissues from the respiratory tract showed earlier positive signals than tissues from other organs for chickens vaccinated with strain 1-2. Conversely, tissues from mainly digestive tract produced earlier positive signals than from respiratory tract and other organs from chickens vaccinated with strain V4. In early infection, strain 1-2 had preferential predilection for the respiratory tract and strain V4 for the digestive tract. Later after vaccination, other organs showed positive results from chickens vaccinated with both 1-2 and V4 strains. The differences in organ tropism observed in this study suggest that 1-2 may perform better than V4 as a live vaccine strain.

Development of a cell culture method for quantal assay of strain I-2 of Newcastle disease virus

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain 1-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain 1-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain 1-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

Genorne sequence of the thermostable Newcastle disease virus (strain I-2) reveals a possible phenotypic locus

The complete genome sequence of the Australian 1-2 heat-tolerant Newcastle disease virus (NDV) vaccine (master seed stocks) was determined and compared to the sequence of the parent virus from which it had been derived after exposure of the parent stock at 56 degrees C for 30 min. Nucleotide changes were observed at a number of positions with synonymous mutations being greater than those observed for non-synonymous mutations. Sequence data for the HN gene of a parental culture of V4 and two heat-tolerant variants of V4 were obtained. These were compared with the data for the 1-2 viruses and with published sequences for parental V4 and for a number of ND vaccine strains. Sequence analyses did not reveal the ARG 303 deletion in the HN protein, previously claimed to be responsible for the thermostable phenotype. No consistent changes were detected that would indicate involvement of the HN protein in heat resistance. The majority of alterations were observed in the L protein of the virus and it is proposed that these alterations were responsible for the heat-tolerant phenotype of the 1-2 NDV vaccine. (c) 2005 Elsevier B.V. All rights reserved.