Adeno-associated virus (AAV) site-specific integration: Formation of AAV–AAVS1 junctions in an in vitro system

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.

Selection and nuclear immobilization of exportable RNAs

The intracellular distribution of RNAs depends on interactions of cis-acting nuclear export elements or nuclear retention elements with trans-acting nuclear transport or retention factors. To learn about the relationship between export and retention, we isolated RNAs that are exported from nuclei of Xenopus laevis oocytes even when most RNA export is blocked by an inhibitor of Ran-dependent nucleocytoplasmic transport, the Matrix protein of vesicular stomatitis virus. Export of the selected RNAs is saturable and specific. When present in chimeric RNAs, the selected sequences acted like nuclear export elements in promoting efficient export of RNAs that otherwise are not exported; the pathway used for export of these chimeric RNAs is that used for the selected RNAs alone. However, these chimeric RNAs, unlike the selected RNAs, were not exported in the presence of Matrix protein; thus, the nonselected sequences can cause retention of the selected RNA sequences under conditions of impaired nucleocytoplasmic transport. We propose that most RNAs are transiently immobilized in the nucleus and that release of these RNAs is an essential and early step in export. Release correlates with functional Ran-dependent transport, and the lack of export of chimeric RNAs may result from interference with the Ran system.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

Nuclear structure in normal and Bloom syndrome cells

Bloom syndrome (BS) is a rare cancer-predisposing disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. BLM, the protein altered in BS, is a RecQ DNA helicase. This report shows that BLM is found in the nucleus of normal human cells in the nuclear domain 10 or promyelocytic leukemia nuclear bodies. These structures are punctate depots of proteins disrupted upon viral infection and in certain human malignancies. BLM is found primarily in nuclear domain 10 except during S phase when it colocalizes with the Werner syndrome gene product, WRN, in the nucleolus. BLM colocalizes with a select subset of telomeres in normal cells and with large telomeric clusters seen in simian virus 40-transformed normal fibroblasts. During S phase, BS cells expel micronuclei containing sites of DNA synthesis. BLM is likely to be part of a DNA surveillance mechanism operating during S phase.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals

Various proteins with different biological activities have been observed to be translocated from the nucleus to the cytoplasm in an energy- and signal-dependent manner in eukaryotic cells. This nuclear export is directed by nuclear export signals (NESs), typically characterized by hydrophobic, primarily leucine, amino acid residues. Moreover, it has been shown that CRM1/exportin 1 is an export receptor for leucine-rich NESs. However, additional NES-interacting proteins have been described. In particular, eukaryotic initiation factor 5A (eIF-5A) has been shown to be a critical cellular cofactor for the nuclear export of the HIV type 1 (HIV-1) Rev trans-activator protein. In this study we compared the nuclear export activity of NESs of different origin. Microinjection of export substrates into the nucleus of somatic cells in combination with specific inhibitors indicated that specific nuclear export pathways exist for different NES-containing proteins. In particular, inhibition of eIF-5A blocked the nuclear export of NESs derived from the HIV-1 Rev and human T cell leukemia virus type I Rex trans-activators, whereas nucleocytoplasmic translocation of the protein kinase inhibitor-NES was unaffected. In contrast, however, inhibition of CRM1/exportin 1 blocked the nuclear export of all NES-containing proteins investigated. Our data confirm that CRM1/exportin 1 is a general export receptor for leucine-rich NESs and suggest that eIF-5A acts either upstream of CRM1/exportin 1 or forms a complex with the NES and CRM1/exportin 1 in the nucleocytoplasmic translocation of the HIV-1 Rev and human T cell leukemia virus type I Rex RNA export factors.

Inhibition of proteasomal degradation by the Gly-Ala repeat of Epstein–Barr virus is influenced by the length of the repeat and the strength of the degradation signal

The Gly-Ala repeat (GAr) of the Epstein–Barr virus nuclear antigen-1 is a transferable element that inhibits in cis ubiquitin/proteasome-dependent proteolysis. We have investigated this inhibitory activity by using green fluorescent protein-based reporters that have been targeted for proteolysis by N end rule or ubiquitin-fusion degradation signals, resulting in various degrees of destabilization. Degradation of the green fluorescent protein substrates was inhibited on insertion of a 25-aa GAr, but strongly destabilized reporters were protected only partially. Protection could be enhanced by increasing the length of the repeat. However, reporters containing the Ub-R and ubiquitin-fusion degradation signals were degraded even in the presence of a 239-aa GAr. In accordance, insertion of a powerful degradation signal relieved the blockade of proteasomal degradation in Epstein–Barr virus nuclear antigen-1. Our findings suggest that the turnover of natural substrates may be finely tuned by GAr-like sequences that counteract targeting signals for proteasomal destruction.

Nuclear localization of prostaglandin E2 receptors

Prostaglandin E2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP1 fused to green fluorescent protein. High-resolution immunostaining of EP1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication

Viruses with RNA genomes often capture and redirect host cell components to assist in mechanisms particular to RNA-dependent RNA synthesis. The nidoviruses are an order of positive-stranded RNA viruses, comprising coronaviruses and arteriviruses, that employ a unique strategy of discontinuous transcription, producing a series of subgenomic mRNAs linking a 5′ leader to distal portions of the genome. For the prototype coronavirus mouse hepatitis virus (MHV), heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has been shown to be able to bind in vitro to the negative strand of the intergenic sequence, a cis-acting element found in the leader RNA and preceding each downstream ORF in the genome. hnRNP A1 thus has been proposed as a host factor in MHV transcription. To test this hypothesis genetically, we initially constructed MHV mutants with a very high-affinity hnRNP A1 binding site inserted in place of, or adjacent to, an intergenic sequence in the MHV genome. This inserted hnRNP A1 binding site was not able to functionally replace, or enhance transcription from, the intergenic sequence. This finding led us to test more directly the role of hnRNP A1 by analysis of MHV replication and RNA synthesis in a murine cell line that does not express this protein. The cellular absence of hnRNP A1 had no detectable effect on the production of infectious virus, the synthesis of genomic RNA, or the quantity or quality of subgenomic mRNAs. These results strongly suggest that hnRNP A1 is not a required host factor for MHV discontinuous transcription or genome replication.

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes

The influenza A virus pandemic of 1918–1919 resulted in an estimated 20–40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Effect of rabies virus infection on gene expression in mouse brain

A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6–7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.

Posttranscriptional Gene Silencing in Transgenic Sugarcane. Dissection of Homology-Dependent Virus Resistance in a Monocot That Has a Complex Polyploid Genome1

RNA-mediated, posttranscriptional gene silencing has been determined as the molecular mechanism underlying transgenic virus resistance in many plant virus-dicot host plant systems. In this paper we show that transgenic virus resistance in sugarcane (Saccharum spp. hybrid) is based on posttranscriptional gene silencing. The resistance is derived from an untranslatable form of the sorghum mosaic potyvirus strain SCH coat protein (CP) gene. Transgenic sugarcane plants challenged with sorghum mosaic potyvirus strain SCH had phenotypes that ranged from fully susceptible to completely resistant, and a recovery phenotype was also observed. Clones derived from the same transformation event or obtained after vegetative propagation could display different levels of virus resistance, suggesting the involvement of a quantitative component in the resistance response. Most resistant plants displayed low or undetectable steady-state CP transgene mRNA levels, although nuclear transcription rates were high. Increased DNA methylation was observed in the transcribed region of the CP transgenes in most of these plants. Collectively, these characteristics indicate that an RNA-mediated, homology-dependent mechanism is at the base of the virus resistance. This work extends posttranscriptional gene silencing and homology-dependent virus resistance, so far observed only in dicots, to an agronomically important, polyploid monocot.

Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes

The genetic basis for virulence in influenza virus is largely unknown. To explore the mutational basis for increased virulence in the lung, the H3N2 prototype clinical isolate, A/HK/1/68, was adapted to the mouse. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose. Thirteen of the 14 amino acid substitutions (93%) detected among clonal isolates were likely instrumental in adaptation because of their positive selection, location in functional regions, and/or independent occurrence in other virulent influenza viruses. Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Experimental adaptation resulted in the selection of three mutations that were in common with the virulent human H5N1 isolate A/HK/156/97 and that may be instrumental in its extreme virulence. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

Multiple vesiculoviral matrix proteins inhibit both nuclear export and import

The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex.

Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.