bluuebunny/crossref_metadata_2025 huggingface dataset:
Secondary Structure Analysis of a Minimal Avian Leukosis-Sarcoma Virus Packaging Signal
Authors: Banks Jennifer D. , Linial Maxine L.
Published: April 2009

We previously identified a 160-nucleotide packaging signal, MΨ, from the 5′ end of the
Rous sarcoma virus
genome. In this study, we determine the secondary structure of MΨ by using phylogenetic analysis with computer modeling and heterologous packaging assays of point mutants. The results of the in vivo studies are in good agreement with the computer model. Additionally, the packaging studies indicate several structures which are important for efficient packaging, including a single-stranded bulge containing the initiation codon for the short open reading frame, uORF3, as well as adjacent stem structures. Finally, we show that the L3 stem-loop at the 3′ end of MΨ is dispensable for packaging, thus identifying an 82-nucleotide minimal packaging signal, μΨ, composed of the O3 stem-loop.
Brain Infection by Neuroinvasive but Avirulent Murine Oncornaviruses
Authors: Ašković Srdjan , McAtee Frank J. , Favara Cynthia , Portis John L.
Published: April 2009

The chimeric murine oncornavirus FrCas
E
causes a rapidly progressive noninflammatory spongiform encephalomyelopathy after neonatal inoculation. The virus was constructed by the introduction of
pol-env
sequences from the wild mouse virus CasBrE into the genome of a neuroinvasive but nonneurovirulent strain of Friend murine leukemia virus (FMuLV), FB29. Although the brain infection by FrCas
E
as well as that by other neurovirulent murine retroviruses has been described in detail, little attention has been paid to the neuroinvasive but nonneurovirulent viruses. The purpose of the present study was to compare brain infection by FrCas
E
with that by FB29 and another nonneurovirulent virus, F43, which contains
pol-env
sequences from FMuLV 57. Both FB29 and F43 infected the same spectrum of cell types in the brain as that infected by FrCas
E
, including endothelial cells, microglia, and populations of neurons which divide postnatally. Viral burdens achieved by the two nonneurovirulent viruses in the brain were actually higher than that of FrCas
E
. The widespread infection of microglia by the two nonneurovirulent viruses is notable because it is infection of these cells by FrCas
E
which is thought to be a critical determinant of its neuropathogenicity. These results indicate that although the sequence of the envelope gene determines neurovirulence, this effect appears to operate through a mechanism which does not influence either viral tropism or viral burden in the brain. Although all three viruses exhibited similar tropism for granule neurons in the cerebellar cortex, there was a striking difference in the distribution of envelope proteins in those cells in vivo. The FrCas
E
envelope protein accumulated in terminal axons, whereas those of FB29 and F43 remained predominantly in the cell bodies. These observations suggest that differences in the intracellular sorting of these proteins may exist and that these differences appear to correlate with neurovirulence.
The UL25 Protein of Pseudorabies Virus Associates with Capsids and Localizes to the Nucleus and to Microtubules
Authors: Kaelin Karin , Dezélée Sybille , Masse Marie Jo , Bras Françoise , Flamand Anne
Published: April 2009

The UL25 gene of pseudorabies virus (PrV) can encode a protein of about 57 kDa which is well conserved among herpesviruses. The UL25 protein of herpes simplex virus type 1 is a capsid constituent involved in virus penetration and capsid maturation. To identify and characterize the UL25 gene product of PrV, polyclonal mouse anti-UL25 antibodies were raised to a bacterially expressed fusion protein. In immunoblotting and immunoprecipitation assays of PrV-infected cell lysates, these anti-UL25 antisera specifically recognized a protein of the expected size with late expression kinetics. This 57-kDa product was also present in purified virions and was found to be associated with all types of capsids. Synthesis of a protein migrating at the same size point was directed from the eukaryotic expression plasmid pCG-UL25. To determine the subcellular localization of UL25, immunofluorescence studies with anti-UL25 antisera were performed on Nonidet P-40-extracted COS-7 cells infected with PrV or transfected with pCG-UL25. In PrV-infected cells, newly synthesized UL25 is directed mainly to distinct nuclear compartments, whereas UL25 expressed in the absence of other viral proteins is distributed more uniformly in the nucleus and colocalizes also with microtubules. To study the fate of UL25 at very early stages of infection, immunofluorescence experiments were performed on invading PrV particles in the presence or absence of drugs that specifically depolymerize components of the cytoskeleton. We found that the incoming nucleocapsids colocalize with microtubules during their transport to the nucleus and that UL25 remains associated with nucleocapsids during this transport.
cisExpression of the F12 Human Immunodeficiency Virus (HIV) Nef Allele Transforms the Highly Productive NL4-3 HIV Type 1 to a Replication-Defective Strain: Involvement of both Env gp41 and CD4 Intracytoplasmic Tails
Authors: Olivetta Eleonora , Pugliese Katherina , Bona Roberta , D'Aloja Paola , Ferrantelli Flavia , Santarcangelo Anna Claudia , Mattia Gianfranco , Verani Paola , Federico Maurizio
Published: April 2009

F12 human immunodeficiency virus type 1 (HIV-1)nefis a naturally occurringnefmutant cloned from the provirus of a nonproductive, nondefective, and interfering HIV-1 variant (F12-HIV). We have already shown that cells stably transfected with a vector expressing the F12-HIVnefallele do not downregulate CD4 receptors and, more peculiarly, become resistant to the replication of wild type (wt) HIV. In order to investigate the mechanism of action of such an HIV inhibition, the F12-HIVnefgene was expressed in the context of the NL4-3 HIV-1 infectious molecular clone by replacing the wtnefgene (NL4-3/chi). Through this experimental approach we established the following. First, NL4-3/chi andnef-defective (Δnef) NL4-3 viral particles behave very similarly in terms of viral entry and HIV protein production during the first replicative cycle. Second, no viral particles were produced from cells infected with NL4-3/chi virions, whatever the multiplicity of infection used. The viral inhibition apparently occurs at level of viral assembling and/or release. Third, this block could not be relieved by in-transexpression of wtnef. Finally, NL4-3/chi reverts to a producer HIV strain when F12-HIV Nef is deprived of its myristoyl residue. Through a CD4 downregulation competition assay, we demonstrated that F12-HIV Nef protein potently inhibits the CD4 downregulation induced by wt Nef. Moreover, we observed a redistribution of CD4 receptors at the cell margin induced by F12-HIV Nef. These observations strongly suggest that F12-HIV Nef maintains the ability to interact with the intracytoplasmic tail of the CD4 receptor molecule. Remarkably, we distinguished the intracytoplasmic tails of Env gp41 and CD4 as, respectively, viral and cellular targets of the F12-HIV Nef-induced viral retention. For the first time, the inhibition of the viral life cycle by means of in-cisexpression of a Nef mutant is here reported. Delineation of the F12-HIV Nef mechanism of action may offer additional approaches to interference with the propagation of HIV infection.
Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus
Authors: Wilson Carolyn A. , Wong Susan , VanBrocklin Matthew , Federspiel Mark J.
Published: April 2009

We previously reported that mitogenic activation of porcine peripheral blood mononuclear cells resulted in production of porcine endogenous retrovirus(es) (PERV[s]) capable of productively infecting human cells (C. Wilson et al., J. Virol. 72:3082–3087, 1998). We now extend that analysis to show that additional passage of isolated virus, named here PERV-NIH, through a human cell line yielded a viral population with a higher titer of infectious virus on human cells than the initial isolate. We show that in a single additional passage on a human cell line, the increase in infectivity for human cells is accounted for by selection against variants carrying pig-tropic envelope sequences (PERV-C) as well as by enrichment for replication-competent genomes. Sequence analysis of the envelope cDNA present in virions demonstrated that the envelope sequence of PERV-NIH is related to but distinct from previously reported PERV envelopes. The in vitro host range of PERV was studied in human primary cells and cell lines, as well as in cell lines from nonhuman primate and other species. This analysis reveals three patterns of susceptibility to infection among these host cells: (i) cells are resistant to infection in our assay; (ii) cells are infected by virus, as viral RNA is detected in the supernatant by reverse transcription-PCR, but the cells are not permissive to productive replication and spread; and (iii) cells are permissive to low-level productive replication. Certain cell lines were permissive for efficient productive infection and spread. These results may prove useful in designing appropriate animal models to assess the in vivo infectivity properties of PERV.
Structural Fingerprinting: Subgrouping of Comoviruses by Structural Studies of Red Clover Mottle Virus to 2.4-Å Resolution and Comparisons with Other Comoviruses
Authors: Lin Tianwei , Clark Anthony J. , Chen Zhongguo , Shanks Michael , Dai Jin-Bi , Li Ying , Schmidt Tim , Oxelfelt Per , Lomonossoff George P. , Johnson John E.
Published: April 2009

Red clover mottle virus
(RCMV) is a member of the comoviruses, a group of picornavirus-like plant viruses. The X-ray structure of RCMV strain S has been determined and refined to 2.4 Å. The overall structure of RCMV is similar to that of two other comoviruses,
Cowpea mosaic virus
(CPMV) and
Bean pod mottle virus
(BPMV). The sequence of the coat proteins of RCMV strain O were modeled into the capsid structure of strain S without causing any distortion, confirming the close resemblance between the two strains. By comparing the RCMV structure with that of other comoviruses, a structural fingerprint at the N terminus of the small subunit was identified which allowed subgrouping of comoviruses into CPMV-like and BPMV-like viruses.
Human Cytomegalovirus Infects Caco-2 Intestinal Epithelial Cells Basolaterally Regardless of the Differentiation State
Authors: Esclatine Audrey , Lemullois Michel , Servin Alain L. , Quero Anne-Marie , Geniteau-Legendre Monique
Published: April 2009

Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.
Avian Reticuloendotheliosis Virus Strain A and Spleen Necrosis Virus Do Not Infect Human Cells
Authors: Gautier R. , Jiang A. , Rousseau V. , Dornburg R. , Jaffredo T.
Published: April 2009

Spleen necrosis virus
(SNV) and
Reticuloendotheliosis virus
strain A (REV-A) belong to the family of reticuloendotheliosis viruses and are 90% sequence related. SNV-derived retroviral vectors produced by the REV-A-based D17.2G packaging cell line were shown to infect human cells (H.-M. Koo, A. M. C. Brown, Y. Ron, and J. P. Dougherty, J. Virol. 65:4769–4776, 1991), while similar vectors produced by another SNV-based packaging cell line, DSH134G, are not infectious in human cells (reviewed by R. Dornburg, Gene Ther. 2:301–310, 1995). Here we describe a careful reevaluation of the infectivity of vectors produced from the most commonly used REV-A- or SNV-based packaging cells obtained from various sources with, among them, one batch of D17.2G packaging cells obtained from the American Type Culture Collection. None of these packaging cells produced vectors able to infect human cells. Thus, contrary to previously published data, we conclude that REV-based vectors are not infectious in human cells.
Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo
Authors: Walters Robert W. , Duan Dongsheng , Engelhardt John F. , Welsh Michael J.
Published: April 2009

Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaP
i
coprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo.
A Heterologous, High-Affinity RNA Ligand for Human Immunodeficiency Virus Gag Protein Has RNA Packaging Activity
Authors: Clever Jared L. , Taplitz Randy A. , Lochrie Michael A. , Polisky Barry , Parslow Tristram G.
Published: April 2009

Retroviral RNA encapsidation depends on the specific binding of Gag proteins to packaging (ψ) signals in genomic RNA. We investigated whether an in vitro-selected, high-affinity RNA ligand for the nucleocapsid (NC) portion of the Gag protein from human immunodeficiency virus type 1 (HIV-1) could mediate packaging into HIV-1 virions. We find that this ligand can functionally substitute for one of the Gag-binding elements (termed SL3) in the HIV-1 ψ locus to support packaging and viral infectivity in
cis
. By contrast, this ligand, which fails to dimerize spontaneously in vitro, is unable to replace a different ψ element (termed SL1) which is required for both Gag binding and dimerization of the HIV-1 genome. A single point mutation within the ligand that eliminates high-affinity in vitro Gag binding also abolishes its packaging activity at the SL3 position. These results demonstrate that specific binding of Gag or NC protein is a critical determinant of genomic RNA packaging.
MxA GTPase Blocks Reporter Gene Expression of Reconstituted Thogoto Virus Ribonucleoprotein Complexes
Authors: Weber Friedemann , Haller Otto , Kochs Georg
Published: April 2009

Human MxA protein accumulates in the cytoplasm of interferon-treated cells and inhibits the multiplication of several RNA viruses, including
Thogoto virus
(THOV), a tick-borne orthomyxovirus that transcribes and replicates its genome in the cell nucleus. The antiviral mechanism of MxA was investigated by using two alternative minireplicon systems in which recombinant viral ribonucleoprotein complexes (vRNPs) of THOV were reconstituted from cloned cDNAs. A chloramphenicol acetyltransferase reporter minigenome RNA was expressed either by T7 RNA polymerase in the cytoplasm of transfected cells or, alternatively, by RNA polymerase I in the nucleus. The inhibitory effect of MxA was studied in both cellular compartments by coexpressing wild-type MxA or TMxA, an artificial nuclear form of MxA. Our results indicate that both MxA proteins recognize the assembled vRNP rather than the newly synthesized unassembled components. The present findings are consistent with previous data which indicated that cytoplasmic MxA prevents transport of vRNPs into the nucleus, whereas nuclear MxA directly inhibits the viral polymerase activity in the nucleus.
Identifying the Target Cell in Primary Simian Immunodeficiency Virus (SIV) Infection: Highly Activated Memory CD4+T Cells Are Rapidly Eliminated in Early SIV Infection In Vivo
Authors: Veazey Ronald S. , Tham Irene C. , Mansfield Keith G. , DeMaria MaryAnn , Forand Amy E. , Shvetz Daniel E. , Chalifoux Laura V. , Sehgal Prabhat K. , Lackner Andrew A.
Published: April 2009

It has recently been shown that rapid and profound CD4+T-cell depletion occurs almost exclusively within the intestinal tract of simian immunodeficiency virus (SIV)-infected macaques within days of infection. Here we demonstrate (by three- and four-color flow cytometry) that this depletion is specific to a definable subset of CD4+T cells, namely, those having both a highly and/or acutely activated (CD69+CD38+HLA-DR+) and memory (CD45RA−Leu8−) phenotype. Moreover, we demonstrate that this subset of helper T cells is found primarily within the intestinal lamina propria. Viral tropism for this particular cell type (which has been previously suggested by various studies in vitro) could explain why profound CD4+T-cell depletion occurs in the intestine and not in peripheral lymphoid tissues in early SIV infection. Furthermore, we demonstrate that an acute loss of this specific subset of activated memory CD4+T cells may also be detected in peripheral blood and lymph nodes in early SIV infection. However, since this particular cell type is present in such small numbers in circulation, its loss does not significantly affect total CD4+T cell counts. This finding suggests that SIV and, presumably, human immunodeficiency virus specifically infect, replicate in, and eliminate definable subsets of CD4+T cells in vivo.
The
S2
Gene of Equine Infectious Anemia Virus Is a Highly Conserved Determinant of Viral Replication and Virulence Properties in Experimentally Infected Ponies
Authors: Li Feng , Leroux Caroline , Craigo Jodi K. , Cook Sheila J. , Issel Charles J. , Montelaro Ronald C.
Published: April 2009

Equine infectious anemia virus (EIAV) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes,
tat
,
rev
, and
S2
. Although serological analyses demonstrate the expression of the S2 protein in persistently infected horses, the role of this viral gene remains undefined. We recently reported that the
S2
gene is not essential for EIAV replication in primary equine macrophages, as EIAV mutants lacking the
S2
gene replicate to levels similar to those of the parental virus (F. Li, B. A. Puffer, and R. C. Montelaro, J. Virol. 72:8344–8348, 1998). We now describe in vivo studies that examine the evolution and role of the
S2
gene in ponies experimentally infected with EIAV. The results of these studies reveal for the first time that the
S2
gene is highly conserved during persistent infection and that deletion of the
S2
gene reduces viral virulence and virus replication levels compared to those of the parental virus containing a functional
S2
gene. These data indicate that the EIAV
S2
gene is in fact an important determinant of viral replication and pathogenic properties in vivo, despite the evident lack of
S2
influence on viral replication levels in vitro. Thus, these observations suggest in vivo functions of EIAV
S2
that are not adequately reflected in simple infections of cultured cells, including natural target macrophages.
Species Specificity of Macaque Rhadinovirus Glycoprotein B Sequences
Authors: Auerbach Marcy R. , Czajak Susan C. , Johnson Welkin E. , Desrosiers Ronald C. , Alexander Louis
Published: April 2009

All members of the
Herpesviridae
family contain sequences for a highly conserved glycoprotein B (gB) gene. We investigated the phylogenetic relationships of gB sequences from eight independent rhadinovirus isolates obtained from three species: rhesus (
Macaca mulatta
), cynomologus (
Macaca fasicularis
), and pig-tailed (
Macaca nemestrina
) macaques. Samples were derived from monkeys housed at four separate facilities. Analysis of these eight independent gB sequences revealed five regions of heterogeneity within the 823- to 829-amino-acid polypeptides: residues 1 to 65, 120 to 185, 255 to 300, 352 to 393, and 412 to 457. The remaining regions of gB were highly conserved among the different macaque isolates. Overall divergence among these gene sequences ranged from 0.1 to 7.2% at the amino acid level. Phylogenetic trees constructed with our macaque rhadinovirus gB sequences and those derived from additional subfamilies or genera (alpha, beta, gamma-1, and gamma-2) revealed that the macaque gB sequences branched with other gamma-2 herpesvirus gB sequences and that within the gamma-2 genera, the macaque gB sequences clustered as a distinct branch. The eight macaque rhadinovirus gB sequences were all approximately equidistant from Kaposi sarcoma-associated herpesvirus (KSHV) gB sequences and had a shorter evolutionary distance to KSHV gB sequences than to any other herpesvirus, including the gamma-2 herpesvirus saimiri (HVS) of New World squirrel monkeys. The macaque gB sequences did not cluster according to the facility of origin, but did cluster according to the species of origin, displaying less intraspecies divergence (0.1 to 2.9%) than interspecies divergence (3.3 to 7.2%). These results demonstrate a close relatedness of rhadinovirus isolates from different macaque species.
Functional Interactions between C/EBP, Sp1, and COUP-TF Regulate Human Immunodeficiency Virus Type 1 Gene Transcription in Human Brain Cells
Authors: Schwartz Christian , Catez Philippe , Rohr Olivier , Lecestre Dominique , Aunis Dominique , Schaeffer Evelyne
Published: April 2009

Human immunodeficiency virus type 1 (HIV-1) infects the central nervous system (CNS) and plays a direct role in the pathogenesis of AIDS dementia. However, mechanisms underlying HIV-1 gene expression in the CNS are poorly understood. The importance of CCAAT/enhancer binding proteins (C/EBP) for HIV-1 expression in cells of the immune system has been recently reported. In this study, we have examined the role and the molecular mechanisms by which proteins of the C/EBP family regulate HIV-1 gene transcription in human brain cells. We found that NF-IL6 acts as a potent activator of the long terminal repeat (LTR)-driven transcription in microglial and oligodendroglioma cells. In contrast, C/EBPγ inhibits NF-IL6-induced activation. Consistent with previous data, our transient expression results show cell-type-specific NF-IL6-mediated transactivation. In glial cells, full activation needs the presence of the C/EBP binding sites; however, NF-IL6 is still able to function via the minimal −40/+80 region. In microglial cells, C/EBP sites are not essential, since NF-IL6 acts through the −68/+80 LTR region, containing two binding sites for the transcription factor Sp1. Moreover, we show that functional interactions between NF-IL6 and Sp1 lead to synergistic transcriptional activation of the LTR in oligodendroglioma and to mutual repression in microglial cells. We further demonstrate that NF-IL6 physically interacts with the nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF), via its DNA binding domain, in vitro and in cells, which results in mutual transcriptional repression. These findings reveal how the interplay of NF-IL6 and C/EBPγ, together with Sp1 and COUP-TF, regulates HIV-1 gene transcription in brain cells.
Respiratory Syncytial Virus That Lacks Open Reading Frame 2 of the M2 Gene (M2-2) Has Altered Growth Characteristics and Is Attenuated in Rodents
Authors: Jin Hong , Cheng Xing , Zhou Helen Z. Y. , Li Shengqiang , Seddiqui Adam
Published: April 2009

The M2 gene of respiratory syncytial virus (RSV) encodes two putative proteins: M2-1 and M2-2; both are believed to be involved in the RNA transcription or replication process. To understand the function of the M2-2 protein in virus replication, we deleted the majority of the M2-2 open reading frame from an infectious cDNA clone derived from the human RSV A2 strain. Transfection of HEp-2 cells with the cDNA clone containing the M2-2 deletion, together with plasmids that encoded the RSV N, P, and L proteins, produced a recombinant RSV that lacked the M2-2 protein (rA2ΔM2-2). Recombinant virus rA2ΔM2-2 was recovered and characterized. The levels of viral mRNA expression for 10 RSV genes examined were unchanged in cells infected with rA2ΔM2-2, except that a shorter M2 mRNA was detected. However, the ratio of viral genomic or antigenomic RNA to mRNA was reduced in rA2ΔM2-2-infected cells. By use of an antibody directed against the bacterially expressed M2-2 protein, the putative M2-2 protein was detected in cells infected with wild-type RSV but not in cells infected with rA2ΔM2-2. rA2ΔM2-2 displayed a small-plaque morphology and grew much more slowly than wild-type RSV in HEp-2 cells. In infected Vero cells, rA2ΔM2-2 exhibited very large syncytium formation compared to that of wild-type recombinant RSV. rA2ΔM2-2 appeared to be a host range mutant, since it replicated poorly in HEp-2, HeLa, and MRC5 cells but replicated efficiently in Vero and LLC-MK2 cells. Replication of rA2ΔM2-2 in the upper and lower respiratory tracts of mice and cotton rats was highly restricted. Despite its attenuated replication in rodents, rA2ΔM2-2 was able to provide protection against challenge with wild-type RSV A2. The genotype and phenotype of the M2-2 deletion mutant were stably maintained after extensive in vitro passages. The attenuated phenotype of rA2ΔM2-2 suggested that rA2ΔM2-2 may be a potential candidate for use as a live attenuated vaccine.
The Disappearance of Cyclins A and B and the Increase in Activity of the G
2
/M-Phase Cellular Kinase cdc2 in Herpes Simplex Virus 1-Infected Cells Require Expression of the α22/U
S
1.5 and U
L
13 Viral Genes
Authors: Advani S. J. , Brandimarti R. , Weichselbaum R. R. , Roizman B.
Published: April 2009

In uninfected cells the G
2
/M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U
L
13 and α22/U
S
1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.
Different Regions of Hepatitis B Virus X Protein Are Required for Enhancement of bZip-Mediated Transactivation versus Transrepression
Authors: Barnabas Sangeeta , Andrisani Ourania M.
Published: April 2009

The hepatitis B virus X protein (pX) interacts directly with the bZip transactivator CREB and the bZip repressors ICERIIγ and ATF3, increasing their DNA-binding affinity in vitro and their transcriptional efficacy in vivo. However, the mechanism of bZip-pX interaction and of the pX-mediated increase in the bZip transcriptional efficacy remains to be understood. In this study with deletion mutants of pX, we delineated a 67-amino-acid region spanning residues 49 to 115 required for direct CREB, ATF3, and ICER IIγ interaction in vitro and in vivo and increased bZip/CRE binding in vitro. Transient transfections of the pX deletion mutants in AML12 hepatocytes demonstrate that pX
49–115
is as effective as the full-length pX in enhancing the ATF3- and ICERIIγ-mediated transrepression. However, this pX region is inactive in increasing the transactivation efficacy of CREB; additional amino acid residues present in pX
49–140
are required to mediate the increased transactivation efficacy of CREB in vivo. This requirement for different regions of pX in affecting CREB transactivation suggests that amino acid residues 115 to 140 integrate additional events in effecting pX-mediated transactivation, such as concomitant interactions with select components of the basal transcriptional apparatus.
The Phosphoprotein of Rabies Virus Is Phosphorylated by a Unique Cellular Protein Kinase and Specific Isomers of Protein Kinase C
Authors: Gupta Ashim K. , Blondel Danielle , Choudhary Suresh , Banerjee Amiya K.
Published: April 2009

The phosphoprotein (P) gene of rabies virus (CVS strain) was cloned and expressed in bacteria. The purified protein was used as the substrate for phosphorylation by the protein kinase(s) present in cell extract prepared from rat brain. Two distinct types of protein kinases, staurosporin sensitive and heparin sensitive, were found to phosphorylate the P protein in vitro by the cell extract. Interestingly, the heparin-sensitive kinase was not the ubiquitous casein kinase II present in a variety of cell types. Further purification of the cell fractions revealed that the protein kinase C (PKC) isomers constitute the staurosporin-sensitive kinases α, β, γ, and ζ, with the PKCγ isomer being the most effective in phosphorylating the P protein. A unique heparin-sensitive kinase was characterized as a 71-kDa protein with biochemical properties not demonstrated by any known protein kinases stored in the protein data bank. This protein kinase, designated RVPK (rabies virus protein kinase), phosphorylates P protein (36 kDa) and alters its mobility in gel to migrate at 40 kDa. In contrast, the PKC isoforms do not change the mobility of unphosphorylated P protein. RVPK appears to be packaged in the purified virions, to display biochemical characteristics similar to those of the cell-purified RVPK, and to similarly alter the mobility of endogenous P protein upon phosphorylation. By site-directed mutagenesis, the sites of phosphorylation of RVPK were mapped at S
63
and S
64
, whereas PKC isomers phosphorylated at S
162
, S
210
, and S
271
. Involvement of a unique protein kinase in phosphorylating rabies virus P protein indicates its important role in the structure and function of the protein and consequently in the life cycle of the virus.
Alteration of Hepatitis A Virus (HAV) Particles by a Soluble Form of HAV Cellular Receptor 1 Containing the Immunoglobulin- and Mucin-Like Regions
Authors: Silberstein Erica , Xing Li , van de Beek Willem , Lu Jinhua , Cheng Holland , Kaplan Gerardo G.
Published: April 2009

Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1). The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobulin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobulin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100- to 125S particles. Negative-stain electron microscopy analysis revealed that the 100- to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100- to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.
Marine tube-dwelling diatoms of the Pacific coast of North America. II. Navicula subg. Navicula and a key to the tube-dwelling diatoms of the region
Authors: Lobban Christopher S.
Published: April 2009

The tube-dwelling cross-lineate Navicula species on the west coast are dominated by a taxon that belongs in or near the N. ramosissima – N. mollis complex. Morphometric data were used to establish that a single, variable population exists. The species was assigned to N. rusticensis. Cells are 28 ± 7 × 5 ± 1 μm and striae number 14 ± 2 in 10 μm; these cells differ from the ramosissima–mollis complex in having 1–3 short central striae that form a narrow rectangular area. The range of size encompasses published limits for both N. ramosissima and N. mollis. A key to tube-dwelling diatoms of the west coast completes this two-part checklist. Key words: diatoms, Pacific, tube-dwelling, Navicula rusticensis, Navicula ramosissima.
Regulation of the fusion of floral organs by temperature and gibberellic acid in the normal and solanifolia mutant of tomato (Lycopersicon esculentum)
Authors: Sekhar K. N. Chandra , Sawhney V. K.
Published: April 2009

The flowers of a single gene homozygous recessive solanifolia (sf/sf) mutant of tomato (Lycopersicon esculentum) possess separate sepals, petals and stamens, and a gynoecium that consists of several carpels with separate styles. In contrast, in the normal tomato (cv. Pearson), floral organs of each whorl are either partially or completely fused. Different temperature conditions and gibberellic acid treatments had no effect on the ontogenetic fusion of sepals and petals of the mutant and normal flowers. However, low temperatures and gibberellic acid induced the separation of stamens and pistil in many of the normal flowers, and they enhanced the separation of carpels in mutant flowers. In contrast, high temperatures and 2-chlo-roethyltrimethyl ammonium chloride, an inhibitor of gibberellin biosynthesis, promoted the fusion of stamens and gynoecia of mutant flowers, but had no apparent effect on the normal flowers. The nonfusion of stamens and carpels, in both genotypes, was associated with an increase in the number of these organs, whereas their fusion was accompanied with a reduction in their number. It is proposed that the nonfusion of floral organs in the sf/sf mutant is partly related to changes in endogenous growth substances which, through an effect on the size of the floral apex, affect the number and fusion of stamens and carpels.
An architectural analysis of Shepherdia canadensis and Shepherdia argentea (Elaeagnaceae): the architectural models
Authors: Hayes P. A. , Steeves T. A. , Neal B. R.
Published: April 2009

The growth patterns of Shepherdia canadensis and S. argentea from seedling stage to adult thicket have been analyzed and compared. In S. canadensis, the seedling axis, originally erect, showed a tendency for early bending towards the horizontal, followed by vertical reorientation of the tip. This was coupled with the basitonic expansion of lateral buds. In contrast, the seedling axis of S. argentea remained erect and branching was predominately acrotonic. As plants of S. canadensis matured, successive branches repeated the seedling pattern, reorienting downward with the tip compensating to remain erect. Basal buds expanded to fill the gap in the canopy and these in turn reoriented downward, repeating the cycle and forming a thicket with the youngest shoots in the centre. Rooting of the horizontal axes was observed and in some cases new thickets could be inititated in this way. In S. argentea, however, shoots remained erect and thicket formation occurred by the expansion of ramets from root buds at the periphery of the thicket. The shoots at the centre were thus the oldest. These features, together with information on branching patterns previously reported, have been used to construct constrasting architectural models for the two species. Key words: Elaeagnaceae, Shepherdia, Buffaloberry, architecture, morphology, development.
Early growth of the root system in Allium cepa
Authors: Birdsall Margaret , MacLeod Ronald D.
Published: April 2009

The development of the root system in Allium cepa was followed for periods of up to 25 days after emergence of the radicle from the seed. Apical meristem activity, in terms of mitotic index, the percentage of cells undergoing DNA synthesis, and cell doubling time, was maximal within several days of emergence in both the primary and adventitious roots, but it gradually decreased in the subsequent period of growth. These changes in proliferative activity in individual roots were paralleled by concurrent changes in elongation rate. Thus, the period of maximum cell production, as well as that of elongation, was of limited duration in each root. Throughout the period of study, the most recently emerged root displayed the highest level of proliferative activity, relative to that found in the other roots on the plant. Changes in the rate of growth of individual roots appeared to be correlated with the early growth of the succeeding root. Key words: Allium, onion, root development, meristem, primordium.
Seed dispersing agents in two regularly flooded river sites
Authors: Skoglund S. Jerry
Published: April 2009

Aspects of seed dispersal, particularly mobility of viable seeds, were investigated in two sites at the lower river Dalälven, southeastern central Sweden, from 1980 to 1985. Three different approaches were used to assess the seed rain. (i) Seed traps were placed at ground level. This component of the seed rain was dominated by seeds from the local vegetation, particularly Betula spp. A total of 30 species were identified, (ii) Drift material was collected in spring and autumn and the seed content was identified through germination trials. Seeds dispersed by drift mainly belonged to species abundant in the study sites, but included a minor element not represented in the local vegetation. A total of 23 species were identified, of which seeds of Potentilla palustris, Carex canescens, and other Carex spp. were most abundant. In the field, seedlings growing on fresh accumulations of drift were dominated by Potentilla palustris. (iii) Samples of snow were collected and the seed content determined visually. Of the seven species represented in the snow samples, Betula spp. dominated. In particular, flooding extended the seed rain beyond the boundaries of many species. It is concluded that the distribution of wet meadow species is not limited by dispersal.
Genealogy and gene flow among annual domesticated species of Cucurbita
Authors: Decker-Walters D. S. , Walters T. W. , Posluszny U. , Kevan P. G.
Published: April 2009

Over the years, workers have gathered data from various sources in the attempt to understand the evolutionary histories and relationships of the domesticated members of Cucurbita. We used allozyme data to address questions of genealogy, interspecific gene flow, and allelic diversity for 23 commercial cultivars and 17 indigenous landraces of C. maxima, C. mixta, C. moschata, and C. pepo. Allelic diversity was greatest in C. pepo (especially ssp. ovifera) and C. moschata, reflecting complex histories for these taxa. Cucurbita maxima and C. mixta were relatively homogeneous despite morphological heterogeneity. Infraspecific classifications based on morphological characters were not supported by the allozyme data for C. maxima, C. mixta, and C. moschata. The allozymes supported morphological evidence of introgression from C. mixta in Oriental cultivars and southwestern United States and northwestern Mexico landraces of C. moschata. Phylogenetic analyses indicated that C. moschata and C. mixta are probably sister species and that C. pepo shares a common ancestor with these species that is not shared with C. maxima.
Habitat relationships and survivorship of tree seedlings in hemlock-hardwood forest
Authors: Collins Scott L.
Published: April 2009

This study was designed to determine (i) if newly germinated and older seedlings of different tree species occupied different microhabitats in the understory of mature hemlock forest; (ii) if different aged seedlings of the same species occupied different microhabitats; and (iii) if initial survivorship of newly germinated seedlings is correlated with microhabitat. In June 1987, 13 environmental variables were measured around different aged individual seedlings of Acer rubrum, Acer saccharum, Betula lutea, Prunus serotina, and Tsuga canadensis. Seedlings of A. rubrum and T. canadensis occupied habitats with more coniferous litter than seedlings of A. saccharum and P. serotina. Different aged seedlings of A. rubrum generally were found in similar habitats, but these microhabitats were different from randomly located samples. Younger seedlings of T. canadensis occupied habitats with significantly shallower litter layers and more coniferous litter than older seedlings and random points. Habitat structure and survivorship of newly germinated seedlings of B. lutea and A. rubrum was monitored from June to September 1988. Initially, A. rubrum had a higher mortality rate than B. lutea. For both species, survivorship was significantly greater in habitats with shallower litter and less woody debris. These differences disappeared later in the growing season. These results imply that seedlings occur in different microhabitats, that habitat differences occur for different aged seedlings, and that microhabitat does initially affect seedling survival. Thus, studies of older seedlings and saplings do not completely incorporate the regeneration strategies of some species. In addition, the "plant's eye view" approach provides a useful method for quantifying habitat factors that affect species distributions both within and between communities. Key words: habitat structure, hemlock forest, litter depth, survivorship, tree seedlings.
Survey and ecological aspects of presumed ectomycorrhizal fungi associated with black spruce in northern Minnesota
Authors: Doudrick Robert L. , Stewart Elwin L. , Alm Alvin A.
Published: April 2009

Twenty-two stands of black spruce, Picea mariana (Mill.) B.S.P., in northern Minnesota were surveyed for ectomycorrhizal fungi. Site selection criteria in black spruce stands included geologic history, soil type, stand origin, age, and productivity. Site preference relationships were noted for several of the 46 species of fungi collected during the 2-year study. Twenty-five isolates were tested in aseptic culture for their ability to form ectomycorrhizae with black spruce seedlings. Ectomycorrhizae were produced by Cenococcum sp., Laccaria bicolor (Maire) Orton, Laccaria laccata var. moelleri Singer, Rhizopogon sp., and Suillus cavipes (Opat.) Smith & Thiers.
Range-wide morphological and anatomical variation in Larix laricina
Authors: Parker William H. , Dickinson Timothy A.
Published: April 2009

To investigate the nature of infraspecific variation in Larix laricina (Du Roi) K. Koch, 60 naturally established stands constituting 44 provenances were sampled from throughout the species' range. Cones were scored for nine traits and short-shoot needle transverse sections were scored for seven traits and 20 radial distances. Variation in all traits was minimal, with the greatest components expressed between trees within stands and within trees. Canonical variates analysis of needle traits demonstrated that provenances were differentiated according to location, whereas canonical variates analysis of cone traits showed a much weaker geographic trend. These results are consistent with the hypothesis that Alaska and northern Yukon provenances resulted from geographically separate migratory waves originating from different locations in the south following glacial recession. While Alaska provenances were distinguished from other provenances based on canonical variates analysis of needle data, but not of cone data, this distinction is insufficient to recognize Alaska tamaracks separately at either the species or varietal level.
Use of immunogold cytochemistry to detect Mn(II)-dependent and lignin peroxidases in wood degraded by the white rot fungi Phanerochaete chrysosporium and Lentinula edodes
Authors: Daniel G. , Pettersson B. , Nllsson T. , Volc J.
Published: April 2009

Using antibodies raised against Mn(II)-dependent peroxidase and transmission electron microscopy immunogold cytochemistry, the spatial distribution of Mn(II)-dependent peroxidase during degradation of wood and wood fragments by Phanerochaete chrysosporium and Lentinula edodes was studied. In P. chrysosporium, the enzyme was localized in peripheral regions of the fungal hyphae on the cell membrane, on membranes of vesicule-like structures, and on the cell wall. The cytoplasmic distribution of Mn(II)-dependent peroxidase appeared similar to that of lignin peroxidase, as determined by double immunogold labeling procedures and antibodies raised against lignin peroxidase. In wood blocks of Betula verrucosa degraded by P. chrysosporium and L. edodes, Mn(II)-dependent peroxidase was detected on all wood cell wall layers showing signs of decay, whether at early or advanced stages of attack. In particular, the enzyme was localized in zones of degradation produced within the S2 wood cell walls. These regions displayed a looser, more open fibrillar structure than unattacked wood cell walls and were readily penetrated by purified preparations of Mn(II)-dependent and lignin peroxidases. With L. edodes, Mn(II)-dependent peroxidase was found to accumulate in middle lamellar regions selectively degraded by the fungus. Mn(II)-dependent peroxidase diffusion into undecayed wood cell walls was not observed. Key words: Mn(II)-dependent peroxidase, Phanerochaete chrysosporium, Lentinula edodes, immunogold cytochemistry, white rot decay, transmission electron microscopy.
Effect of intercellular washing fluids on the interactions between bean plants and fungi nonpathogenic on beans
Authors: Li Aimin , Heath Michèle C.
Published: April 2009

Intercellular washing fluids from various compatible plant–parasite interactions were tested for their ability to reduce silica deposition on plant walls and to increase haustorium production in bean leaves following inoculation with the normally incompatible cowpea rust fungus. Only fluids from bean, wheat, and sunflower plants infected with their respective compatible rust fungi exhibited such activity, and no fluid had any similar effect on the nonhost interaction between bean leaves and other rust fungi. Fluids from rusted bean leaves did not increase the intercellular growth of a saprophytic fungus or other fungal parasites nonpathogenic on beans, nor did they affect silica deposition induced by these fungi. These data suggest that some feature of silica induction in bean by the cowpea rust fungus is unique and is affected by components in the intercellular washing fluids from only certain rust-infected plants. Fluids from uninoculated healthy wheat or sunflower plants and a Driselase solution that induces haustoria in vitro all increased haustorium formation by the cowpea rust fungus, but to a lesser extent than fluids from infected plants; silica deposition was not affected in these cases, suggesting that fluids from uninoculated plants may promote haustorium formation rather than prevent silica deposition.