bluuebunny/crossref_metadata_2025 huggingface dataset:
The
HTR1
Gene Is a Dominant Negative Mutant Allele of
MTH1
and Blocks Snf3- and Rgt2-Dependent Glucose Signaling in Yeast
Authors: Schulte Frank , Wieczorke Roman , Hollenberg Cornelis P. , Boles Eckhard
Published: July 2002

Saccharomyces cerevisiae HTR1
mutants are severely impaired in the uptake of glucose. We have cloned dominant
HTR1
mutant alleles and show that they encode mutant forms of the Mth1 protein. Mth1 is shown to be involved in carbon source-dependent regulation of its own, invertase and hexose transporter gene expression. The mutant forms block the transduction of the Snf3- and Rgt2-mediated glucose signals upstream of the Rgt1 transcriptional regulator.
Disruption of
aldA
Influences the Developmental Process in
Myxococcus xanthus
Authors: Ward Mandy J. , Lew Helen , Zusman David R.
Published: July 2002

Previously, we identified a gene (
aldA
) from
Myxococcus xanthus
, which we suggested encoded the enzyme alanine dehydrogenase on the basis of similarity to known Ald protein sequences (M. J. Ward, H. Lew, A. Treuner-Lange, and D. R. Zusman, J. Bacteriol. 180:5668–5675, 1998). In this study, we have confirmed that
aldA
does encode a functional alanine dehydrogenase, since it catalyzes the reversible conversion of alanine to pyruvate and ammonia. Whereas an
aldA
gene disruption mutation did not significantly influence the rate of growth or spreading on a rich medium, AldA was required for growth on a minimal medium containing
l
-alanine as the major source of carbon. Under developmental conditions, the
aldA
mutation caused delayed aggregation in both wild-type (DZ2) and FB (DZF1) strains. Poorly formed aggregates and reduced levels of spores were apparent in the DZ2
aldA
mutant, even after prolonged development.
A Second [2Fe-2S] Ferredoxin from
Sphingomonas
sp. Strain RW1 Can Function as an Electron Donor for the Dioxin Dioxygenase
Authors: Armengaud Jean , Gaillard Jacques , Timmis Kenneth N.
Published: July 2002

The first step in the degradation of dibenzofuran and dibenzo-
p
-dioxin by
Sphingomonas
sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (
fdx3
) that could potentially specify a new ferredoxin has been identified downstream of
dxnA1A2
, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954–3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in
Escherichia coli
. This third ferredoxin thus far identified in
Sphingomonas
sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E′
0
= −247 ± 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (−245 mV), a homologous ferredoxin previously characterized in
Sphingomonas
sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from
Sphingomonas
sp. strain RW1. RedA2 exhibits a

K
m

value of 3.2 ± 0.3 μM for Fdx3. In vivo coexpression of
fdx3
and
redA2
with
dxnA1A2
confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.
Azotobacter vinelandii
Aldehyde Dehydrogenase Regulated by ς
54
: Role in Alcohol Catabolism and Encystment
Authors: Gama-Castro Socorro , Núñez Cinthia , Segura Daniel , Moreno Soledad , Guzmán Josefina , Espı́n Guadalupe
Published: July 2002

Encystment in
Azotobacter vinelandii
is induced by
n
-butanol or β-hydroxybutyrate (BHB). We identified a gene, encoding an aldehyde dehydrogenase, that was named
aldA
. An
aldA
mutation impaired bacterial growth on
n
-butanol, ethanol, or hexanol as the sole carbon source. Expression of
aldA
increased in cells shifted from sucrose to
n
-butanol and was shown to be dependent on the alternative ς
54
factor. A mutation in
rpoN
encoding the ς
54
factor also impaired growth on alcohols. Encystment on
n
-butanol, but not on BHB, was impaired in
aldA
or
rpoN
mutants, indicating that
n
-butanol is not an inducer of encystment by itself but must be catabolized in order to induce encystment.
The Alternative Electron Acceptor Tetrathionate Supports B
12
-Dependent Anaerobic Growth of
Salmonella enterica
Serovar Typhimurium on Ethanolamine or 1,2-Propanediol
Authors: Price-Carter Marian , Tingey Justin , Bobik Thomas A. , Roth John R.
Published: July 2002

Synthesis of cobalamin de novo by
Salmonella enterica
serovar Typhimurium strain LT2 and the absence of this ability in
Escherichia coli
present several problems. This large synthetic pathway is shared by virtually all salmonellae and must be maintained by selection, yet no conditions are known under which growth depends on endogenous B
12
. The cofactor is required for degradation of 1,2-propanediol and ethanolamine. However, cofactor synthesis occurs only anaerobically, and neither of these carbon sources supports anaerobic growth with any of the alternative electron acceptors tested thus far. This paradox is resolved by the electron acceptor tetrathionate, which allows
Salmonella
to grow anaerobically on ethanolamine or 1,2-propanediol by using endogenously synthesized B
12
. Tetrathionate provides the only known conditions under which simple
cob
mutants (unable to make B
12
) show a growth defect. Genes involved in this metabolism include the
ttr
operon, which encodes tetrathionate reductase. This operon is globally regulated by OxrA (Fnr) and induced anaerobically by a two-component system in response to tetrathionate.
Salmonella
reduces tetrathionate to thiosulfate, which it can further reduce to H
2
S, by using enzymes encoded by the genes
phs
and
asr
. The genes for 1,2-propanediol degradation (
pdu
) and B
12
synthesis (
cob
), along with the genes for sulfur reduction (
ttr
,
phs,
and
asr
), constitute more than 1% of the
Salmonella
genome and are all absent from
E. coli
. In diverging from
E. coli
,
Salmonella
acquired some of these genes unilaterally and maintained others that are ancestral but have been lost from the
E. coli
lineage.
Expression of Herpes Simplex Virus ICP47 and Human Cytomegalovirus US11 Prevents Recognition of Transgene Products by CD8+Cytotoxic T Lymphocytes
Authors: Berger Carolina , Xuereb Suzanne , Johnson David C. , Watanabe Kathe S. , Kiem Hans-Peter , Greenberg Philip D. , Riddell Stanley R.
Published: July 2002

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8+CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.
An Evolutionarily Conserved Positively Charged Amino Acid in the Putative Membrane-Spanning Domain of the Foamy Virus Envelope Protein Controls Fusion Activity
Authors: Pietschmann Thomas , Zentgraf Hanswalter , Rethwilm Axel , Lindemann Dirk
Published: July 2002

Foamy viruses (FVs) are highly fusogenic, and their replication induces massive syncytium formation in infected cell cultures which is believed to be mediated by expression of the envelope (Env) protein. The FV Env is essential for virus particle egress. The unusually long putative membrane-spanning domain (MSD) of the transmembrane subunit carries dispersed charged amino acids and has an important function for particle envelopment. To better understand the capsid-envelope interaction and Env-mediated cell fusion, we generated a variety of FV MSD mutations. C-terminal deletions revealed the cytoplasmic domain to be dispensable but the full-length MSD to be required for fusogenic activity. The N-terminal 15 amino acids of the MSD were found to be sufficient for membrane anchorage and promotion of FV particle release. Expression of wild-type Env protein rarely induced syncytia due to intracellular retention. Coexpression with FV Gag-Pol resulted in particle export and a dramatic increase in fusion activity. A nonconservative mutation of K
959
in the middle of the putative MSD resulted in increased fusogenic activity of Env in the absence of Gag-Pol due to enhanced cell surface expression as well as structural changes in the mutant proteins. Coexpression with Gag-Pol resulted in a further increase in the fusion activity of mutant FV Env proteins. Our results suggest that an interaction between the viral capsid and Env is required for FV-induced giant-cell formation and that the positive charge in the MSD is an important determinant controlling intracellular transport and fusogenic activity of the FV Env protein.
Multiple Antiviral Activities of Cyanovirin-N: Blocking of Human Immunodeficiency Virus Type 1 gp120 Interaction with CD4 and Coreceptor and Inhibition of Diverse Enveloped Viruses
Authors: Dey Barna , Lerner Danica L. , Lusso Paolo , Boyd Michael R. , Elder John H. , Berger Edward A.
Published: July 2002

Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.
Successful Vaccine-Induced Seroconversion by Single-Dose Immunization in the Presence of Measles Virus-Specific Maternal Antibodies
Authors: Schlereth Bernd , Rose John K. , Buonocore Linda , ter Meulen Volker , Niewiesk Stefan
Published: July 2002

In humans, maternal antibodies inhibit successful immunization against measles, because they interfere with vaccine-induced seroconversion. We have investigated this problem using the cotton rat model (
Sigmodon hispidus
). As in humans, passively transferred antibodies inhibit the induction of measles virus (MV)-neutralizing antibodies and protection after immunization with MV. In contrast, a recombinant vesicular stomatitis virus (VSV) expressing the MV hemagglutinin (VSV-H) induces high titers of neutralizing antibodies to MV in the presence of MV-specific antibodies. The induction of neutralizing antibodies increased with increasing virus dose, and all doses gave good protection from subsequent challenge with MV. Induction of antibodies by VSV-H was observed in the presence of passively transferred human or cotton rat antibodies, which were used as the models of maternal antibodies. Because MV hemagglutinin is not a functional part of the VSV-H envelope, MV-specific antibodies only slightly inhibit VSV-H replication in vitro. This dissociation of function and antigenicity is probably key to the induction of a neutralizing antibody in the presence of a maternal antibody.
Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease
Authors: Lin Ying-Chuan , Beck Zachary , Lee Taekyu , Le Van-Duc , Morris Garrett M. , Olson Arthur J. , Wong Chi-Huey , Elder John H.
Published: July 2002

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.
Pegylated Alpha Interferon Is an Effective Treatment for Virulent Venezuelan Equine Encephalitis Virus and Has Profound Effects on the Host Immune Response to Infection
Authors: Lukaszewski Roman A. , Brooks Timothy J. G.
Published: July 2002

Venezuelan equine encephalitis virus (VEEV) is a highly infectious alphavirus endemic in parts of Central and South America. The disease is transmitted by mosquitoes, and the natural reservoir is the small rodent population, with epidemics occurring in horses and occasionally humans. Following infection, VEEV replicates in lymphoid tissues prior to invasion of the central nervous system. Treatment of VEEV-infected BALB/c mice with polyethylene glycol-conjugated alpha interferon (PEG IFN-α) results in a greatly enhanced survival from either a subcutaneous or an aerosol infection. Virus is undetectable within PEG IFN-α-treated individuals by day 30 postinfection (p.i.). Treatment results in a number of changes to the immune response characteristics normally associated with VEEV infection. Increased macrophage activation occurs in PEG IFN-α-treated BALB/c mice infected with VEEV. The rapid activation of splenic CD4, CD8, and B cells by day 2 p.i. normally associated with VEEV infection is absent in PEG IFN-α-treated mice. The high tumor necrosis factor alpha production by macrophages from untreated mice is greatly diminished in PEG IFN-α-treated mice. These results suggest key immunological mechanisms targeted by this lethal alphavirus that can be modulated by prolonged exposure to IFN-α.
Differential Activity of Two Non-
hr
Origins during Replication of the Baculovirus
Autographa californica
Nuclear Polyhedrosis Virus Genome
Authors: Habib Saman , Hasnain Seyed E.
Published: July 2002

The identification of potential baculovirus origins of replication (
ori
) has involved the generation and characterization of defective interfering particles that contain major genomic deletions yet retain their capability to replicate by testing the replication ability of transiently transfected plasmids carrying viral sequences in infected cells. So far, there has not been any evidence to demonstrate the actual utilization of these putative origins in
Autographa californica
multinucleocapsid nuclear polyhedrosis virus (
Ac
MNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for the
ori
activity of the
Hin
dIII-K region and the
ie
-1 promoter sequence in
Ac
MNPV. We also provide evidence for differential activity of the two
ori
in the context of the viral genome through the replication phase of viral infection. Comparison of the number of molecules representing the
Hin
dIII-K and
ie-1
origins vis-à-vis the non-
ori polH
region in a size-selected nascent DNA preparation revealed that the
Hin
dIII-K
ori
is utilized ∼14 times more efficiently than the
ie-1
region during the late phase of infection.
Hin
dIII-K also remains the more active
ori
through the early and middle replication phases. Our results provide in vivo evidence in support of the view that
Ac
MNPV replication involves multiple
ori
that are activated with vastly different efficiencies during the viral infection cycle.
The Predicted Metal-Binding Region of the Arterivirus Helicase Protein Is Involved in Subgenomic mRNA Synthesis, Genome Replication, and Virion Biogenesis
Authors: van Dinten Leonie C. , van Tol Hans , Gorbalenya Alexander E. , Snijder Eric J.
Published: July 2002

Equine arteritis virus
(EAV), the prototype
Arterivirus
, is a positive-stranded RNA virus that expresses its replicase in the form of two large polyproteins of 1,727 and 3,175 amino acids. The functional replicase subunits (nonstructural proteins), which drive EAV genome replication and subgenomic mRNA transcription, are generated by extensive proteolytic processing. Subgenomic mRNA transcription involves an unusual discontinuous step and generates the mRNAs for structural protein expression. Previously, the phenotype of mutant EAV030F, which carries a single replicase point mutation (Ser-2429→Pro), had implicated the nsp10 replicase subunit (51 kDa) in viral RNA synthesis, and in particular in subgenomic mRNA transcription. nsp10 contains an N-terminal (putative) metal-binding domain (MBD), located just upstream of the Ser-2429→Pro mutation, and a helicase activity in its C-terminal part. We have now analyzed the N-terminal domain of nsp10 in considerable detail. A total of 38 mutants, most of them carrying specific single point mutations, were tested in the context of an EAV infectious cDNA clone. Variable effects on viral genome replication and subgenomic mRNA transcription were observed. In general, our results indicated that the MBD region, and in particular a set of 13 conserved Cys and His residues that are assumed to be involved in zinc binding, is essential for viral RNA synthesis. On the basis of these data and comparative sequence analyses, we postulate that the MBD may employ a rather unusual mode of zinc binding that could result in the association of up to four zinc cations with this domain. The region containing residue Ser-2429 may play the role of “hinge spacer,” which connects the MBD to the rest of nsp10. Several mutations in this region specifically affected subgenomic mRNA synthesis. Furthermore, one of the MBD mutants was replication and transcription competent but did not produce infectious progeny virus. This suggests that nsp10 is involved in an as yet unidentified step of virion biogenesis.
Topoisomerase I Associates Specifically with Simian Virus 40 Large-T-Antigen Double Hexamer–Origin Complexes
Authors: Gai Dahai , Roy Rupa , Wu Chunxiao , Simmons Daniel T.
Published: July 2002

Topoisomerase I (topo I) is required for releasing torsional stress during simian virus 40 (SV40) DNA replication. Recently, it has been demonstrated that topo I participates in initiation of replication as well as in elongation. Although T antigen and topo I can bind to one another in vitro, there is no direct evidence that topo I is a component of the replication initiation complex. We demonstrate in this report that topo I associates with T-antigen double hexamers bound to SV40 origin DNA (T
DH
) but not to single hexamers. This association has the same nucleotide and DNA requirements as those for the formation of double hexamers on DNA. Interestingly, topo I prefers to bind to fully formed T
DH
complexes over other oligomerized forms of T antigen associated with the origin. High ratios of topo I to origin DNA destabilize T
DH
. The partial unwinding of a small-circular-DNA substrate is dependent on the presence of both T antigen and topo I but is inhibited at high topo I concentrations. Competition experiments with a topo I-binding fragment of T antigen indicate that an interaction between T antigen and topo I occurs during the unwinding reaction. We propose that topo I is recruited to the initiation complex after the assembly of T
DH
and before unwinding to facilitate DNA replication.
Hepatitis Delta Virus Replication Generates Complexes of Large Hepatitis Delta Antigen and Antigenomic RNA That Affiliate with and Alter Nuclear Domain 10
Authors: Bell Peter , Brazas Robert , Ganem Donald , Maul Gerd G.
Published: July 2002

Hepatitis delta virus (HDV), a single-stranded RNA virus, bears a single coding region whose product, the hepatitis delta antigen (HDAg), is expressed in two isoforms, small (S-HDAg) and large (L-HDAg). S-HDAg is required for replication of HDV, while L-HDAg inhibits viral replication and is required for the envelopment of the HDV genomic RNA by hepatitis B virus proteins. Here we have examined the spatial distribution of HDV RNA and proteins in infected nuclei, with particular reference to specific nuclear domains. We found that L-HDAg was aggregated in specific nuclear domains and that over half of these domains were localized beside nuclear domain 10 (ND10). At later times, ND10-associated proteins like PML were found in larger HDAg complexes that had developed into apparently hollow spheres. In these larger complexes, PML was found chiefly in the rims of the spheres, while the known ND10 components Sp100, Daxx, and NDP55 were found in the centers of the spheres. Thus, ND10 proteins that normally are closely linked separate within HDAg-associated complexes. Viral RNA of antigenomic polarity, whether expressed from genomic RNA or directly from introduced plasmids, colocalizes with L-HDAg and the transcriptional repressor PML. In contrast, HDV genomic RNA was distributed more uniformly throughout the nucleus. These results suggest that different host protein complexes may assemble on viral RNA strands of different polarities, and they also suggest that this RNA virus, like DNA viruses, can alter the distribution of ND10-associated proteins. The fact that viral components specifically linked to repression of replication can associate with one of the ND10-associated proteins (PML) raises the possibility that this host protein may play a role in the regulation of HDV RNA synthesis.
Ubiquitination of the Human Immunodeficiency Virus Type 1 Env Glycoprotein
Authors: Bültmann Andreas , Eberle Josef , Haas Jürgen
Published: July 2002

Expression of the human immunodeficiency virus type 1 (HIV-1) Env glycoprotein is stringently regulated in infected cells. The majority of the glycoprotein does not reach the cell surface but rather is retained in the endoplasmic reticulum or acis-Golgi compartment and subsequently degraded. We here report that Env of various HIV-1 isolates is ubiquitinated at the extracellular domain of gp41 and that Env expression could be increased by lactacystin, a specific proteasome inhibitor, suggesting that the ubiquitin/proteasome system is involved in control of expression and degradation.
Human Cytomegalovirus Replicates Abortively in Polymorphonuclear Leukocytes after Transfer from Infected Endothelial Cells via Transient Microfusion Events
Authors: Gerna Giuseppe , Percivalle Elena , Baldanti Fausto , Sozzani Silvano , Lanzarini Paolo , Genini Emilia , Lilleri Daniele , Revello Maria Grazia
Published: July 2002

Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA, and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains. The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls. Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture. Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs is likely to be mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild-type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i.e., endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.
Characterization of Stable, Soluble Trimers Containing Complete Ectodomains of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins
Authors: Yang Xinzhen , Farzan Michael , Wyatt Richard , Sodroski Joseph
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins function as a membrane-anchored trimer of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. Previously, we reported three approaches to stabilize soluble trimers containing parts of the gp41 ectodomains: addition of GCN4 trimeric helices, disruption of the cleavage site between gp120 and gp41, and introduction of cysteines in the gp41 coiled coil to form intersubunit disulfide bonds. Here, we applied similar approaches to stabilize soluble gp140 trimers including the complete gp120 and gp41 ectodomains. A combination of fusion with the GCN4 trimeric sequences and disruption of the gp120-gp41 cleavage site resulted in relatively homogeneous gp140 trimers with exceptional stability. The gp120 epitopes recognized by neutralizing antibodies are intact and exposed on these gp140 trimers. By contrast, the nonneutralizing antibody epitopes on the gp120 subunits of the soluble trimers are relatively occluded compared with those on monomeric gp120 preparations. This antigenic similarity to the functional HIV-1 envelope glycoproteins and the presence of the complete gp41 ectodomain should make the soluble gp140 trimers useful tools for structural and immunologic studies.
DNA Vaccination Prevents and/or Delays Carcinoma Development of Papillomavirus-Induced Skin Papillomas on Rabbits
Authors: Han Ricai , Cladel Nancy M. , Reed Cynthia A. , Peng Xuwen , Budgeon Lynn R. , Pickel Martin , Christensen Neil D.
Published: July 2002

Malignant progression is a life-threatening consequence of human papillomavirus-associated lesions. In this study, we tested the efficacy of papillomavirus early-gene-based vaccines for prevention of carcinoma development of papillomavirus-induced skin papillomas on rabbits. Rabbit skin papillomas were initiated by infection with cottontail rabbit papillomavirus (CRPV). The papillomas were allowed to grow for 3 months without any treatment intervention. Rabbits were then immunized by gene gun-mediated intracutaneous administration of four DNA plasmids encoding CRPV E1, E2, E6, and E7 genes, respectively. All eight control rabbits receiving vector alone developed invasive carcinoma within 8 to 13 months. In contrast, only two of eight vaccinated rabbits developed carcinoma at 12 and 15 months, respectively. Papilloma growth was suppressed in the majority of vaccinated rabbits but not completely eradicated. These results indicate that gene gun-mediated immunization with papillomavirus early genes may be a promising strategy for prevention of malignant progression of human papillomavirus-associated lesions in humans.
Cytoplasmic Dynein LC8 Interacts with Lyssavirus Phosphoprotein
Authors: Jacob Yves , Badrane Hassan , Ceccaldi Pierre-Emmanuel , Tordo Noël
Published: July 2002

Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon “retrograde transport” and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.
Toward a Comprehensive Phylogeny for Mammalian and Avian Herpesviruses
Authors: McGeoch Duncan J. , Dolan Aidan , Ralph Adam C.
Published: July 2002

With the aim of deriving a definitive phylogenetic tree for as many mammalian and avian herpesvirus species as possible, alignments were made of amino acid sequences from eight conserved and ubiquitously present genes of herpesviruses, with 48 virus species each represented by at least one gene. Phylogenetic trees for both single-gene and concatenated alignments were evaluated thoroughly by maximum-likelihood methods, with each of the three herpesvirus subfamilies (the
Alpha
-,
Beta
-, and
Gammaherpesvirinae
) examined independently. Composite trees were constructed starting with the top-scoring tree based on the broadest set of genes and supplemented by addition of virus species from trees based on narrower gene sets, to give finally a 46-species tree; branching order for three regions within the tree remained unresolved. Sublineages of the
Alpha
- and
Betaherpesvirinae
showed extensive cospeciation with host lineages by criteria of congruence in branching patterns and consistency in extent of divergence. The
Gammaherpesvirinae
presented a more complex picture, with both higher and lower substitution rates in different sublineages. The final tree obtained represents the most detailed view to date of phylogenetic relationships in any family of large-genome viruses.
Enhancement of Immunoglobulin G2a and Cytotoxic T-Lymphocyte Responses by a Booster Immunization with Recombinant Hepatitis C Virus E2 Protein in E2 DNA-Primed Mice
Authors: Song Man Ki , Lee Seung Woo , Suh You Suk , Lee Ki Jeong , Sung Young Chul
Published: July 2002

The induction of strong cytotoxic T-lymphocyte (CTL) and humoral responses appear to be essential for the elimination of persistently infecting viruses, such as hepatitis C virus (HCV). Here, we tested several vaccine regimens and demonstrate that a combined vaccine regimen, consisting of HCV E2 DNA priming and boosting with recombinant E2 protein, induces the strongest immune responses to HCV E2 protein. This combined vaccine regimen augments E2-specific immunoglobulin G2a (IgG2a) and CD8+CTL responses to a greater extent than immunizations with recombinant E2 protein and E2 DNA alone, respectively. In addition, the data showed that a protein boost following one DNA priming was also effective, but much less so than those following two DNA primings. These data indicate that sufficient DNA priming is essential for the enhancement of DNA encoded antigen-specific immunity by a booster immunization with recombinant E2 protein. Furthermore, the enhanced CD8+CTL and IgG2a responses induced by our combined vaccine regimens are closely associated with the protection of BALB/c mice from challenge with modified CT26 tumor cells expressing HCV E2 protein. Together, our results provide important implications for vaccine development for many pathogens, including HCV, which require strong antibody and CTL responses.
Kinetics of Recombinant Adeno-Associated Virus-Mediated Gene Transfer
Authors: Malik Ajay K. , Monahan Paul E. , Allen David L. , Chen Bing-Guan , Samulski R. Jude , Kurachi Kotoku
Published: July 2002

Recombinant adeno-associated virus (rAAV) vectors have been shown to be useful for efficient gene delivery to a variety of dividing and nondividing cells. Mechanisms responsible for the long-term, persistent expression of the rAAV transgene are not well understood. In this study we investigated the kinetics of rAAV-mediated human factor IX (hFIX) gene transfer into human primary myoblasts and myotubes. Transduction of both myoblasts and myotubes occured with a similar and high efficiency. After 3 to 4 weeks of transduction, rAAV with a cytomegalovirus (CMV) promoter showed 10- to 15-fold higher expression than that with a muscle-specific creatine kinase enhancer linked to β-actin promoter. Factor IX expression from transduced myoblasts as well as myotubes reached levels as high as approximately 2 μg of hFIX/10
6
cells/day. Southern blot analyses of high-molecular-weight (HMW) cellular genomic and Hirt DNAs isolated from rAAV/CMVhFIXm1-transduced cells showed that the conversion of single-stranded vector genomes to double-stranded DNA forms, but not the level of the integrated forms in HMW DNA, correlated with increasing expression of the transgene. Together, these results indicate that rAAV can transduce both proliferating and terminally differentiated muscle cells at about the same efficiency, that expression of transgenes increases linearly over their lifetime with no initial lag phase, and that increasing expression correlates with the appearance of double-stranded episomal rAAV genomes. Evidence showing that the rAAV virions can copackage hFIX, presumably nonspecifically, was also obtained.
Identification of Additional Coat-Scaffolding Interactions in a Bacteriophage P22 Mutant Defective in Maturation
Authors: Thuman-Commike Pamela A. , Greene Barrie , Jakana Joanita , McGough Amy , Prevelige Peter E. , Chiu Wah
Published: July 2002

Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation.
Kaposi's Sarcoma-Associated Herpesvirus LANA2 Is a B-Cell-Specific Latent Viral Protein That Inhibits p53
Authors: Rivas Carmen , Thlick Ai-En , Parravicini Carlo , Moore Patrick S. , Chang Yuan
Published: July 2002

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, is associated with three proliferative diseases ranging from viral cytokine-induced hyperplasia to monoclonal neoplasia: multicentric Castleman's disease (CD), Kaposi's sarcoma (KS), and primary effusion lymphoma (PEL). Here we report a new latency-associated 1,704-bp KSHV spliced gene belonging to a cluster of KSHV sequences having homology to the interferon regulatory factor (IRF) family of transcription factors. ORFK10.5 encodes a protein, latency-associated nuclear antigen 2 (LANA2), which is expressed in KSHV-infected hematopoietic tissues, including PEL and CD but not KS lesions. LANA2 is abundantly expressed in the nuclei of cultured KSHV-infected B cells. Transcription of K10.5 in PEL cell cultures is not inhibited by DNA polymerase inhibitors nor significantly induced by phorbol ester treatment. Unlike LANA1, LANA2 does not elicit a serologic response from patients with KS, PEL, or CD as measured by Western blot hybridization. Both KSHV vIRF1 (ORFK9) and LANA2 (ORFK10.5) appear to have arisen through gene duplication of a captured cellular IRF gene. LANA2 is a potent inhibitor of p53-induced transcription in reporter assays. LANA2 antagonizes apoptosis due to p53 overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicin treatment of wild-type p53 U2OS cells. While LANA2 specifically interacts with amino acids 290 to 393 of p53 in glutathione
S
-transferase pull-down assays, we were unable to demonstrate LANA2-p53 interaction in vivo by immunoprecipitation. These findings show that KSHV has tissue-specific latent gene expression programs and identify a new latent protein which may contribute to KSHV tumorigenesis in hematopoietic tissues via p53 inhibition.
Reduction of Simian-Human Immunodeficiency Virus 89.6P Viremia in Rhesus Monkeys by Recombinant Modified Vaccinia Virus Ankara Vaccination
Authors: Barouch Dan H. , Santra Sampa , Kuroda Marcelo J. , Schmitz Jörn E. , Plishka Ronald , Buckler-White Alicia , Gaitan Alicia E. , Zin Rebekah , Nam Jae-Hwan , Wyatt Linda S. , Lifton Michelle A. , Nickerson Christine E. , Moss Bernard , Montefiori David C. , Hirsch Vanessa M. , Letvin Norman L.
Published: July 2002

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239gag-poland HIV-1 89.6envelicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.
Mink Cell Focus-Forming Murine Leukemia Virus Killing of Mink Cells Involves Apoptosis and Superinfection
Authors: Yoshimura Fayth K. , Wang Tao , Nanua Suparna
Published: July 2002

Induction of apoptosis by different types of pathogenic retroviruses is an important step in disease development. We have observed that infection of thymic lymphocytes by the mink cell focus-forming murine leukemia virus (MCF MLV) during the preleukemic period resulted in an enhancement of apoptosis of these cells. To further study the ability of MCF MLVs to induce apoptosis and the role of this process in viral pathogenesis, we have developed an in vitro system of virus-induced apoptosis. MCF13 MLV infection of mink epithelial cells resulted in the production of cytopathic foci. In contrast, infection of mink cells with the 4070A amphotropic MLV did not produce any cytopathic effects. Staining of MCF13 MLV-infected cells with propidium iodide and annexin V-fluorescein isothiocyanate indicated that virus-induced cell death was due to apoptosis. At 6 days postinfection, the percentage of apoptotic MCF13 MLV-infected cells was 27% compared with 2 to 3% for mock- or amphotropic MLV-infected cells, representing a 9- to 14-fold difference. Assays for caspase-3 activation confirmed the detection by flow cytometry of apoptosis of MCF13 MLV-infected cells. Large amounts of unintegrated linear viral DNA were detectable by Southern blot analysis during the acute phase of infection, which indicated that MCF13 MLV is able to superinfect mink cells. Unintegrated viral DNA of only the linear form was detectable in thymic lymphocytes isolated from MCF13 MLV-inoculated mice during the preleukemic period. These results indicated that the ability of MCF13 MLV to induce apoptosis is correlated with its ability to superinfect cells and that this occurs as an early step in thymic lymphoma development.
Characterization of a Late Entry Event in the Replication Cycle of Human Immunodeficiency Virus Type 2
Authors: McKnight Áine , Griffiths David J. , Dittmar Matthias , Clapham Paul , Thomas Elaine
Published: July 2002

Certain human cell lines and primary macrophage cultures are restricted to infection by some primary isolates of human immunodeficiency virus type 2 (HIV-2), although early steps of the viral life cycle such as fusion at the plasma membrane and reverse transcription are fully supported. The late postintegration events, transcription, translation, assembly, budding, and maturation into infectious virions are functional in restrictive cells. Apart from primary macrophages, the restrictive cell types are actively dividing, and nuclear import of preintegration complexes (PICs) is not required for infection. We therefore postulate that the PICs are trapped in a cellular compartment, preventing subsequent steps in the replication cycle that lead to integration of the provirus. To test this we showed that HIV-2 particles pseudotyped with vesicular stomatitis virus envelope G protein, which delivers HIV into an endocytic compartment, could overcome the block to infection. We suggest that delivery of the viral core into an appropriate cellular compartment is a critical step during the entry process of HIV.
Immunization with a Pentameric L1 Fusion Protein Protects against Papillomavirus Infection
Authors: Yuan Hang , Estes Patricia A. , Chen Yan , Newsome Joseph , Olcese Vanessa A. , Garcea Robert L. , Schlegel Richard
Published: July 2002

The prophylactic papillomavirus vaccines currently in clinical trials are composed of viral L1 capsid protein that is synthesized in eukaryotic expression systems and purified in the form of virus-like particles (VLPs). To evaluate whether VLPs are necessary for effective vaccination, we expressed the L1 protein as a glutathione
S
-transferase (GST) fusion protein in
Escherichia coli
and assayed its immunogenic activity in an established canine oral papillomavirus (COPV) model that previously validated the efficacy of VLP vaccines. The GST-COPV L1 fusion protein formed pentamers, but these capsomere-like structures did not assemble into VLPs. Despite the lack of VLP formation, the GST-COPV L1 protein retained its native conformation as determined by reactivity with conformation-specific anti-COPV antibodies. Most importantly, the GST-COPV L1 pentamers completely protected dogs from high-dose viral infection of their oral mucosa. L1 fusion proteins expressed in bacteria represent an economical alternative to VLPs as a human papillomavirus vaccine.
Highly Productive Infection with Pseudotyped Human Immunodeficiency Virus Type 1 (HIV-1) Indicates No Intracellular Restrictions to HIV-1 Replication in Primary Human Astrocytes
Authors: Canki Mario , Thai Janice Ngee Foong , Chao Wei , Ghorpade Anuja , Potash Mary Jane , Volsky David J.
Published: July 2002

Human astrocytes can be infected with human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo, but, in contrast to T lymphocytes and macrophages, virus expression is inefficient. To investigate the HIV-1 life cycle in human fetal astrocytes, we infected cells with HIV-1 pseudotyped with envelope glycoproteins of either amphotropic murine leukemia virus or vesicular stomatitis virus. Infection by both pseudotypes was productive and long lasting and reached a peak of 68% infected cells and 1.7 μg of viral p24 per ml of culture supernatant 7 days after virus inoculation and then continued with gradually declining levels of virus expression through 7 weeks of follow-up. This contrasted with less than 0.1% HIV-1 antigen-positive cells and 400 pg of extracellular p24 per ml at the peak of astrocyte infection with native HIV-1. Cell viability and growth kinetics were similar in infected and control cells. Northern blot analysis revealed the presence of major HIV-1 RNA species of 9, 4, and 2 kb in astrocytes exposed to pseudotyped (but not wild-type) HIV-1 at 2, 14, and 28 days after infection. Consistent with productive infection, the 9- and 4-kb viral transcripts in astrocytes infected by pseudotyped HIV-1 were as abundant as the 2-kb mRNA during 4 weeks of follow-up, and both structural and regulatory viral proteins were detected in infected cells by immunoblotting or cell staining. The progeny virus released by these cells was infectious. These results indicate that the major barrier to HIV-1 infection of primary astrocytes is at virus entry and that astrocytes have no intrinsic intracellular restriction to efficient HIV-1 replication.