bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Translocation in Photoreceptor Light Adaptation: A Common Theme in Vertebrate and Invertebrate Vision
Authors: Arshavsky Vadim Y.
Published: December 2003

How do our eyes adjust to daily light levels that vary by almost 11 orders of magnitude? Research shows that, in both vertebrates and invertebrates, signaling proteins are translocated in a light-dependent manner between the photoreceptor cellular compartments where visual transduction takes place, and the rest of the photoreceptor cell. Protein translocation is likely to contribute to photoreceptor light adaptation by adjusting the sensitivity and speed of photoresponse to ever-changing conditions of ambient illumination.
Is Reelin the Answer to Synapse Elimination at the Neuromuscular Junction?
Authors: Chih Ben , Scheiffele Peter
Published: December 2003

The formation of mature neuronal circuits during development involves elimination of a large number of synapses by activity-dependent processes. A recent study suggests that synapse elimination at the neuromuscular junction is impaired in
reeler
mutant mice, which are lacking the extracellular matrix protein Reelin. In this process, Reelin acts through an unexpected, proteolytic mechanism that is independent of Disabled 1, a cytoplasmic factor that mediates Reelin signaling in the central nervous system. This Perspective discusses possible models for Reelin function in the framework of activity-dependent synapse elimination at the neuromuscular junction.
Monitoring Signaling Processes in Living Cells Using Biosensors
Authors: Hahn Klaus
Published: December 2003

This collection of nine animations shows how different types of biosensors report changes in cellular processes through the production of a visually detectable signal. Biosensors can be created by attaching one or more fluorescent proteins (such as green fluorescent protein) to a target protein or peptide or by attaching a fluorescent dye that is sensitive to its environment to a protein or peptide. Conformational changes in proteins in response to ligand binding, changes in the concentration of cellular metabolites or signaling messengers, changes in protein localization, and changes in protein activity or covalent modification can all be detected with biosensors. These animations can be used separately or together to illustrate how molecular biology, chemistry, and microscopy have converged to allow cellular processes to be visualized in living cells. Several of the animations describe the production of a fluorescent resonance energy transfer (FRET) signal.
Microarray Analysis in Drug Discovery: An Uplifting View of Depression
Authors: Levy Shawn E.
Published: December 2003

Genomic profiling provides insights into drug evaluation for diseases without defined molecular mechanisms or cellular assays. Levy provides a brief background in the development of microarray analysis and discussion of the application of this technique to pharmacogenomics. Highlighted is the microarray analysis of primary human neurons treated with antidepressants, antipsychotics, or opioid receptor agonists, demonstrating that these classes of drugs can be properly categorized by using two different statistical analysis methods: classification tree and random forest. Not only is microarray analysis valuable for drug evaluation and leading candidate development, but the genes identified as markers for the various drug classifications point to new directions for research into the underlying pathways responsible for human diseases, such as depression and psychosis.
Therapeutic Potential of Antisense Nucleic Acid Molecules
Authors: Opalinska Joanna B. , Gewirtz Alan M.
Published: December 2003

Elucidation of many disease-related signal transduction and gene expression pathways has provided unparalleled opportunities for the development of targeted therapeutics. The types of molecules in development are increasingly varied and include small-molecule enzyme inhibitors, humanized antibodies to cell surface receptors, and antisense nucleic acids for silencing the expression of specific genes. This Perspective reviews the basis for various antisense strategies for modulating gene expression, including RNA interference, and discusses the prospects for their clinical use.
Huntingtin: Alive and Well and Working in Middle Management
Authors: MacDonald Marcy E.
Published: December 2003

A decade after the discovery of the
HD
gene, huntingtin is implicated in signal transduction in neuronal and nonneuronal cells. Its predicted physical properties, dynamic subcellular locations, and variable protein associations suggest that huntingtin may act to organize components of signal transduction complexes. This middle management position befits a ubiquitous, conserved protein and provides the opportunity for some subtle piece of mischief that leads eventually to a devastating inherited human disorder.
Signaling Pathways Take Aim at Neurotransmitter Transporters
Authors: Robinson Michael B.
Published: December 2003

Neurotransmitter transporters are the target of various pharmacological agents used to treat psychological or cognitive conditions, such as depression and attention-deficit disorder. In addition, some of the effects of stimulant-type drugs of abuse result from inhibition of neurotransmitter transporters. Robinson describes the intersection between neurotransmitter transporters and signaling pathways. Neurotransmitter transporters can be regulated by altering the rate of internalization and insertion into the plasma membrane to control cell surface expression or by altering the activity of the transporters within the membrane. As the mechanisms governing regulation of these transporters become elucidated, new potential therapeutic targets may be revealed, given the many processes affected by the activity of neurotransmitter transporters.
Computational Design of Variant TNF Molecules: A Novel Methodology for Inhibition of Proinflammatory Cascades
Authors: Abraham Edward
Published: December 2003

Site-directed mutagenesis of tumor necrosis factor (TNF) based on prediction of the interaction of specific residues with TNF receptors generated dominant-negative constructs, in which single- or double-amino acid changes result in decreased receptor binding and cellular activation. These dominant-negatives not only provide a novel manner to block the proinflammatory effects of TNF, but also can be used as a tool to examine ligand-receptor interactions and their importance in signaling. Because these TNF mutant molecules are smaller than those used for conventional anti-TNF therapies, such as etanercept or infliximab, they are likely to achieve greater tissue concentrations and may provide enhanced therapeutic effect. However, the immunogenicity, as well as efficacy, of the dominant-negative TNF constructs must be more completely examined.
Oscillatory Mechanisms Underlying the
Drosophila
Circadian Clock
Authors: Van Gelder Russell N.
Published: December 2003

This animation depicts a schematic of the
Drosophila
circadian clock mechanism in a single, pacemaking lateral brain neuron. Circadian clocks allow organisms to exhibit rhythmic behaviors and processes based on a nearly 24-hour cycle in the absence of any light stimulation. In the fruit fly, the basic molecular mechanism consists of two intertwined transcription-translation negative feedback loops. One loop--the "positive loop"--controls the rhythmic expression of a positive transcription factor gene,
Clock
. The second loop--the "negative loop"--controls the transcription of
period
and
timeless
, two genes that encode repressor proteins. The loops are intertwined because Period and Timeless repress transcription mediated by the transcription factors Clock and Cycle, whereas the Clock:Cycle heterodimer drives transcription of
period
and
timeless
, as well as that of
vrille
, a repressor of
Clock
expression. Light cycles can synchronize the
Drosophila
circadian pathway by directly stimulating
cryptochrome
-dependent degradation of Timeless.
Oscillatory Mechanisms Underlying the Murine Circadian Clock
Authors: Van Gelder Russell N. , Herzog Erik D.
Published: December 2003

This animation depicts a schematic of the murine circadian clock mechanism in a single, pacemaking neuron in the suprachiasmatic nucleus (SCN) of the hypothalamus, where the master pacemaker is located. Circadian clocks allow organisms to display behaviors and processes with a 24-hour rhythm even in the absence of light input. The basic molecular mechanism consists of two intertwined transcription-translation negative feedback loops. One loop--the "positive loop"--controls the rhythmic expression of a positive transcription factor gene,
Bmal1
(also called
Mop3
). The second loop--the "negative loop"--controls the transcription of genes in the
Period
and
Cryptochrome
families, which encode repressor proteins. The loops are intertwined because the proteins PERIOD and CRYPTOCHROME directly repress transcription mediated by the transcription factors CLOCK and BMAL1, whereas the CLOCK:BMAL1 heterodimer drives transcription of the
Period
and
Cryptochrome
genes, as well as that of
Rev-erb-alpha
, which represses
Bmal1
expression. Other proteins, such as casein kinase I ϵ (CKIϵ) play essential modulatory roles in mammalian circadian timekeeping.
Potential Synergy: Voltage-Driven Steps in Receptor-G Protein Coupling and Beyond
Authors: Bolton Thomas B. , Zholos Alexander V.
Published: December 2003

G protein-coupled receptor responses are not generally considered to show voltage dependence, but recently, the sensitivity of an oocyte to acetylcholine, which activates the m2 subtype of muscarinic receptors, was shown to be affected by changes in the membrane potential. Another example is the m2 receptor-evoked cation current in mammalian smooth muscle cells, which shows strong voltage dependence even when the receptor is bypassed by activating the G protein directly by intracellular application of GTPγS, a hydrolysis-resistant guanosine triphosphate (GTP) analog. Even among the few examples of voltage-dependent behavior known from among the plethora of G protein-coupled responses, the mechanism underlying the voltage dependence seems to differ.
Growth Factors Bind Receptor Tyrosine Kinases to Stimulate Cell Survival, Cell Division, Cell Growth, and Cytoskeletal Rearrangement
Authors: Cantley Lewis C.
Published: December 2003

These two animations show some of the molecular events associated with the growth factors activation of receptor tyrosine kinases that leads to the stimulation of phosphoinositide 3-kinase. In Animation 1, growth factors stimulate cell survival and entry into the S phase of the cell cycle. In Animation 2, growth factors stimulate the activation of proteins that regulate the cytoskeleton, stimulation of protein synthesis, and stimulation of the Tec family of tyrosine kinases. Animation 2 also shows how the action of phosphatases, such as PTEN, terminates signaling and resets the system to receive a new stimulus. The cellular processes illustrated include protein and lipid phosphorylation and dephosphorylation, protein degradation, protein translocation, and regulation of gene expression.
EH and UIM: Endocytosis and More
Authors: Polo Simona , Confalonieri Stefano , Salcini Anna Elisabetta , Di Fiore Pier Paolo
Published: December 2003

Exogenously and endogenously originated signals are propagated within the cell by functional and physical networks of proteins, leading to numerous biological outcomes. Many protein-protein interactions take place between binding domains and short peptide motifs. Frequently, these interactions are inducible by upstream signaling events, in which case one of the two binding surfaces may be created by a posttranslational modification. Here, we discuss two protein networks. One, the EH-network, is based on the Eps15 homology (EH) domain, which binds to peptides containing the sequence Asp-Pro-Phe (NPF). The other, which we define as the monoubiquitin (mUb) network, relies on monoubiquitination, which is emerging as an important posttranslational modification that regulates protein function. Both networks were initially implicated in the control of plasma membrane receptor endocytosis and in the regulation of intracellular trafficking routes. The ramifications of these two networks, however, appear to extend into many other aspects of cell physiology as well, such as transcriptional regulation, actin cytoskeleton remodeling, and DNA repair. The focus of this review is to integrate available knowledge of the EH- and mUb networks with predictions of genetic and physical interactions stemming from functional genomics approaches.
Mucosal and Systemic Immune Responses to a Human Immunodeficiency Virus Type 1 Epitope Induced upon Vaginal Infection with a Recombinant Influenza A Virus
Authors: Garulli Bruno , Kawaoka Yoshihiro , Castrucci Maria R.
Published: December 2003

The humoral and cellular immune responses in the genital mucosa likely play an important role in the prevention of sexually transmitted infections, including infection with human immunodeficiency virus type 1 (HIV-1). Here we show that vaginal infection of progesterone-treated BALB/c mice with a recombinant influenza virus bearing the immunodominant P18IIIB cytotoxic T-lymphocyte (CTL) epitope of the gp160 envelope protein from an HIV-1 IIIB isolate (P18IIIB; RIQRGPGRAFVTIGK) can induce a specific immune response in regional mucosal lymph nodes, as well as in a systemic site (the spleen). A single inoculation of mice with the recombinant influenza virus induced long-lasting (at least 5 months) antigen-specific CTL memory detectable as a rapid recall of effector CTLs upon vaginal infection with recombinant vaccinia virus expressing HIV-1 IIIB envelope gene products. Long-term antigen-specific CTL memory was also induced and maintained in distant mucosal tissues when mice were intranasally immunized with the recombinant influenza virus. These results indicate that mucosal immunization and, in particular, local vaginal immunization with recombinant influenza virus can provide strong, durable immune responses in the female genital tract of mice.
Regulation of Human Immunodeficiency Virus Type 1 Env-Mediated Membrane Fusion by Viral Protease Activity
Authors: Murakami Tsutomu , Ablan Sherimay , Freed Eric O. , Tanaka Yuetsu
Published: December 2003

We and others have presented evidence for a direct interaction between the matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) Gag protein and the cytoplasmic tail of the transmembrane envelope (Env) glycoprotein gp41. In addition, it has been postulated that the MA domain of Gag undergoes a conformational change following Gag processing, and the cytoplasmic tail of gp41 has been shown to modulate Env-mediated membrane fusion activity. Together, these results raise the possibility that the interaction between the gp41 cytoplasmic tail and MA is regulated by protease (PR)-mediated Gag processing, perhaps affecting Env function. To examine whether Gag processing affects Env-mediated fusion, we compared the ability of wild-type (WT) HIV-1 Env and a mutant lacking the gp41 cytoplasmic tail to induce fusion in the context of an active (PR
+
) or inactive (PR
−
) viral PR. We observed that PR
−
virions bearing WT Env displayed defects in cell-cell fusion. Impaired fusion did not appear to be due to differences in the levels of virion-associated Env, in CD4-dependent binding to target cells, or in the formation of the CD4-induced gp41 six-helix bundle. Interestingly, truncation of the gp41 cytoplasmic tail reversed the fusion defect. These results suggest that interactions between unprocessed Gag and the gp41 cytoplasmic tail suppress fusion.
Analysis of the Requirement for Glycoprotein M in Herpes Simplex Virus Type 1 Morphogenesis
Authors: Browne Helena , Bell Susanne , Minson Tony
Published: December 2003

A mutant of herpes simplex virus type 1 lacking both glycoprotein M and glycoprotein E was marginally compromised in terms of its in vitro growth characteristics. This finding is in marked contrast to a similar mutant of the related alphaherpesvirus, pseudorabies virus (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol.
73:
5364-5372, 1999), and suggests that the glycoprotein requirements for virion assembly may vary among different members of this family of viruses.
Incorporation of Pol into Human Immunodeficiency Virus Type 1 Gag Virus-Like Particles Occurs Independently of the Upstream Gag Domain in Gag-Pol
Authors: Cen Shan , Niu Meijuan , Saadatmand Jenan , Guo Fei , Huang Yue , Nabel Gary J. , Kleiman Lawrence
Published: December 2003

By using particle-associated reverse transcriptase (RT) activity as an assay for Pol incorporation into human immunodeficiency virus type 1 (HIV-1) Gag virus-like particles (VLPs), it has been found that truncated, protease-negative, Gag-Pol missing
cis
Gag sequences is still incorporated into Gag VLPs, albeit at significantly reduced levels (10 to 20% of the level of wild-type Gag-Pol). In this work, we have directly measured the incorporation of truncated Gag-Pol species into Gag VLPs and have found that truncated Gag-Pol that is missing all sequences upstream of RT is still incorporated into Gag VLPs at levels approximating 70% of that achieved by wild-type Gag-Pol. Neither protease nor integrase regions in Pol are required for its incorporation, implying an interaction between Gag and RT sequences in the Pol protein. While the incorporation of Gag-Pol into Gag VLPs is reduced 12-fold by the replacement of the nucleocapsid within Gag with a leucine zipper motif, this mutation does not affect Pol incorporation. However, the deletion of p6 in Gag reduces Pol incorporation into Gag VLPs four- to fivefold. Pol shows the same ability as Gag-Pol to selectively package tRNA
Lys
into Gag VLPs, and primer tRNA
3
Lys
is found annealed to the viral genomic RNA. These data suggest that after the initial separation of Gag from Pol during cleavage of Gag-Pol by viral protease, the Pol species still retains the capacity to bind to both Gag and tRNA
3
Lys
, which may be required for Pol and tRNA
3
Lys
to be retained in the assembling virion until budding is completed.
RIII/Sa Mice with a High Incidence of Mammary Tumors Express Two Exogenous Strains and One Potential Endogenous Strain of Mouse Mammary Tumor Virus
Authors: Sarkar Nurul H. , Golovkina Tatyana , Uz-Zaman Taher
Published: December 2003

The inbred mouse strain RIII has long been known for shedding large amounts of mouse mammary tumor virus (MMTV) particles in milk and for the development of hormone-dependent early mammary tumors at a very high incidence (>90%). We have established one RIII subline (RIII/Sa) that shows a pattern of virus expression and tumor incidence similar to that in RIII mice. In the present study, we analyzed the milk and mammary tumors of RIII/Sa mice for virus characterization by reverse transcriptase PCR (RT-PCR) cloning and sequencing of the open reading frame (ORF) of the MMTV long terminal repeats (LTRs). Our results show that these mice express a mixture of at least three different MMTV strains, two of which, designated here as RIII/Sa MMTV-1 and RIII/Sa MMTV-2, are exogenous. The third virus, RIII/Sa MMTV-3, appears to carry the signature of an endogenous provirus,
Mtv
-
17
. Similar studies done with the milk and mammary glands of another subline, RIIIS/J, revealed that they do not express MMTV in their milk. The RIII/Sa and RIIIS/J mice also exhibited differences in their endogenous proviral contents. Twelve spontaneously developed mammary tumors of RIII/Sa mice were examined for possible
Wnt
-
1
and/or
int
-
2
/
Fgf
3 mutations that are usually found to occur in most mouse mammary tumors as a consequence of MMTV proviral integration. This work led to the isolation of one MMTV-
Wnt
-
1
junction fragment and one MMTV-
int
-
2
/
Fgf
3 junction fragment from 2 of the 12 tumors. Further analyses showed that both junction fragments contained the RIII/Sa MMTV-2-specific LTR ORF, indicating that this virus was involved in the development of both tumors. Whether RIII/Sa MMTV-1 and/or RIII/Sa MMTV-3 plays any role in mammary tumor development in RIII/Sa mice remains to be established. Overall, the present study demonstrates, to our surprise, that (i) RIII/Sa mice express, unlike other native mouse strains, three strains of MMTVs; and (ii) the virions are completely different from the virus expressed by another subline of RIII mice, the BR6 mice.
Important Role for the CA-NC Spacer Region in the Assembly of Bovine Immunodeficiency Virus Gag Protein
Authors: Guo Xiaofeng , Hu Jing , Whitney James B. , Russell Rodney S. , Liang Chen
Published: December 2003

Lentiviral Gag proteins contain a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain. This short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (HIV-1). We have now extended this finding to the CA-NC spacer motif within the Gag protein of bovine immunodeficiency virus (BIV). Mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to the severely disrupted association of the mutated Gag with the plasma membrane, as shown by the results of membrane flotation assays and confocal microscopy. Detailed mutagenesis analysis of the BIV CA-NC spacer region for virus assembly determinants led to the identification of two key residues, L368 and M372, which are separated by three amino acids, 369-VAA-371. Incidentally, the same two residues are present within the HIV-1 CA-NC spacer region at positions 364 and 368 and have also been shown to be crucial for HIV-1 assembly. Regardless of this conservation between these two viruses, the BIV CA-NC spacer could not be replaced by its HIV-1 counterpart without decreasing virus production, as opposed to its successful replacement by the CA-NC spacer sequences from the nonprimate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus and visna virus, with the sequence from FIV showing the highest effectiveness in this regard. Taken together, these data suggest a pivotal role for the CA-NC spacer region in the assembly of BIV Gag; however, the mechanism involved therein may differ from that for the HIV-1 CA-NC spacer.
Toll-Like Receptor 4-Dependent Activation of Dendritic Cells by a Retrovirus
Authors: Burzyn Dalia , Rassa John C. , Kim David , Nepomnaschy Irene , Ross Susan R. , Piazzon Isabel
Published: December 2003

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.
A Point Mutation within Conserved Region VI of Herpes Simplex Virus Type 1 DNA Polymerase Confers Altered Drug Sensitivity and Enhances Replication Fidelity
Authors: Hwang Ying T. , Zuccola Harmon J. , Lu Qiaosheng , Hwang Charles B. C.
Published: December 2003

Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the
supF
marker gene, which was integrated within the thymidine kinase locus (
tk
), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.
Characterization of the RNA Components of a Putative Molecular Switch in the 3′ Untranslated Region of the Murine Coronavirus Genome
Authors: Goebel Scott J. , Hsue Bilan , Dombrowski Todd F. , Masters Paul S.
Published: December 2003

RNA virus genomes containcis-acting sequence and structural elements that participate in viral replication. We previously identified a bulged stem-loop secondary structure at the upstream end of the 3′ untranslated region (3′ UTR) of the genome of the coronavirus mouse hepatitis virus (MHV). This element, beginning immediately downstream of the nucleocapsid gene stop codon, was shown to be essential for virus replication. Other investigators discovered an adjacent downstream pseudoknot in the 3′ UTR of the closely related bovine coronavirus (BCoV). This pseudoknot was also shown to be essential for replication, and it has a conserved counterpart in every group 1 and group 2 coronavirus. In MHV and BCoV, the bulged stem-loop and pseudoknot are, in part, mutually exclusive, because of the overlap of the last segment of the stem-loop and stem 1 of the pseudoknot. This led us to hypothesize that they form a molecular switch, possibly regulating a transition occurring during viral RNA synthesis. We have now performed an extensive genetic analysis of the two components of this proposed switch. Our results define essential and nonessential components of these structures and establish the limits to which essential parts of each element can be destabilized prior to loss of function. Most notably, we have confirmed the interrelationship of the two putative switch elements. Additionally, we have identified a pseudoknot loop insertion mutation that appears to point to a genetic interaction between the pseudoknot and a distant region of the genome.
The Spacing between Cysteines Two and Three of the LDL-A Module of Tva Is Important for Subgroup A Avian Sarcoma and Leukosis Virus Entry
Authors: Rai Tia , Marble Deborah , Rihani Kayla , Rong Lijun
Published: December 2003

Rong et al. have demonstrated previously that with a few substitutions, the fourth repeat of human low-density lipoprotein (hLDL-A4) receptor can functionally replace the LDL-A module of Tva, the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), in viral entry (L. Rong, K. Gendron, and P. Bates, Proc. Natl. Acad. Sci. USA
95:
8467-8472, 1998). Here we have shown that swapping the amino terminus of hLDL repeat 5 (hLDL-A5) with that of Tva, in addition to the corresponding substitutions made in human LDL-A4, was required to convert hLDL-A5 into an efficient ASLV-A receptor. These results substantiated our previous findings regarding the role of the specific residues in the viral interaction domain of Tva and demonstrated the critical role of the amino terminus of the Tva LDL-A module in ASLV-A infection. Furthermore, we have shown that the residues between cysteines 2 and 3 of the Tva LDL-A module in a Tva/LDL-A5 chimeric protein can be functionally replaced by the corresponding region of another LDL-A module, human LDL receptor-related protein repeat 22 (LDL-A22), to mediate efficient ASLV-A entry. Since the only conserved feature between the C2-C3 region of LDL-A22 and the Tva LDL-A module is that both contain nine amino acids of which none are conserved, we conclude that the spacing between C2 and C3 of the LDL-A module of Tva is an important determinant for ASLV-A entry. Thus, the present study provides strong evidence to support our hypothesis that one role of the N terminus of the LDL-A module of Tva is to allow proper folding and conformation of the protein for optimal interaction with the viral glycoprotein EnvA in ASLV-A entry.
Role of a Mutation in Human Cytomegalovirus Gene UL104 in Resistance to Benzimidazole Ribonucleosides
Authors: Komazin Gloria , Townsend Leroy B. , Drach John C.
Published: December 2003

The benzimidazole
d
-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV “terminase” and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA.
Late Domain-Dependent Inhibition of Equine Infectious Anemia Virus Budding
Authors: Shehu-Xhilaga Miranda , Ablan Sherimay , Demirov Dimiter G. , Chen Chaoping , Montelaro Ronald C. , Freed Eric O.
Published: December 2003

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5′) inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3′) potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5′, TSG-3′, and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5′ correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3′, whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.
Efficient Intracellular Assembly of Papillomaviral Vectors
Authors: Buck Christopher B. , Pastrana Diana V. , Lowy Douglas R. , Schiller John T.
Published: December 2003

Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.
Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production
Authors: Wu Ru Feng , Gu Ying , Xu You Cheng , Mitola Stefania , Bussolino Federico , Terada Lance S.
Published: December 2003

Human immunodeficiency virus type 1 Tat exerts prominent angiogenic effects which may lead to a variety of vasculopathic conditions in AIDS patients. Because endothelial cells undergo prominent cytoskeletal rearrangement during angiogenesis, we investigated the specific effects of Tat on the endothelial cell actin cytoskeleton. Glutathione
S
-transferase (GST)-Tat, at a level of 200 ng/ml (equivalent to 52 ng of Tat/ml), caused stress fiber disassembly, peripheral retraction, and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells. At 600 ng of GST-Tat/ml (157 ng of Tat/ml), actin structures were lost, and severe cytoskeletal collapse occurred. In contrast, GST-Tat harboring mutations within either the cysteine-rich or basic domains exerted minimal effects on the endothelial cytoskeleton. HUVEC expressing a DsRed-Tat fusion protein displayed similar actin rearrangements, followed by actin collapse, whereas neighboring nontransfected cells retained normal actin structures. Because active mutants of p21-activated kinase 1 (PAK1) induce identical changes in actin dynamics, we hypothesized that Tat exerts its cytoskeletal effects through PAK1. GST-Tat activated PAK1 within 5 min, and adenovirus delivery of a kinase-dead PAK1 [PAK1(K298A)] completely prevented cytoskeletal collapse induced by GST-Tat or DsRed-Tat and also blocked downstream activation of c-Jun N-terminal kinase. Further, GST-Tat increased phosphorylation of the NADPH oxidase subunit p47

phox

and caused its rapid redistribution to membrane ruffles. PAK1(K298A) blocked p47

phox

phosphorylation, and interference with NADPH oxidase function through superoxide scavenging or through expression of a transdominant inhibitor, p67(V204A), prevented GST-Tat-induced alterations in the actin cytoskeleton. We conclude that Tat induces actin cytoskeletal rearrangements through PAK1 and downstream activation of the endothelial NADPH oxidase.
NS-3 Protein of the
Junonia coenia
Densovirus Is Essential for Viral DNA Replication in an Ld 652 Cell Line and
Spodoptera littoralis
Larvae
Authors: Abd-Alla Adly , Jousset Françoise-Xavière , Li Yi , Fédière Gilles , Cousserans François , Bergoin Max
Published: December 2003

The genome of
Junonia coenia
densovirus (JcDNV) shares with members of the genus
Densovirus
the property of possessing structural (VP) and nonstructural (NS) genes in opposite orientations. The three NS genes located in the 5′ half on one strand encode three NS proteins assumed to be involved in viral DNA replication: NS-1 and NS-2, which are common to all DNVs, and a 28-kDa polypeptide, NS-3, with a unique sequence. Whereas the essential role played by JcDNV NS-1 in viral DNA replication has been clearly established (C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol.
76:
338-345, 2002), nothing is known of the biological function(s) of NS-3. To investigate this function, we designed constructs derived from pBRJ, a plasmid encompassing an infectious sequence of JcDNV DNA (M. Jourdan, F. X. Jousset, M. Gervais, S. Skory, M. Bergoin, and B. Dumas, Virology
179:
403-409, 1990), with partial or complete deletion of NS-3 sequence or with the ATG initiation codon mutated by site-directed mutagenesis. Transfection of these constructs to sensitive Ld 652 cells or
Spodoptera littoralis
larvae prevented the accomplishment of a productive cycle. We clearly established that the blocking of the replicative cycle in the absence of NS-3 expression occurred at the level of viral DNA replication. Replication of viral DNA could be restored by cotransfecting Ld 652 cells with a plasmid expressing JcDNV-NS-3 protein in
trans
. Time course analysis showed that NS-3 is produced early (6 h posttransfection) in the replicative cycle, and its production parallels that of replicative-form viral DNA. Finally, we present evidence that NS-1 and NS-2 proteins are synthesized at apparently the same levels whether or not NS-3 is expressed.
Functional Analysis of the Human Papillomavirus Type 16 E1
∧
E4 Protein Provides a Mechanism for In Vivo and In Vitro Keratin Filament Reorganization
Authors: Wang Qian , Griffin Heather , Southern Shirley , Jackson Deborah , Martin Ana , McIntosh Pauline , Davy Clare , Masterson Phillip J. , Walker Philip A. , Laskey Peter , Omary M. Bishr , Doorbar John
Published: December 2003

High-risk human papillomaviruses, such as human papillomavirus type 16 (HPV16), are the primary cause of cervical cancer. The HPV16 E1
∧
E4 protein associates with keratin intermediate filaments and causes network collapse when expressed in epithelial cells in vitro. Here, we show that keratin association and network reorganization also occur in vivo in low-grade cervical neoplasia caused by HPV16. The 16E1
∧
E4 protein binds to keratins directly and interacts strongly with keratin 18, a member of the type I intermediate-filament family. By contrast, 16E1
∧
E4 bound only weakly to keratin 8, a type II intermediate-filament protein, and showed no detectable affinity for the type III protein, vimentin. The N-terminal 16 amino acids of the 16E1
∧
E4 protein, which contains the YPLLXLL motif that is conserved among supergroup A viruses, were sufficient to target green fluorescent protein to the keratin network. When expressed in the SiHa cervical epithelial cell line, the full-length 16E1
∧
E4 protein caused an almost total inhibition of keratin dynamics, despite the phosphorylation of keratin 18 at serine 33, which normally leads to 14-3-3-mediated keratin solubilization. Mutant 16E1
∧
E4 proteins which lack the LLKLL motif, or which have lost amino acids from their C termini, and which were compromised in the ability to associate with keratins did not disturb normal keratin dynamics. 16E1
∧
E4 was found to exist as dimers and hexamers, whereas a C-terminal deletion mutant (16E1
∧
E4Δ87-92) existed as monomers and formed multimeric structures only poorly. Considered together, our results suggest that by associating with keratins through its N terminus, and by associating with itself through its C terminus, 16E1
∧
E4 may act as a keratin cross-linker and prevent the movement of keratins between the soluble and insoluble compartments. The increase in avidity associated with multimeric binding may contribute to the ability of 16E1
∧
E4 to sequester its cellular targets in the cytoplasm.
Nipah Virus: Vaccination and Passive Protection Studies in a Hamster Model
Authors: Guillaume V. , Contamin H. , Loth P. , Georges-Courbot M.-C. , Lefeuvre A. , Marianneau P. , Chua K. B. , Lam S. K. , Buckland R. , Deubel V. , Wild T. F.
Published: December 2003

Nipah virus, a member of the paramyxovirus family, was first isolated and identified in 1999 when the virus crossed the species barrier from fruit bats to pigs and then infected humans, inducing an encephalitis with up to 40% mortality. At present there is no prophylaxis for Nipah virus. We investigated the possibility of vaccination and passive transfer of antibodies as interventions against this disease. We show that both of the Nipah virus glycoproteins (G and F) when expressed as vaccinia virus recombinants induced an immune response in hamsters which protected against a lethal challenge by Nipah virus. Similarly, passive transfer of antibody induced by either of the glycoproteins protected the animals. In both the active and passive immunization studies, however, the challenge virus was capable of hyperimmunizing the vaccinated animals, suggesting that although the virus replicates under these conditions, the immune system can eventually control the infection.
Evidence for Frequent Recombination within SpeciesHuman Enterovirus BBased on Complete Genomic Sequences of All Thirty-Seven Serotypes
Authors: Oberste M. Steven , Maher Kaija , Pallansch Mark A.
Published: December 2003

The speciesHuman enterovirus B(HEV-B) in the familyPicornaviridaeconsists of coxsackievirus A9; coxsackieviruses B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. We have determined complete genome sequences for the remaining 22 HEV-B serotypes whose sequences were not represented in public databases and analyzed these in conjunction with previously available complete sequences in GenBank. Members of HEV-B were monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5′-nontranslated region (NTR), where they are known to cluster with members of HEV-A. Within HEV-B, phylogenies constructed from the structural (P1) and nonstructural regions of the genome (P2 and P3) are incongruent, suggesting that recombination had occurred. Similarity plots and bootscanning analysis across the complete genome identified multiple sites at which the phylogeny of a given strain's sequence shifted, indicating potential recombination points. These points are distributed in the 5′-NTR and throughout P2 and P3, but no sites with >80% bootstrap support were identified within the capsid. Individual sequence comparisons and phylogenetic analyses suggest that members of HEV-B have recombined with one another on multiple occasions, resulting in a complex mosaic of sequences derived from multiple parental viruses in the nonstructural regions of the genome. We conclude that RNA recombination is a common mechanism for enterovirus evolution and that recombination within the nonstructural regions of the genome (P2 and P3) has been observed only among members of the same species.
Differential Effects of Actin Cytoskeleton Dynamics on Equine Infectious Anemia Virus Particle Production
Authors: Chen Chaoping , Weisz Ora A. , Stolz Donna B. , Watkins Simon C. , Montelaro Ronald C.
Published: December 2003

Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.
A Reovirus of the Fungus
Cryphonectria parasitica
That Is Infectious as Particles and Related to the
Coltivirus
Genus of Animal Pathogens
Authors: Hillman Bradley I. , Supyani S. , Kondo Hideki , Suzuki Nobuhiro
Published: December 2003

RNA viruses of filamentous fungi fall into two broad categories, those that contain double-stranded RNA (dsRNA) genomes in rigid particles and those that are more closely related to positive-sense, single-stranded RNA viruses with dsRNA replicative intermediates found within lipid vesicles. Effective infectivity systems have been described for the latter, using RNA transcripts, but not for the former. We report the characterization of a reovirus from
Cryphonectria parasitica
, the filamentous fungus that causes chestnut blight disease. The virus substantially reduces the virulence of the fungus and results in dramatically altered colony morphology, as well as changes in other associated fungal traits, relative to the virus-free isogenic strain. Virus particles from infected mycelium contained 11 segments of dsRNA and showed characteristics typical of the family
Reoviridae
. Sequences of the largest three segments revealed that the virus is closely related to the
Coltivirus
genus of animal pathogens, which includes the human pathogen
Colorado tick fever virus
. The introduction of purified virus particles into protoplasts from virus-free isolates of the fungus resulted in a newly infected mycelium with the same morphology and virus composition as the original virus-infected isolate. This represents the completion of Koch's postulates for a true dsRNA virus from a filamentous fungus and the description of a definitive fungal member of the family
Reoviridae
.
Genome Structure and Thymic Expression of an Endogenous Retrovirus in Zebrafish
Authors: Shen Ching-Hung , Steiner Lisa A.
Published: December 2003

In a search for previously unknown genes that are required for lymphocyte development in zebrafish, a retroviral sequence was identified in a subtracted thymus cDNA library and in genomic DNA libraries. The provirus is 11.2 kb and contains intact open reading frames for the
gag
,
pol
, and
env
genes, as well as nearly identical flanking long terminal repeat sequences. As determined by in situ hybridization, the thymus appears to be a major tissue for retroviral expression in both larval and adult fish. Several viral transcripts were found by Northern blotting in the adult thymus. The provirus was found at the same genomic locus in sperm from four fish, suggesting that it is an endogenous retrovirus. Phylogenetic analysis indicates that it is closest to, yet distinct from, the cluster of murine leukemia virus-related retroviruses, suggesting that this virus represents a new group of retroviruses.