bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroendocrine regulation of sebocytes – a pathogenetic link between stress and acne
Authors: Zouboulis Christos C. , Böhm Markus
Published: October 2004

Abstract:  A causative link between emotional stress and acne has long been postulated. There is mounting evidence that the molecular mechanism underlying this observation is related to the expression of receptors for several neuroendocrine mediators by the sebaceous gland. Recent and ongoing studies have indicated that human sebocytes express functional receptors for corticotropin‐releasing hormone, melanocortins, β‐endorphin, vasoactive intestinal polypeptide, neuropeptide Y and calcitonin gene‐related peptide. After ligand binding, these receptors modulate the production of inflammatory cytokines, proliferation, differentiation, lipogenesis and androgen metabolism in sebocytes. By means of their autocrine, paracrine and endocrine actions, these neuroendocrine factors appear to mediate centrally and topically induced stress towards the sebaceous gland, ultimately affecting the clinical course of acne.
Melanocortins in fibroblast biology – current update and future perspective for dermatology
Authors: Böhm Markus , Luger Thomas A.
Published: October 2004

Abstract: The skin is a target organ and source for proopiomelanocortin (POMC)‐derived peptides, such as α‐melanocyte‐stimulating hormone (α‐MSH), which acts by binding to melanocortin receptors (MC‐Rs). Recent progress in our understanding of the cutaneous POMC system has demonstrated that human dermal fibroblasts (HDFs) are a novel target for α‐MSH. MC‐1R is expressed by HDFsin vitroandin situ. MC‐1R expression is also detectable in human connective tissue sheath fibroblasts (CTSFs) and in dermal papilla cells (DPCs) of the hair follicle, the latter concomitantly expressing MC‐1R and MC‐4Rin vitroandin situ. Both HDFs and DPCs are capable of generating POMC‐derived peptides, although cell‐specific differences exist in the expression of prohormone convertases and the amounts of POMC‐derived peptides generated. Functional studies have shown that α‐MSH exerts anti‐inflammatory actions in human fibroblastic skin cells by suppressing interleukin‐1 (IL‐1)‐induced IL‐8 production, activation of the transcription factor activator protein‐1 (AP‐1) and induction of intercellular adhesion molecule‐1 by interferon‐α. In addition, α‐MSH antagonizes the effect of transforming growth factor‐β1(TGF‐β1) on collagen synthesis in HDFsin vitroand exerts antifibrogenic activity in a mouse model of cutaneous fibrosis. These findings indicate that fibroblastic cells participate in the cutaneous POMC system in which α‐MSH appears to act as a modulator of inflammatory and fibrogenic responses. The biological activities of α‐MSH in fibroblastic cells of the skin point towards novel clues in our understanding of the pathophysiology of fibrotic skin disorders and inflammatory diseases of the hair follicle and, finally, suggest innovative therapeutic options for the treatment of these conditions.
Upregulation of RANTES Gene Expression in Neuroglia by Japanese Encephalitis Virus Infection
Authors: Chen Chun-Jung , Chen Jian-Hong , Chen Shih-Yun , Liao Su-Lan , Raung Shue-Ling
Published: October 2004

Infection with Japanese encephalitis virus (JEV) causes cerebral inflammation and stimulates inflammatory cytokine expression. Glial cells orchestrate immunocyte recruitment to focal sites of viral infection within the central nervous system (CNS) and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since immune cell infiltration is prominent, we investigated the production of a responding chemoattractant, RANTES (regulated upon activation, normal T-cell expressed and secreted), in response to JEV infection of glial cells. Infection with JEV was found to elicit the production of RANTES from primary neurons/glia, mixed glia, microglia, and astrocytes but not from neuron cultures. The production of RANTES did not seem to be directly responsible for JEV-induced neuronal death but instead contributed to the recruitment of immune cells. RANTES expression required viral replication and the activation of extracellular signal-regulated kinase (ERK) as well as transcription factors, including nuclear factor kappa B (NF-κB) and nuclear factor IL-6 (NF-IL-6). The induction of RANTES expression by JEV infection in glial cells needed the coordinate activation of NF-κB and NF-IL-6. Using enzymatic inhibitors, we demonstrated a strong correlation between the ERK signaling pathway and RANTES expression. However, JEV replication was not dependent on the activation of ERK, NF-κB, and NF-IL-6. Altogether, these results demonstrated that infection of glial cells by JEV provided the early ERK-, NF-κB-, and NF-IL-6-mediated signals that directly activated RANTES expression, which might be involved in the initiation and amplification of inflammatory responses in the CNS.
New Class of Orthopoxvirus Antiviral Drugs That Block Viral Maturation
Authors: Byrd Chelsea M. , Bolken Tove' C. , Mjalli Adnan M. , Arimilli Murty N. , Andrews Robert C. , Rothlein Robert , Andrea Tariq , Rao Mohan , Owens Katrina L. , Hruby Dennis E.
Published: October 2004

By using a homology-based bioinformatics approach, a structural model of the vaccinia virus (VV) I7L proteinase was developed. A unique chemical library of ∼51,000 compounds was computationally queried to identify potential active site inhibitors. The resulting biased subset of compounds was assayed for both toxicity and the ability to inhibit the growth of VV in tissue culture cells. A family of chemotypically related compounds was found which exhibits selective activity against orthopoxviruses, inhibiting VV with 50% inhibitory concentrations of 3 to 12 μM. These compounds exhibited no significant cytotoxicity in the four cell lines tested and did not inhibit the growth of other organisms such as
Saccharomyces cerevisiae
,
Pseudomonas aeruginosa
, adenovirus, or encephalomyocarditis virus. Phenotypic analyses of virus-infected cells were conducted in the presence of active compounds to verify that the correct biochemical step (I7L-mediated core protein processing) was being inhibited. Electron microscopy of compound-treated VV-infected cells indicated a block in morphogenesis. Compound-resistant viruses were generated and resistance was mapped to the I7L open reading frame. Transient expression with the mutant I7L gene rescued the ability of wild-type virus to replicate in the presence of compound, indicating that this is the only gene necessary for resistance. This novel class of inhibitors has potential for development as an efficient antiviral drug against pathogenic orthopoxviruses, including smallpox.
Identification and Characterization of Persistent Human Erythrovirus Infection in Blood Donor Samples
Authors: Candotti Daniel , Etiz Nermin , Parsyan Armen , Allain Jean-Pierre
Published: October 2004

The presence of human erythrovirus DNA in 2,440 blood donations from the United Kingdom and sub-Saharan Africa (Ghana, Malawi, and South Africa) was screened. Sensitive qualitative and real-time quantitative PCR assays revealed a higher prevalence of persistent infection with the simultaneous presence of immunoglobulin G (IgG) and viral DNA (0.55 to 1.3%) than previously reported. This condition was characterized by a low viral load (median, 558 IU/ml; range, 42 to 135,000 IU/ml), antibody-complexed virus, free specific IgG, and potentially infectious free virus. Human erythrovirus genotype 1 (formerly parvovirus B19) was prevalent in the United Kingdom, Malawi, and South Africa. In contrast, only human erythrovirus genotype 3 (erythrovirus variant V9) was prevalent in Ghana. Genotype 3 had considerable genetic diversity, clustering in two probable subtypes. Genotype 1-based antibody assays failed to detect 38.5% of Ghanaian samples containing antibodies to genotype 3 virus but did not fail to detect cases of persistent infection. This study indicates a potential African origin of genotype 3 human erythrovirus and considerable shortcomings in the tools currently used to diagnose erythrovirus infection.
Novel Host Range and Cytopathic Variant of Ecotropic Friend Murine Leukemia Virus
Authors: Jung Yong Tae , Wu Tiyun , Kozak Christine A.
Published: October 2004

A variant ecotropic Friend murine leukemia virus, F-S MLV, is capable of inducing the formation of large multinucleated syncytia in
Mus dunni
cells. This cytopathicity resembles that of Spl574 MLV, a novel variant recently isolated from the spleen of a
Mus spicilegus
mouse neonatally inoculated with Moloney MLV. F-S MLV is an N-tropic Friend MLV that also has the unusual ability to infect hamster cells, which are normally resistant to mouse ecotropic MLVs. Syncytium induction by both F-S MLV and Spl574 is accompanied by the accumulation of large amounts of unintegrated viral DNA, a hallmark of pathogenic retroviruses, but not previously reported for mouse ecotropic gammaretroviruses. Sequencing and site-specific mutagenesis determined that the syncytium-inducing phenotype of F-S MLV can be attributed to a single amino acid substitution (S84A) in the VRA region of the viral
env
gene. This site corresponds to that of the single substitution previously shown to be responsible for the cytopathicity of Spl574, S82F. The S84A substitution in F-S MLV also contributes to the ability of this virus to infect hamster cells, but Spl574 MLV is unable to infect hamster cells. Because this serine residue is one of the critical amino acids that form the CAT-1 receptor binding site, and because
M. dunni
and hamster cells have variant CAT-1 receptors, these results suggest that syncytium formation as well as altered host range may be a consequence of altered interaction between virus and receptor.
Cell Cycle Arrest in G
2
Induces Human Immunodeficiency Virus Type 1 Transcriptional Activation through Histone Acetylation and Recruitment of CBP, NF-κB, and c-Jun to the Long Terminal Repeat Promoter
Authors: Thierry Sylvain , Marechal Vincent , Rosenzwajg Michelle , Sabbah Michèle , Redeuilh Gérard , Nicolas Jean-Claude , Gozlan Joël
Published: October 2004

In human immunodeficiency virus type 1 (HIV-1)-infected cells, a cell cycle arrest in G
2
increases viral expression and may represent a strategy for the virus to optimize its expression. In latently infected cells, balance between viral silencing and reactivation relies on the nucleosomal organization of the integrated long terminal repeat (LTR). It is shown here that nucleosome nuc-1, which is located downstream of the TATA box, is specifically modified when latently infected cells are arrested in G
2
by chemical inducers. Notably, histones H3 and H4 are hyperacetylated, and this modification is associated with an increased LTR-driven transcription. nuc-1 hyperacetylation is also associated with the recruitment of histone acetyltransferase CBP and transcription factors NF-κB and c-Jun. NF-κB and/or c-Jun binding to the LTR in G
2
-arrested cells appears to be required for CBP recruitment as well as for nuc-1 remodeling and viral reactivation.
The Severe Acute Respiratory Syndrome Coronavirus Nsp15 Protein Is an Endoribonuclease That Prefers Manganese as a Cofactor
Authors: Bhardwaj Kanchan , Guarino Linda , Kao C. Cheng
Published: October 2004

Nonstructural protein 15 (Nsp15) of the severe acute respiratory syndrome coronavirus (SARS-CoV) produced in
Escherichia coli
has endoribonuclease activity that preferentially cleaved 5′ of uridylates of RNAs. Blocking either the 5′ or 3′ terminus did not affect cleavage. Double- and single-stranded RNAs were both substrates for Nsp15 but with different kinetics for cleavage. Mn
2+
at 2 to 10 mM was needed for optimal endoribonuclease activity, but Mg
2+
and several other divalent metals were capable of supporting only a low level of activity. Concentrations of Mn
2+
needed for endoribonuclease activity induced significant conformation change(s) in the protein, as measured by changes in tryptophan fluorescence. A similar endoribonucleolytic activity was detected for the orthologous protein from another coronavirus, demonstrating that the endoribonuclease activity of Nsp15 may be common to coronaviruses. This work presents an initial biochemical characterization of a novel coronavirus endoribonuclease.
Vesicular Stomatitis Virus Evolution during Alternation between Persistent Infection in Insect Cells and Acute Infection in Mammalian Cells Is Dominated by the Persistence Phase
Authors: Zárate Selene , Novella Isabel S.
Published: October 2004

Vesicular stomatitis virus has the potential for very rapid evolution in the laboratory, but like many other arboviruses, it evolves at a relatively slow rate in the natural environment. Previous work showed that alternating replication in different cell types does not promote stasis. In order to determine whether other factors promote stasis, we compared the fitness trajectories of populations evolving during acute infections in mammalian cells, populations evolving during persistent infections in insect cells, and populations evolving during alternating acute and persistent infection cycles. Populations evolving under constant conditions increased in fitness in the environment in which they replicated. An asymmetric trade-off was observed such that acute infection had no cost for persistence but persistent replication had a dramatic cost for acute infection in mammalian cells. After an initial period of increase, fitness remained approximately constant in all the populations that included persistent replication, but fitness continuously increased in populations evolving during acute infections. Determination of the consensus sequence of the genes encoding the N, P, M, and G proteins showed that the pattern of mutation accumulation was coherent with fitness changes during persistence so that once fitness reached a maximum, the rate of mutation accumulation dropped. Persistent replication dominated both the genetic and the phenotypic evolution of the populations that alternated between acute infection of mammalian cells and persistence in insect cells, and fitness loss was observed in the mammalian environment despite periodic replication in mammalian cells. These results show that stasis can be achieved without good levels of adaptation to both the mammalian and the insect environments.
Uptake Pathway of Polyomavirus via Ganglioside GD1a
Authors: Gilbert Joanna , Benjamin Thomas
Published: October 2004

The pathway of entry of polyomavirus (Py) has been investigated with glycolipid-deficient C6 cells and added ganglioside GD1a as a specific virus receptor. Unsupplemented C6 cells show a low basal level of infection but become highly infectable by Py following preincubation with the sialic acid-containing ganglioside GD1a (38). Addition of GD1a has no effect on the overall level of virus binding but mediates the internalization and transit of virus to the endoplasmic reticulum (ER). This pathway of entry is cholesterol and caveola dependent and requires intact microtubules as well as a dynamic state of the microfilament system. In contrast to vesicular transport of other cargo via glycolipids, Py particles do not appear to pass through the Golgi apparatus. Colcemid and brefeldin A block transport of the virus to the ER in GD1a-supplemented cells and lead to accumulation of virus in a caveolin-1-containing environment. Several features distinguish the efficient GD1a-mediated pathway of virus uptake from the less-efficient pathway of basal infection in C6 cells.
Entry of Herpes Simplex Virus Mediated by Chimeric Forms of Nectin1 Retargeted to Endosomes or to Lipid Rafts Occurs through Acidic Endosomes
Authors: Gianni Tatiana , Campadelli-Fiume Gabriella , Menotti Laura
Published: October 2004

Herpes simplex virus (HSV) enters cells by fusion with target membranes, commonly the plasma membrane. In some cells, including CHO cells expressing the nectin1 or herpesvirus entry mediator receptors, entry occurs through an endocytic route. We report the following results. (i) When expressed in J cells, nectin1 and HVEM mediated a pathway of entry insensitive to endosome acidification inhibitors. (ii) A chimeric nectin1 receptor competent for endosomal uptake by fusion of the nectin1 ectodomain with the transmembrane sequence and cytoplasmic tail of the epidermal growth factor receptor (EGFR1) (nectin1-EGFR1) and chimeric nectin1 sorted to lipid rafts by a glycosylphosphatidylinositol anchor mediated endocytic entry blocked by the early endosome inhibitor wortmannin and by the endosome acidification inhibitors bafilomycin and NH
4
Cl. (iii) Entry mediated by nectin1-EGFR1 was selectively inhibited by AG1478, a tyrosine phosphorylation inhibitor that targets the EGFR1 cytoplasmic tail and blocks the signaling pathway that culminates in clathrin-dependent uptake of the receptor into endosomes. We draw the following conclusions. (i) The same receptor may initiate different routes of infection, depending on the cell in which it is expressed. Hence, the cell is a determinant that controls whether a given receptor initiates a plasma membrane or an endocytic route of entry. (ii) Receptors whose physiology involves uptake into endosomes or sorting to lipid rafts are suitable to serve as HSV receptors. (iii) Structural features of the receptors are additional determinants that control whether HSV entry occurs at the plasma membrane or at endosomes. These findings are relevant to studies of HSV retargeting to specific receptors.
CXCR4-Dependent Infection of CD8
+
, but Not CD4
+
, Lymphocytes by a Primary Human Immunodeficiency Virus Type 1 Isolate
Authors: Zerhouni Bouchra , Nelson Julie A. E. , Saha Kunal
Published: October 2004

We recently isolated from an infant an X4-syncytium-inducing (SI) human immunodeficiency virus type 1 (HIV-1) variant (92US143-T8) that was able to infect CD8
+
lymphocytes independently of CD4. Although it was CD4 independent, the 92US143-T8 isolate also maintained the ability to infect CD4
+
cells. In the present study, we investigated the role of CXCR4 in the infection of CD4
+
and CD8
+
cells by this primary isolate. The expression of CXCR4 was down modulated in CD8
+
lymphocytes after infection with the 93US143-T8 isolate. Infection of CD8
+
lymphocytes by the 93US143-T8 isolate was prevented by treatment with AMD3100, a specific antagonist for CXCR4, indicating CXCR4-dependent infection. Interestingly, AMD3100 treatment had no inhibitory role in the infection of purified CD4
+
lymphocytes by the same isolate. Furthermore, AMD3100 treatment failed to prevent infection of known CD4
+
CXCR4
+
T-cell lines (MT-2 and CEM) by the 93US143-T8 isolate. In fact, virus replication in the CD4
+
cells was often enhanced in the presence of AMD3100. Viruses produced from the infected CD4
+
cells in the presence of AMD3100 maintained an unchanged envelope genotype and an SI phenotype. For the first time, these results provide evidence of CXCR4-dependent infection of CD8
+
lymphocytes by a primary HIV-1 isolate. This study also shows a different mode of infection for the CD4
+
and CD8
+
lymphocytes by the same HIV-1 variant. Finally, our findings suggest that a more careful evaluation is necessary before the random use of AMD3100 as a new entry inhibitor in patients harboring SI HIV-1 strains.
Adenovirus E3-6.7K Protein Is Required in Conjunction with the E3-RID Protein Complex for the Internalization and Degradation of TRAIL Receptor 2
Authors: Lichtenstein Drew L. , Doronin Konstantin , Toth Karoly , Kuppuswamy Mohan , Wold William S. M. , Tollefson Ann E.
Published: October 2004

Adenoviruses (Ads) encode several proteins within the early region 3 (E3) transcription unit that help protect infected cells from elimination by the immune system. Among these immunomodulatory proteins, the receptor internalization and degradation (RID) protein complex, which is composed of the RIDα (formerly E3-10.4K) and RIDβ (formerly E3-14.5K) subunits, stimulates the internalization and degradation of certain members of the tumor necrosis factor (TNF) receptor superfamily, thus blocking apoptosis initiated by Fas and TNF-related apoptosis-inducing ligand (TRAIL). The experiments reported here show that TRAIL receptor 2 (TR2) is cleared from the cell surface in Ad-infected cells. Virus mutants containing deletions that span E3 were used to show that the RID and E3-6.7K proteins are both necessary for the internalization and degradation of TR2, whereas only the RID protein is required for TRAIL receptor 1 downregulation. In addition, replication-defective Ad vectors that express individual E3 proteins were used to establish that the RID and E3-6.7K proteins are sufficient to clear TR2. These data demonstrate that E3-6.7K is an important component of the antiapoptosis arsenal encoded by the E3 transcription unit of subgroup C Ads.
Postentry Neutralization of Adenovirus Type 5 by an Antihexon Antibody
Authors: Varghese Robin , Mikyas Yeshi , Stewart Phoebe L. , Ralston Robert
Published: October 2004

Antibodies against hexon, the major coat protein of adenovirus (Ad), are an important component of the neutralizing activity in serum from naturally infected humans and experimentally infected animals. The mechanisms by which antihexon antibodies neutralize the virus have not been defined. As a model system, murine monoclonal antibodies raised against Ad type 5 (Ad5) were screened for antihexon binding and neutralization activity; one monoclonal antibody, designated 9C12, was selected for further characterization. The minimum ratio of 9C12 to Ad5 required for neutralization was 240 antibody molecules per virus particle, or 1 antibody per hexon trimer. Analysis of antibody-virus complexes by dynamic light scattering and negative-stain electron microscopy (EM) showed that the virus particles were coated with electron-dense material but not aggregated at neutralizing ratios. Cryo-EM image reconstruction of the antibody-virus complex showed that the surface of the virus particle was covered by a meshwork of 9C12 antibody density, consistent with bivalent binding at multiple sites. Confocal analysis revealed that viral attachment, cell entry, and intracellular transport to the nuclear periphery still occur in the presence of neutralizing levels of 9C12. A model is presented for neutralization of Ad by an antihexon antibody in which the hexon capsid is cross-linked by antibodies, thus preventing virus uncoating and nuclear entry of viral DNA.
Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles
Authors: Finke Stefan , Brzózka Krzysztof , Conzelmann Karl-Klaus
Published: October 2004

Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD ΔP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.
Distinct Human Immunodeficiency Virus Type 1 Subtype A Virus Circulating in West Africa: Sub-Subtype A3
Authors: Meloni Seema Thakore , Kim Bohye , Sankalé Jean-Louis , Hamel Donald J. , Tovanabutra Sodsai , Mboup Souleymane , McCutchan Francine E. , Kanki Phyllis J.
Published: October 2004

Phylogenetic analyses demonstrate significant diversity in worldwide circulating strains of human immunodeficiency virus type 1 (HIV-1). Detailed studies have revealed a complex pattern of intersubtype recombinations, as well as evidence of sub-subtypes circulating in various populations. In this study, we characterized an HIV-1 strain that had previously been identified as a distinct subcluster within the subtype A radiation based on partial sequence data. These viruses were of particular interest given that we recently found that their prevalence was significantly higher in dually infected individuals compared to women who were singly infected with HIV-1. Five viruses isolated from commercial sex workers in Dakar, Senegal, were full-length PCR amplified and sequenced. Phylogenetic analyses indicated that, whereas three of these viruses were closely related and clustered overall within the HIV-1 subtype A radiation, they were distinct from previously characterized sub-subtype A1 and A2 viruses. The clustering pattern was maintained in the individual
gag
,
pol
, and
env
regions of the genome. Distance calculations between these viruses, which we termed A3, and other reference sub-subtype A1 and A2 viruses fell in the range of distances between previously characterized sub-subtype groups. In addition, we found evidence of two A3-containing recombinants in our cohort. These recombinants are mosaics composed of sequence from both sub-subtype A3 and CRF02_AG, the major circulating recombinant form in this West African population. Based on phylogenetic analyses, we propose that the group of viruses found in the Dakar sex worker cohort, previously referred to as HIV-1 A subcluster 2, be referred to as HIV-1 sub-subtype A3.
Structural Basis for Coevolution of a Human Immunodeficiency Virus Type 1 Nucleocapsid-p1 Cleavage Site with a V82A Drug-Resistant Mutation in Viral Protease
Authors: Prabu-Jeyabalan Moses , Nalivaika Ellen A. , King Nancy M. , Schiffer Celia A.
Published: October 2004

Maturation of human immunodeficiency virus (HIV) depends on the processing of Gag and Pol polyproteins by the viral protease, making this enzyme a prime target for anti-HIV therapy. Among the protease substrates, the nucleocapsid-p1 (NC-p1) sequence is the least homologous, and its cleavage is the rate-determining step in viral maturation. In the other substrates of HIV-1 protease, P1 is usually either a hydrophobic or an aromatic residue, and P2 is usually a branched residue. NC-p1, however, contains Asn at P1 and Ala at P2. In response to the V82A drug-resistant protease mutation, the P2 alanine of NC-p1 mutates to valine (AP2V). To provide a structural rationale for HIV-1 protease binding to the NC-p1 cleavage site, we solved the crystal structures of inactive (D25N) WT and V82A HIV-1 proteases in complex with their respective WT and AP2V mutant NC-p1 substrates. Overall, the WT NC-p1 peptide binds HIV-1 protease less optimally than the AP2V mutant, as indicated by the presence of fewer hydrogen bonds and fewer van der Waals contacts. AlaP2 does not fill the P2 pocket completely; PheP1′ makes van der Waals interactions with Val82 that are lost with the V82A protease mutation. This loss is compensated by the AP2V mutation, which reorients the peptide to a conformation more similar to that observed in other substrate-protease complexes. Thus, the mutant substrate not only binds the mutant protease more optimally but also reveals the interdependency between the P1′ and P2 substrate sites. This structural interdependency results from coevolution of the substrate with the viral protease.
Novel Origin of the 1918 Pandemic Influenza Virus Nucleoprotein Gene
Authors: Reid Ann H. , Fanning Thomas G. , Janczewski Thomas A. , Lourens Raina M. , Taubenberger Jeffery K.
Published: October 2004

The nucleoprotein (NP) gene of the 1918 pandemic influenza A virus has been amplified and sequenced from archival material. The NP gene is known to be involved in many aspects of viral function and to interact with host proteins, thereby playing a role in host specificity. The 1918 NP amino acid sequence differs at only six amino acids from avian consensus sequences, consistent with reassortment from an avian source shortly before 1918. However, the nucleotide sequence of the 1918 NP gene has more than 170 differences from avian strain consensus sequences, suggesting substantial evolutionary distance from known avian strain sequences. Both the gene and protein sequences of the 1918 NP fall within the mammalian clade upon phylogenetic analysis. The evolutionary distance of the 1918 NP sequences from avian and mammalian strain sequences is examined, using several different parameters. The results suggest that the 1918 strain did not retain the previously circulating human NP. Nor is it likely to have obtained its NP by reassortment with an avian strain similar to those now characterized. The results are consistent with the existence of a currently unknown host for influenza, with an NP similar to current avian strain NPs at the amino acid level but with many synonymous nucleotide differences, suggesting evolutionary isolation from the currently characterized avian influenza virus gene pool.
Transition from Acute to Persistent Theiler's Virus Infection Requires Active Viral Replication That Drives Proinflammatory Cytokine Expression and Chronic Demyelinating Disease
Authors: Trottier Mark , Schlitt Brian P. , Kung Aisha Y. , Lipton Howard L.
Published: October 2004

The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10- to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.
ChimeriVax-West Nile Virus Live-Attenuated Vaccine: Preclinical Evaluation of Safety, Immunogenicity, and Efficacy
Authors: Arroyo Juan , Miller Chuck , Catalan John , Myers Gwendolyn A. , Ratterree Marion S. , Trent Dennis W. , Monath Thomas P.
Published: October 2004

The availability of ChimeriVax vaccine technology for delivery of flavivirus protective antigens at the time West Nile (WN) virus was first detected in North America in 1999 contributed to the rapid development of the vaccine candidate against WN virus described here. ChimeriVax-Japanese encephalitis (JE), the first live- attenuated vaccine developed with this technology has successfully undergone phase I and II clinical trials. The ChimeriVax technology utilizes yellow fever virus (YF) 17D vaccine strain capsid and nonstructural genes to deliver the envelope gene of other flaviviruses as live-attenuated chimeric viruses. Amino acid sequence homology between the envelope protein (E) of JE and WN viruses facilitated targeting attenuating mutation sites to develop the WN vaccine. Here we discuss preclinical studies with the ChimeriVax-WN virus in mice and macaques. ChimeriVax-WN virus vaccine is less neurovirulent than the commercial YF 17D vaccine in mice and nonhuman primates. Attenuation of the virus is determined by the chimeric nature of the construct containing attenuating mutations in the YF 17D virus backbone and three point mutations introduced to alter residues 107, 316, and 440 in the WN virus E protein gene. The safety, immunogenicity, and efficacy of the ChimeriVax-WN
02
vaccine in the macaque model indicate the vaccine candidate is expected to be safe and immunogenic for humans.
Granzymes and Caspase 3 Play Important Roles in Control of Gammaherpesvirus Latency
Authors: Loh Joy , Thomas Dori A. , Revell Paula A. , Ley Timothy J. , Virgin Herbert W.
Published: October 2004

Gammaherpesviruses can establish lifelong latent infections in lymphoid cells of their hosts despite active antiviral immunity. Identification of the immune mechanisms which regulate gammaherpesvirus latent infection is therefore essential for understanding how gammaherpesviruses persist for the lifetime of their host. Recently, an individual with chronic active Epstein-Barr virus infection was found to have mutations in perforin, and studies using murine gammaherpesvirus 68 (γHV68) as a small-animal model for gammaherpesvirus infection have similarly revealed a critical role for perforin in regulating latent infection. These results suggest involvement of the perforin/granzyme granule exocytosis pathway in immune regulation of gammaherpesvirus latent infection. In this study, we examined γHV68 infection of knockout mice to identify specific molecules within the perforin/granzyme pathway which are essential for regulating gammaherpesvirus latent infection. We show that granzymes A and B and the granzyme B substrate, caspase 3, are important for regulating γHV68 latent infection. Interestingly, we show for the first time that orphan granzymes encoded in the granzyme B gene cluster are also critical for regulating viral infection. The requirement for specific granzymes differs for early versus late forms of latent infection. These data indicate that different granzymes play important and distinct roles in regulating latent gammaherpesvirus infection.
Murine T Cells Potently Restrict Human Immunodeficiency Virus Infection
Authors: Baumann Jörg G. , Unutmaz Derya , Miller Michael D. , Breun Sabine K. J. , Grill Stacy M. , Mirro Jane , Littman Dan R. , Rein Alan , KewalRamani Vineet N.
Published: October 2004

Development of a mouse model for human immunodeficiency virus type 1 (HIV-1) infection has advanced through the progressive identification of host cell factors required for HIV-1 replication. Murine cells lack HIV-1 receptor molecules, do not support efficient viral gene expression, and lack factors necessary for the assembly and release of virions. Many of these blocks have been described using mouse fibroblast cell lines. Here we identify a postentry block to HIV-1 infection in mouse T-cell lines that has not been detected in mouse fibroblasts. While murine fibroblastic lines are comparable to human T-cell lines in permissivity to HIV-1 transduction, infection of murine T cells is 100-fold less efficient. Virus entry occurs efficiently in murine T cells. However, reduced efficiency of the completion of reverse transcription and nuclear transfer of the viral preintegration complex are observed. Although this block has similarities to the restriction of murine retroviruses by Fv1, there is no correlation of HIV-1 susceptibility with cellular Fv1 genotypes. In addition, the block to HIV-1 infection in murine T-cell lines cannot be saturated by a high virus dose. Further studies of this newly identified block may lend insight into the early events of retroviral replication and reveal new targets for antiretroviral interventions.
Coxsackievirus Group B Type 3 Infection Upregulates Expression of Monocyte Chemoattractant Protein 1 in Cardiac Myocytes, Which Leads to Enhanced Migration of Mononuclear Cells in Viral Myocarditis
Authors: Shen Yan , Xu Wei , Chu Yi-Wei , Wang Ying , Liu Quan-Sheng , Xiong Si-Dong
Published: October 2004

Coxsackievirus group B type 3 (CVB3) is an important cause of viral myocarditis. The infiltration of mononuclear cells into the myocardial tissue is one of the key events in viral myocarditis. Immediately after CVB3 infects the heart, the expression of chemokine(s) by infected myocardial cells may be the first trigger for inflammatory infiltration and immune response. However, it is unknown whether CVB3 can induce the chemokine expression in cardiac myocytes. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemokine that stimulates the migration of mononuclear cells. The objective of the present study was to investigate the effect of CVB3 infection on MCP-1 expression in murine cardiac myocytes and the role of MCP-1 in migration of mononuclear cells in viral myocarditis. Our results showed that the expression of MCP-1 was significantly increased in cardiac myocytes after wild-type CVB3 infection in a time- and dose-dependent manner, which resulted in enhanced migration of mononuclear cells in mice with viral myocarditis. The migration of mononuclear cells was partially abolished by antibodies specific for MCP-1 in vivo and in vitro. Administration of anti-MCP-1 antibody prevented infiltration of mononuclear cells bearing the MCP-1 receptor CCR2 in mice with viral myocarditis. Infection by UV-irradiated CVB3 induced rapid and transient expression of MCP-1 in cardiac myocytes. In conclusion, our results indicate that CVB3 infection stimulates the expression of MCP-1 in myocardial cells, which subsequently leads to migration of mononuclear cells in viral myocarditis.
Enhanced Cellular Receptor Usage by a Bioselected Variant of Coxsackievirus A21
Authors: Johansson E. Susanne , Xing Li , Cheng R. Holland , Shafren Darren R.
Published: October 2004

Decay-accelerating factor (DAF) functions as cell attachment receptor for a wide range of human enteroviruses. The Kuykendall prototype strain of coxsackievirus A21 (CVA21) attaches to DAF but requires interactions with intercellular cell adhesion molecule 1 (ICAM-1) to infect cells. We show here that a bioselected variant of CVA21 (CVA21-DAFv) generated by multiple passages in DAF-expressing, ICAM-1-negative rhabdomyosarcoma (RD) cells acquired the capacity to induce rapid and complete lysis of ICAM-1-deficient cells while retaining the capacity to bind ICAM-1. CVA21-DAFv binding to DAF on RD cells mediated lytic infection and was inhibited by either antibody blockade with a specific anti-DAF SCR1 monoclonal antibody (MAb) or soluble human DAF. Despite being bioselected in RD cells, CVA21-DAFv was able to lytically infect an additional ICAM-1-negative cancer cell line via DAF interactions alone. The finding that radiolabeled CVA21-DAFv virions are less readily eluted from surface-expressed DAF than are parental CVA21 virions during a competitive epitope challenge by an anti-DAF SCR1 MAb suggests that interactions between CVA21-DAFv and DAF are of higher affinity than those of the parental strain. Nucleotide sequence analysis of the capsid-coding region of the CVA21-DAFv revealed the presence of two amino acid substitutions in capsid protein VP3 (R96H and E101A), possibly conferring the enhanced DAF-binding phenotype of CVA21-DAFv. These residues are predicted to be embedded at the interface of VP1, VP2, and VP3 and are postulated to enhance the affinity of DAF interaction occurring outside the capsid canyon. Taken together, the data clearly demonstrate an enhanced DAF-using phenotype and expanded receptor utilization of CVA21-DAFv compared to the parental strain, further highlighting that capsid interactions with DAF alone facilitate rapid multicycle lytic cell infection.
Evolutionary Dynamics of the Glycan Shield of theHuman Immunodeficiency Virus Envelope during Natural Infection andImplications for Exposure of the 2G12Epitope
Authors: Dacheux Laurent , Moreau Alain , Ataman-Önal Yasemin , Biron François , Verrier Bernard , Barin Francis
Published: October 2004

Elucidation
of the kinetics of exposure of neutralizing epitopes on the envelope of
human immunodeficiency virus type 1 (HIV-1) during the course of
infection may provide key information about how HIV escapes the immune
system or why its envelope is such a poor immunogen to induce broadly
efficient neutralizing antibodies. We analyzed the kinetics of exposure
of the epitopes corresponding to the broadly neutralizing human
monoclonal antibodies immunoglobulin G1b12 (IgG1b12), 2G12, and 2F5 at
the quasispecies level during infection. We studied the antigenicity
and sequences of 94 full-length envelope clones present during primary
infection and at least 4 years later in four HIV-1 clade B-infected
patients. No or only minor exposure differences were observed for the
2F5 and IgG1b12 epitopes between the early and late clones. Conversely,
the envelope glycoproteins of the HIV-1 quasispecies present during
primary infection did not expose the 2G12 neutralizing epitope, unlike
those present after several years in three of the four patients.
Sequence analysis revealed major differences at potential N-linked
glycosylation sites between early and late clones, particularly at
positions known to be important for 2G12 binding. Our study, in natural
mutants, confirms that the glycosylation sites N295, N332, and N392 are
essential for 2G12 binding. This study demonstrates the relationship
between the evolving “glycan shield ” of HIV and the
kinetics of exposure of the 2G12 epitope during the course of natural
infection.
Serotype-Specific Entry of Dengue Virus into Liver Cells: Identification of the 37-Kilodalton/67-Kilodalton High-Affinity Laminin Receptor as a Dengue Virus Serotype 1 Receptor
Authors: Thepparit Chutima , Smith Duncan R.
Published: October 2004

Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.
Novel Role for Decay-Accelerating Factor in Coxsackievirus A21-Mediated Cell Infectivity
Authors: Newcombe Nicole G. , Beagley Leone G. , Christiansen Dale , Loveland Bruce E. , Johansson E. Susanne , Beagley Ken W. , Barry Richard D. , Shafren Darren R.
Published: October 2004

Decay-accelerating factor (DAF) is involved in the cell membrane attachment of many human enteroviruses. Presently, further specific active roles of DAF in mediating productive cell infection and in the pathogenesis of natural enterovirus infection are poorly understood. In an attempt to more fully understand the role of DAF in lytic cell infection we examined the specific interactions of the prototype strain of coxsackievirus A21 (CVA21) with surface-expressed DAF. Investigations into discrete DAF-CVA21 interactions focused on viral binding; viral particle elution with respect to the parameters of time, temperature, and pH; and subsequent cell infection. Radiolabeled-virus binding assays revealed that peak elution of CVA21 from DAF occurred within 15 min of initial attachment and that the DAF-eluted virus increased in a linear fashion with respect to temperature and pH. CVA21 eluted from endogenous surface-expressed DAF was highly infectious, in contrast to CVA21 eluted from intercellular adhesion molecule 1 (ICAM-1), which retained little to no infectivity. Using an adenovirus transduction system, we demonstrate that CVA21 can remain infectious for up to 24 h after DAF binding and is capable of initiating a multicycle lytic infection upon delayed ICAM-1 surface expression. Taken together, the data suggest that a major role of DAF in cell infection by the prototype strain of CVA21 is to provide membrane concentration of infectious virions, effectively increasing viral interactions with endogenous or induced ICAM-1.
The Nucleoprotein Is Required for Efficient Coronavirus Genome Replication
Authors: Almazán Fernando , Galán Carmen , Enjuanes Luis
Published: October 2004

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in
cis
or in
trans
. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.
Characterization of a Novel Close-to-Root Papillomavirus from a Florida Manatee by Using Multiply Primed Rolling-Circle Amplification:
Trichechus manatus latirostris
Papillomavirus Type 1
Authors: Rector Annabel , Bossart Gregory D. , Ghim Shin-Je , Sundberg John P. , Jenson A. Bennett , Van Ranst Marc
Published: October 2004

By using an isothermal multiply primed rolling-circle amplification protocol, the complete genomic DNA of a novel papillomavirus was amplified from a skin lesion biopsy of a Florida manatee (
Trichechus manatus latirostris
), one of the most endangered marine mammals in United States coastal waters. The nucleotide sequence, genome organization, and phylogenetic position of the
Trichechus manatus latirostris
papillomavirus type 1 (TmPV-1) were determined. TmPV-1 is the first virus isolated from the order of
Sirenia
. A phylogenetic analysis shows that TmPV-1 is only distantly related to other papillomavirus sequences, and it appears in our phylogenetic tree as a novel close-to-root papillomavirus genus.
The Phylogeography of Orangutan Foamy Viruses Supports the Theory of Ancient Repopulation of Sumatra
Authors: Verschoor Ernst J. , Langenhuijzen Susan , Bontjer Ilja , Fagrouch Zahra , Niphuis Henk , Warren Kristin S. , Eulenberger K. , Heeney Jonathan L.
Published: October 2004

Phylogenetic analysis of foamy virus sequences obtained from Bornean and Sumatran orangutans showed a distinct clustering pattern. One subcluster was represented by both Bornean and Sumatran orangutan simian foamy viruses (SFV). Combined analysis of host mitochondrial DNA and SFV phylogeny provided evidence for the hypothesis of the repopulation of Sumatra by orangutans from Borneo.