bluuebunny/crossref_metadata_2025 huggingface dataset:
Wild-Type Levels of Nuclear Localization and Human Immunodeficiency Virus Type 1 Replication in the Absence of the Central DNA Flap
Authors: Limón Ana , Nakajima Noriko , Lu Richard , Ghory Hina Z. , Engelman Alan
Published: November 2002

Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F
−
) HIV-1
LAI
was completely defective for viral spread in the MT-4 T-cell line, yet F
−
HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1
NL4-3
and a derivative of HIV-1
HXBc2
deficient for both Vpr and Nef. In stark contrast to the previous report, F
−
derivatives of both strains replicated efficiently in MT-4 cells. F
−
HIV-1
NL4-3
also spread like wild-type HIV-1
NL4-3
in infected Jurkat and primary T-cell cultures. In contrast, F
−
HIV-1
HXBc2
was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F
−
HIV-1
HXBc2
entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F
−
HIV-1
HXBc2
growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type
nef
restored replication to F
−
HIV-1
HXBc2
in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.
Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import
Authors: Dvorin Jeffrey D. , Bell Peter , Maul Gerd G. , Yamashita Masahiro , Emerman Michael , Malim Michael H.
Published: November 2002

Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.
Toward Testing the Hypothesis that Group B Coxsackieviruses (CVB) Trigger Insulin-Dependent Diabetes: Inoculating Nonobese Diabetic Mice with CVB Markedly Lowers Diabetes Incidence
Authors: Tracy S. , Drescher K. M. , Chapman N. M. , Kim K.-S. , Carson S. D. , Pirruccello S. , Lane P. H. , Romero J. R. , Leser J. S.
Published: November 2002

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.
Characterization of a Novel Murine Retrovirus Mixture That Facilitates Hematopoiesis
Authors: Hook Lauren M. , Jude Brooke A. , Ter-Grigorov Victor S. , Hartley Janet W. , Morse Herbert C. , Trainin Zeev , Toder Vladimir , Chervonsky Alexander V. , Golovkina Tatyana V.
Published: November 2002

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med.
3:
37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1
+
Lin
−
hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments.
Emv1
was expressed in the spleens of multiparous mice but not in those of virgin mice, and
Bxv1Emv1
-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.
Antigenic Properties of the Human Immunodeficiency Virus Transmembrane Glycoprotein during Cell-Cell Fusion
Authors: Finnegan Catherine M. , Berg Werner , Lewis George K. , DeVico Anthony L.
Published: November 2002

Human immunodeficiency virus (HIV) entry is triggered by interactions between a pair of heptad repeats in the gp41 ectodomain, which convert a prehairpin gp41 trimer into a fusogenic three-hairpin bundle. Here we examined the disposition and antigenic nature of these structures during the HIV-mediated fusion of HeLa cells expressing either HIV
HXB2
envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various lengths of time and then arrested. Fusion intermediates were then examined for reactivity with various monoclonal antibodies (MAbs) against immunogenic cluster I and cluster II epitopes in the gp41 ectodomain. All of these MAbs produced similar staining patterns indicative of reactivity with prehairpin gp41 intermediates or related structures. MAb staining was seen on Env cells only upon exposure to soluble CD4, CD4-positive, coreceptor-negative cells, or stromal cell-derived factor-treated target cells. In the fusion system, the MAbs reacted with the interfaces of attached Env and target cells within 10 min of coculture. MAb reactivity colocalized with the formation of gp120-CD4-coreceptor tricomplexes after longer periods of coculture, although reactivity was absent on cells exhibiting cytoplasmic dye transfer. Notably, the MAbs were unable to inhibit fusion even when allowed to react with soluble-CD4-triggered or temperature-arrested antigens prior to initiation of the fusion process. In comparison, a broadly neutralizing antibody, 2F5, which recognizes gp41 antigens in the HIV envelope spike, was immunoreactive with free Env cells and Env-target cell clusters but not with fused cells. Notably, exposure of the 2F5 epitope required temperature-dependent elements of the HIV envelope structure, as MAb binding occurred only above 19°C. Overall, these results demonstrate that immunogenic epitopes, both neutralizing and nonneutralizing, are accessible on gp41 antigens prior to membrane fusion. The 2F5 epitope appears to depend on temperature-dependent elements on prefusion antigens, whereas cluster I and cluster II epitopes are displayed by transient gp41 structures. Such findings have important implications for HIV vaccine approaches based on gp41 intermediates.
Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence
Authors: Noris Emanuela , Zannetti Claudia , Demurtas Anna , Sinclair John , De Andrea Marco , Gariglio Marisa , Landolfo Santo
Published: November 2002

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display β-galactosidase (β-Gal) activity at neutral pH (senescence-associated β-Gal [SA-β-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16
INK4a
, a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21
CIP1/WAF
. Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early (α) genes are sufficient for the induction of SA-β-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16
INK4a
(observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-β-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16
INK4a
and p53 pathways.
Noninvasive Bioluminescence Imaging of Herpes Simplex Virus Type 1 Infection and Therapy in Living Mice
Authors: Luker Gary D. , Bardill J. Patrick , Prior Julie L. , Pica Christina M. , Piwnica-Worms David , Leib David A.
Published: November 2002

Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (
Photinus pyralis
) and
Renilla
(
Renilla reniformis
) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of
Renilla
luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.
The Human Papillomavirus Type 16 E5 Protein Impairs TRAIL- and FasL-Mediated Apoptosis in HaCaT Cells by Different Mechanisms
Authors: Kabsch Kirsten , Alonso Angel
Published: November 2002

The effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis was investigated by using the polyclonal HaCaT-cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference. Apoptosis was triggered either by Fas ligand (FasL) or by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and was monitored by detection of cleavage of procaspase-8 and procaspase-3, as well as their substrate poly(ADP-ribose) polymerase (PARP). In contrast to the HaCaT/pMSG control cells we found that apoptosis induced by either of the two ligands is strongly suppressed in the E5-expressing keratinocytes. Fas expression is reduced by about a factor of two in HaCaT/E5 cells, which could be part of the mechanisms that protect the cells from FasL-induced apoptosis. For the TRAIL receptors, no such downregulation was observed. Here, E5 impairs the formation of the death-inducing signaling complex triggered by TRAIL. Apparently, E5 employs different mechanisms to inhibit death receptor signaling. This effect is not restricted to HaCaT/E5 cells since we found that the mouse fibroblast cell line A31-E5 is protected from TRAIL-induced apoptosis, as well but not the E5-lacking control cells A31-Neo. However, no such protection was observed upon FasL-induced apoptosis. Presumably, some of the antiapoptotic mechanisms employed by E5 of the human pathogenic HPV-16 are cell type specific. We propose that inhibition of ligand-mediated apoptosis in human keratinocytes is a primary function of the HPV-16 E5 protein needed to prevent apoptosis at early stages of viral infection.
Direct Binding of Human Immunodeficiency Virus Type 1 Nef to the Major Histocompatibility Complex Class I (MHC-I) Cytoplasmic Tail Disrupts MHC-I Trafficking
Authors: Williams Maya , Roeth Jeremiah F. , Kasper Matthew R. , Fleis Rebekah I. , Przybycin Chris G. , Collins Kathleen L.
Published: November 2002

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins.
Suppression of Tetradecanoyl Phorbol Acetate-Induced Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus by K1 Signal Transduction
Authors: Lee Bok-Soo , Paulose-Murphy Mini , Chung Young-Hwa , Connlole Michelle , Zeichner Steven , Jung Jae U.
Published: November 2002

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region and elicits cellular signal transduction through this motif. To investigate the role of K1 signal transduction in KSHV replication, we expressed full-length K1 and CD8-K1 chimeras in BCBL1 cells. Unlike its strong signaling activity in uninfected B lymphocytes, K1 did not induce intracellular calcium mobilization or NF-AT activation at detectable levels in KSHV-infected BCBL1 cells. Instead, K1 signaling dramatically suppressed KSHV lytic reactivation induced by tetradecanoyl phorbol acetate (TPA) stimulation, but not by ORF50 ectopic expression. Mutational analysis showed that the cytoplasmic ITAM sequence of K1 was required for this suppression. Viral microarray and immunoblot analyses demonstrated that K1 signaling suppressed the TPA-mediated increase in the expression of a large subset of viral lytic genes in KSHV-infected BCBL1 cells. Furthermore, electrophoretic mobility shift assays demonstrated that TPA-induced activation of AP-1, NF-κB, and Oct-1 activities was severely diminished in BCBL1 cells expressing the K1 cytoplasmic domain. The reduced activities of these transcription factors may confer the observed reduction in viral lytic gene expression. These results demonstrate that K1-mediated signal transduction in KSHV-infected cells is profoundly different from that in KSHV-negative cells. Furthermore, K1 signal transduction efficiently suppresses TPA-mediated viral reactivation in an ITAM-dependent manner, and this suppression may contribute to the establishment and/or maintenance of KSHV latency in vivo.
Characterization of the Golgi Retention Motif of Rift Valley Fever Virus G
N
Glycoprotein
Authors: Gerrard Sonja R. , Nichol Stuart T.
Published: November 2002

As
Rift Valley fever
(RVF)
virus
, and probably all members of the family
Bunyaviridae
, matures in the Golgi apparatus, the targeting of the virus glycoproteins to the Golgi apparatus plays a pivotal role in the virus replication cycle. No consensus Golgi localization motif appears to be shared among the glycoproteins of these viruses. The viruses of the family
Bunyaviridae
synthesize their glycoproteins, G
N
and G
C
, as a polyprotein. The Golgi localization signal of RVF virus has been shown to reside within the G
N
protein by use of a plasmid-based transient expression system to synthesize individual G
N
and G
C
proteins. While the distribution of individually expressed G
N
significantly overlaps with cellular Golgi proteins such as β-COP and GS-28, G
C
expressed in the absence of G
N
localizes to the endoplasmic reticulum. Further analysis of expressed G
N
truncated proteins and green fluorescent protein/G
N
chimeric proteins demonstrated that the RVF virus Golgi localization signal mapped to a 48-amino-acid region of G
N
encompassing the 20-amino-acid transmembrane domain and the adjacent 28 amino acids of the cytosolic tail.
The Hydrophilic Amino-Terminal Arm of Reovirus Core Shell Protein λ1 Is Dispensable for Particle Assembly
Authors: Kim Jonghwa , Zhang Xing , Centonze Victoria E. , Bowman Valorie D. , Noble Simon , Baker Timothy S. , Nibert Max L.
Published: November 2002

The reovirus core particle is a molecular machine that mediates synthesis, capping, and export of the viral plus strand RNA transcripts. Its assembly and structure-function relationships remain to be well understood. Following the lead of previous studies with other
Reoviridae
family members, most notably orbiviruses and rotaviruses, we used recombinant baculoviruses to coexpress reovirus core proteins λ1, λ2, and σ2 in insect cells. The resulting core-like particles (CLPs) were purified and characterized. They were found to be similar to cores with regard to their sizes, morphologies, and protein compositions. Like cores, they could also be coated in vitro with the two major outer-capsid proteins, μ1 and σ3, to produce virion-like particles. Coexpression of core shell protein λ1 and core nodule protein σ2 was sufficient to yield CLPs that could withstand purification, whereas expression of λ1 alone was not, indicating a required role for σ2 as a previous study also suggested. In addition, CLPs that lacked λ2 (formed from λ1 and σ2 only) could not be coated with μ1 and σ3, indicating a required role for λ2 in the assembly of these outer-capsid proteins into particles. To extend the use of this system for understanding the core and its assembly, we addressed the hypothesis that the hydrophilic amino-terminal region of λ1, which adopts an extended arm-like conformation around each threefold axis in the reovirus core crystal structure, plays an important role in assembling the core shell. Using a series of λ1 deletion mutants, we showed that the amino-terminal 230 residues of λ1, including its zinc finger, are dispensable for CLP assembly. Residues in the 231-to-259 region of λ1, however, were required. The core crystal structure suggests that residues in the 231-to-259 region are necessary because they affect the interaction of λ1 with the threefold and/or fivefold copies of σ2. An effective system for studies of reovirus core structure, assembly, and functions is hereby established.
Borna Disease Virus Accelerates Inflammation and Disease Associated with Transgenic Expression of Interleukin-12 in the Central Nervous System
Authors: Freude Susanna , Hausmann Jürgen , Hofer Markus , Pham-Mitchell Ngan , Campbell Iain L. , Staeheli Peter , Pagenstecher Axel
Published: November 2002

Targeted expression of biologically active interleukin-12 (IL-12) in astrocytes of the central nervous system (CNS) results in spontaneous neuroimmunological disease of aged mice. Borna disease virus (BDV) can readily multiply in the mouse CNS but does not trigger disease in most strains. Here we show that a large percentage of IL-12 transgenic mice developed severe ataxia within 5 to 10 weeks after infection with BDV. By contrast, no disease developed in mock-infected IL-12 transgenic and wild-type mice until 4 months of age. Neurological symptoms were rare in infected wild-type animals, and if they occurred, these were milder and appeared later. Histological analyses showed that the cerebellum of infected IL-12 transgenic mice, which is the brain region with strongest transgene expression, contained large numbers of CD4+and CD8+T cells as well as lower numbers of B cells, whereas other parts of the CNS showed only mild infiltration by lymphocytes. The cerebellum of diseased mice further showed severe astrogliosis, calcifications and signs of neurodegeneration. BDV antigen and nucleic acids were present in lower amounts in the inflamed cerebellum of infected transgenic mice than in the noninflamed cerebellum of infected wild-type littermates, suggesting that IL-12 or IL-12-induced cytokines exhibited antiviral activity. We propose that BDV infection accelerates the frequency by which immune cells such as lymphocytes and NK cells enter the CNS and then respond to IL-12 present in the local milieu causing disease. Our results illustrate that infection of the CNS with a virus that is benign in certain hosts can be harmful in such normally disease-resistant hosts if the tissue is unfavorably preconditioned by proinflammatory cytokines.
Regulation of Indoleamine 2,3-Dioxygenase Expression in Simian Immunodeficiency Virus-Infected Monkey Brains
Authors: Burudi E. M. E. , Marcondes M. Cecilia G. , Watry Debbie D. , Zandonatti Michelle , Taffe Michael A. , Fox Howard S.
Published: November 2002

The human immunodeficiency virus type 1-associated cognitive-motor disorder, including the AIDS dementia complex, is characterized by brain functional abnormalities that are associated with injury initiated by viral infection of the brain. Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in tryptophan catabolism in extrahepatic tissues, can lead to neurotoxicity through the generation of quinolinic acid and immunosuppression and can alter brain chemistry via depletion of tryptophan. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we demonstrate that cells of the macrophage lineage are the main source for expression of IDO in the SIV-infected monkey brain. Animals with SIV encephalitis have the highest levels of IDO mRNA, and the level of IDO correlates with gamma interferon (IFN-γ) and viral load levels. In vitro studies on mouse microglia reveal that IFN-γ is the primary inducer of IDO expression. These findings demonstrate the link between IDO expression, IFN-γ levels, and brain pathology signs observed in neuro-AIDS.
Virus Replication and Cytokine Production in Dengue Virus-Infected Human B Lymphocytes
Authors: Lin Yu-Wen , Wang Kuan-Ju , Lei Huan-Yao , Lin Yee-Shin , Yeh Trai-Ming , Liu Hsiao-Sheng , Liu Ching-Chuan , Chen Shun-Hua
Published: November 2002

Dengue virus (DV) replication, antibody-enhanced viral infection, and cytokine responses of human primary B lymphocytes (cells) were characterized and compared with those of monocytes. The presence of a replication template (negative-strand RNA intermediate), viral antigens including core and nonstructural proteins, and increasing amounts of virus with time postinfection indicated that DV actively replicated in B cells. Virus infection also induced B cells to produce interleukin-6 and tumor necrosis factor alpha, which have been previously implicated in virus pathogenesis. In addition, a heterologous antibody was able to enhance both virus and cytokine production in B cells. Furthermore, the levels of virus replication, antibody-enhanced virus replication, and cytokine responses observed in B cells were not statistically different from those in monocytes. These results suggest that B cells may play an important role in DV pathogenesis.
An Antibody to the Putative Aphid Recognition Site on Cucumber Mosaic Virus Recognizes Pentons but Not Hexons
Authors: Bowman Valorie D. , Chase Elaine S. , Franz Alexander W. E. , Chipman Paul R. , Zhang Xing , Perry Keith L. , Baker Timothy S. , Smith Thomas J.
Published: November 2002

Cucumber mosaic virus (CMV), the type member of the genus
Cucumovirus
(family
Bromoviridae
), is transmitted by aphids in a nonpersistent manner. Mutagenesis experiments identified the βH-βI loop of the capsid subunit as a potential key motif responsible for interactions with the insect vector. To further examine the functional characteristics of this motif, we generated monoclonal antibodies that bound to native virions but not to βH-βI mutants. Fab fragments from these antibodies were complexed with wild-type CMV and the virus-Fab structure was determined to 12-Å resolution by using electron cryomicroscopy and image reconstruction techniques. The electron density attributed to the bound antibody has a turret-like appearance and protrudes from each of the 12 fivefold axes of the icosahedral virus. Thus, the antibody binds only to the pentameric clusters (pentons) of A subunits of the T=3 quasisymmetric virus and does not appear to bind to any of the B and C subunits that occur as hexameric clusters (hexons) at the threefold (quasi-sixfold) axes. Modeling and electron density comparisons were used to analyze the paratope-epitope interface and demonstrated that the antibody binds to three βH-βI loops in three adjacent A subunits in each penton. This antibody can discriminate between A and B/C subunits even though the βH-βI loop adopts the same structure in all 180 capsid subunits and is therefore recognizing differences in subunit arrangements. Antibodies with such character have potential use as probes of viral assembly. Our results may provide an additional rationale for designing synthetic vaccines by using symmetrical viral particles.
Processing and Degradation of Exogenous Prion Protein by CD11c+Myeloid Dendritic Cells In Vitro
Authors: Luhr Katarina M. , Wallin Robert P. A. , Ljunggren Hans-Gustaf , Löw Peter , Taraboulos Albert , Kristensson Krister
Published: November 2002

The immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. CD11c+myeloid dendritic cells (DC) could, due to their subepithelial location and their migratory capacity, be early targets for prion infection and contribute to the spread of infection. In order to analyze mechanisms by which these cells may affect prion propagation, we studied in vitro the effect of exposing such DC to scrapie-infected GT1-1 cells, which produce the scrapie prion protein PrPSc. In this system, the DC efficiently engulfed the infected GT1-1 cells. Unexpectedly, PrPSc, which is generally resistant to protease digestion, was processed and rapidly degraded. Based on this observation we speculate that CD11c+DC may play a dual role in prion infections: on one hand they may facilitate neuroinvasion by transfer of the infectious agent as suggested from in vivo studies, but on the other hand they may protect against the infection by causing an efficient degradation of PrPSc. Thus, the migrating and highly proteolytic CD11c+myeloid DC may affect the balance between propagation and clearance of PrPScin the organism.
Saccharomyces cerevisiaeIs Permissive for Replication of Bovine Papillomavirus Type 1
Authors: Zhao Kong-Nan , Frazer Ian H.
Published: November 2002

We recently demonstrated thatSaccharomyces cerevisiaeprotoplasts can take up bovine papillomavirus type 1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPV virus-like particles are assembled in infectedS. cerevisiaecultures from newly synthesized capsid proteins and also package newly synthesized DNA, including full-length and truncated viral DNA andS. cerevisiae-derived DNA. Virus particles prepared inS. cerevisiaeare able to convey packaged DNA to Cos1 cells and to transform C127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude thatS. cerevisiaeis permissive for the replication of BPV1 virus.
Antibody Epitopes on the Neuraminidase of a Recent H3N2 Influenza Virus (A/Memphis/31/98)
Authors: Gulati Upma , Hwang Chi-Ching , Venkatramani Lalitha , Gulati Shelly , Stray Stephen J. , Lee Janis T. , Laver W. Graeme , Bochkarev Alexey , Zlotnick Adam , Air Gillian M.
Published: November 2002

We have characterized monoclonal antibodies raised against the neuraminidase (NA) of a Sydney-like influenza virus (A/Memphis/31/98, H3N2) in a reassortant virus A/NWS/33HA-A/Mem/31/98NA(H1N2) and nine escape mutants selected by these monoclonal antibodies. Five of the antibodies use the same heavy chain VDJ genes and may not be independent. Another antibody, Mem5, uses the same VHand J genes with a different D gene and different isotype. Sequence changes in escape mutants selected by these antibodies occur in two loops of the NA, at amino acid 198, 199, 220, or 221. These amino acids are located on the opposite side of the NA monomer to the major epitopes found in N9 and early N2 NAs. Escape mutants with a change at 198 have reduced NA activity compared to the wild-type virus. Asp198 points toward the substrate binding pocket, and we had previously found that a site-directed mutation of this amino acid resulted in a loss of enzyme activity (M. R. Lentz, R. G. Webster, and G. M. Air, Biochemistry26:5351-5358, 1987). Mutations at residue 199, 220, or 221 did not alter the NA activity significantly compared to that of wild-type NA. A 3.5-Å structure of Mem5 Fab complexed with the Mem/98 NA shows that the Mem5 antibody binds at the sites of escape mutation selected by the other antibodies.
Identification of Six
Autographa californica
Multicapsid Nucleopolyhedrovirus Early Genes That Mediate Nuclear Localization of G-Actin
Authors: Ohkawa Taro , Rowe Annette R. , Volkman Loy E.
Published: November 2002

Nuclear filamentous actin (F-actin) is required for nucleopolyhedrovirus (NPV) progeny production in NPV-infected, cultured lepidopteran cells. We have determined that monomeric G-actin is localized within the nuclei of host cells during the early stage of infection by
Autographa californica
multicapsid nucleopolyhedrovirus (Ac
M
NPV). With a library of cloned Ac
M
NPV genomic fragments, along with a plasmid engineered to express enhanced green fluorescent protein-
Bombyx mori
G-actin in transient transfection experiments, we identified six Ac
M
NPV early genes that mediate nuclear localization of G-actin in TN-368 cells:
ie-1
,
pe38
,
he65
,
Ac004, Ac102,
and
Ac152
. Within this subset,
ie-1
and
pe38
encode immediate-early transcriptional transactivators,
he65
encodes a delayed-early product, and the products encoded by
Ac004, Ac102,
and
Ac152
have not been characterized. We found that when driven by foreign promoters,
ie-1
,
pe38,
and
Ac004
had to be expressed prior to
Ac102
or
he65
for nuclear G-actin to accumulate and that expression of
Ac152
was no longer required. These results and others suggested that the product of
Ac152
was a transactivator (directly or indirectly) of both
Ac102
and
he65
and that recruitment of G-actin to the nucleus was a temporally regulated process. Determining the functions of each of the six Ac
M
NPV gene products with respect to our assay should provide valuable clues to basic cellular mechanisms of actin regulation and how Ac
M
NPV infection affects them.
Mutant Human Cytomegalovirus Lacking the Immediate-Early TRS1 Coding Region Exhibits a Late Defect
Authors: Blankenship Catherine A. , Shenk Thomas
Published: November 2002

The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. AD
sub
IRS1 failed to produce the related IRS1 and IRS1
263
proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in
trans
of the IRS1
263
protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. AD
sub
TRS1 failed to produce the TRS1 protein, and it generated an ∼200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in AD
sub
TRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within AD
sub
TRS1-infected cells. Further, infectious AD
sub
TRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the AD
sub
TRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.
The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion
Authors: Jeetendra E. , Robison Clinton S. , Albritton Lorraine M. , Whitt Michael A.
Published: November 2002

Recently we showed that the membrane-proximal stem region of the vesicular stomatitis virus (VSV) G protein ectodomain (G stem [GS]), together with the transmembrane and cytoplasmic domains, was sufficient to mediate efficient VSV budding (C. S. Robison and M. A. Whitt, J. Virol. 74:2239-2246, 2000). Here, we show that GS can also potentiate the membrane fusion activity of heterologous viral fusion proteins when GS is coexpressed with those proteins. For some fusion proteins, there was as much as a 40-fold increase in syncytium formation when GS was coexpressed compared to that seen when the fusion protein was expressed alone. Fusion potentiation by GS was not protein specific, since it occurred with both pH-dependent as well as pH-independent fusion proteins. Using a recombinant vesicular stomatitis virus encoding GS that contained an N-terminal hemagglutinin (HA) tag (GS
HA
virus), we found that the GS
HA
virus bound to cells as well as the wild-type virus did at pH 7.0; however, the GS
HA
virus was noninfectious. Analysis of cells expressing GS
HA
in a three-color membrane fusion assay revealed that GS
HA
could induce lipid mixing but not cytoplasmic mixing, indicating that GS can induce hemifusion. Treatment of GS
HA
virus-bound cells with the membrane-destabilizing drug chlorpromazine rescued the hemifusion block and allowed entry and subsequent replication of GS
HA
virus, demonstrating that GS-mediated hemifusion was a functional intermediate in the membrane fusion pathway. Using a series of truncation mutants, we also determined that only 14 residues of GS, together with the VSV G transmembrane and cytoplasmic tail, were sufficient for fusion potentiation. To our knowledge, this is the first report which shows that a small domain of one viral glycoprotein can promote the fusion activity of other, unrelated viral glycoproteins.
The p23 Protein of Hibiscus Chlorotic Ringspot Virus Is Indispensable for Host-Specific Replication
Authors: Liang Xiao-Zhen , Lucy Andrew P. , Ding Shou-Wei , Wong Sek-Man
Published: November 2002

Hibiscus chlorotic ringspot virus (HCRSV) possesses a novel open reading frame (ORF) which encodes a putative 23-kDa protein (p23). We report here the in vivo detection of p23 and demonstrate its essential role in viral replication. The expression of p23 could be detected in protein extracts from transfected kenaf (
Hibiscus cannabinus
L.) protoplasts and in HCRSV-infected leaves. Further, direct immunoblotting of infected kenaf leaves also showed the presence of p23, and transient expression in onion and kenaf cells demonstrated that the protein is distributed throughout the cell. Site-directed mutagenesis showed that mutations introduced into the ORF of p23 abolished viral replication in kenaf protoplasts and plants but not in
Chenopodium quinoa
L. The loss of function of the p23 mutant M23/S33-1 could be complemented in
trans
upon the induced expression of p23 from an infiltrated construct bearing the ORF (pCam23). Altogether, these results demonstrate that p23 is a bona fide HCRSV protein that is expressed in vivo and suggest that p23 is indispensable for the host-specific replication of HCRSV. In addition, we show that p23 does not bind nucleic acids in vitro and does not act as a suppressor of posttranscriptional gene silencing in transgenic tobacco carrying a green fluorescent protein.
Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector
Authors: Ezelle Heather J. , Markovic Dubravka , Barber Glen N.
Published: November 2002

Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.
Binding of Norwalk Virus-Like Particles to ABH Histo-Blood Group Antigens Is Blocked by Antisera from Infected Human Volunteers or Experimentally Vaccinated Mice
Authors: Harrington Patrick R. , Lindesmith Lisa , Yount Boyd , Moe Christine L. , Baric Ralph S.
Published: November 2002

Attachment of Norwalk (NV), Snow Mountain (SMV), and Hawaii (HV) virus-like particles (VLPs) to specific ABH histo-blood group antigens was investigated by using human saliva and synthetic biotinylated carbohydrates. The three distinct Norwalk-like viruses (NLVs) have various capacities for binding ABH histo-blood group antigens, suggesting that different mechanisms for NLV attachment likely exist. Importantly, antisera from NV-infected human volunteers, as well as from mice inoculated with packaged Venezuelan equine encephalitis virus replicons expressing NV VLPs, blocked the ability of NV VLPs to bind synthetic H type 1, Le
b
, and H type 3, suggesting a potential mechanism for antibody-mediated neutralization of NV.
Viral Evolution during Structured Treatment Interruptions in Chronically Human Immunodeficiency Virus-Infected Individuals
Authors: Martinez-Picado Javier , Frost Simon D. W. , Izquierdo Nuria , Morales-Lopetegi K. , Marfil Silvia , Puig Teresa , Cabrera Cecilia , Clotet Bonaventura , Ruiz Lidia
Published: November 2002

We analyzed the evolution of the human immunodeficiency virus type 1 (HIV-1)
env
gene in 12 chronically infected individuals who underwent structured treatment interruptions (STIs). Analyses of length variation and of clonal sequences demonstrated highly unpredictable evolution, which may limit the strengthening of HIV-specific immune responses by STIs because of the variability in exposure to viral antigens.
Targeted Derepression of the Human Immunodeficiency Virus Type 1 Long Terminal Repeat by Pyrrole-Imidazole Polyamides
Authors: Coull Jason J. , He Guocheng , Melander Christian , Rucker Victor C. , Dervan Peter B. , Margolis David M.
Published: November 2002

The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.
Mucosal Immunization with Live Recombinant Bovine Respiratory Syncytial Virus (BRSV) and Recombinant BRSV Lacking the Envelope Glycoprotein G Protects against Challenge with Wild-Type BRSV
Authors: Schmidt Ulrike , Beyer Jörg , Polster Ulf , Gershwin Laurel J. , Buchholz Ursula J.
Published: November 2002

Recombinant bovine respiratory syncytial virus (rBRSV) and an rBRSV deletion mutant lacking the G gene (rBRSVΔG) were characterized in calves with respect to replication competence, attenuation, and protective efficacy as live-attenuated BRSV vaccines. Both recombinant viruses were safe and induced protection against a BRSV challenge infection. rBRSV replicated efficiently in the upper respiratory tract. Intranasal immunization with rBRSVΔG led to infection but not to mucosal virus replication. Neutralizing antibodies were induced by rBRSV and rBRSVΔG. Thus, the BRSV attachment glycoprotein G seems to be dispensable in vaccinating calves against BRSV.
Comparison of Abnormal Prion Protein Glycoform Patterns from Transmissible Spongiform Encephalopathy Agent-Infected Deer, Elk, Sheep, and Cattle
Authors: Race Richard E. , Raines Anne , Baron Thierry G. M. , Miller Michael W. , Jenny Allen , Williams Elizabeth S.
Published: November 2002

Analysis of abnormal prion protein glycoform patterns from chronic wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and cattle, and cattle with bovine spongiform encephalopathy failed to identify patterns capable of reliably distinguishing these transmissible spongiform encephalopathy diseases. However, PrP-res patterns sometimes differed among individual animals, suggesting infection by different or multiple CWD strains in some species.
Host Range and Receptor Binding Properties of Vectors Bearing Feline Leukemia Virus Subgroup B Envelopes Can Be Modulated by Envelope Sequences outside of the Receptor Binding Domain
Authors: Faix Peggy Ho , Feldman Steven A. , Overbaugh Julie , Eiden Maribeth V.
Published: November 2002

To evaluate host range differences between two different strains of feline leukemia virus subgroup B (FeLV-B), we compared the binding and infectivity patterns of retrovirus vectors bearing either FeLV-B-90Z or FeLV-B-GA envelopes. We report here that the ability of these envelopes to utilize different Pit1 orthologs is mediated primarily by the receptor binding domain; however, in the case of FeLV-B-90Z, the C terminus also contributes to the recognition of certain Pit1 orthologs.
Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes
Authors: Suzuki Youichi , Craigie Robert
Published: November 2002

Retroviral integration is mediated by a preintegration complex (PIC) which contains the viral DNA made by reverse transcription together with associated protein factors. Prior to association with target DNA, the PIC must avoid suicidal intramolecular integration of its viral DNA (autointegration). We have demonstrated that barrier-to-autointegration factor (BAF) blocks the autointegration of Moloney murine leukemia virus (MoMLV) PICs in vitro. In this study, we show that BAF is an authentic component of MoMLV. Analysis of the sedimentation properties of initial, salt-stripped, and BAF-reconstituted PICs reveals that the viral DNA within the PIC is reversibly compacted by BAF, consistent with the functional role of BAF in protecting the viral DNA from autointegration. Furthermore, we find that BAF can promote the association of PICs with target DNA. Thus, our data suggest that BAF plays critical roles in promoting preferential intermolecular integration by both blocking autointegration and stimulating the capture of target DNA.