bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity-Tagged Miniprion Derivatives Spontaneously Adopt Protease-Resistant Conformations
Authors: Supattapone Surachai , Nguyen Hoang-Oanh B. , Muramoto Tamaki , Cohen Fred E. , DeArmond Stephen J. , Prusiner Stanley B. , Scott Michael
Published: July 2002

An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His
6
) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Δ214–220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP
Sc
. We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP
Sc
. These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.
Avian Endogenous Retrovirus EAV-HP Shares Regions of Identity with Avian Leukosis Virus Subgroup J and the Avian Retrotransposon ART-CH
Authors: Sacco M. A. , Flannery D. M. J. , Howes K. , Venugopal K.
Published: July 2002

The existence of novel endogenous retrovirus elements in the chicken genome, designated EAV-HP, with close sequence identity to the
env
gene of avian leukosis virus (ALV) subgroup J has been reported (L. M. Smith, A. A. Toye, K. Howes, N. Bumstead, L. N. Payne, and K. Venugopal, J. Gen. Virol. 80:261–268, 1999). To resolve the genome structure of these retroviral elements, we have determined the complete sequence of two proviral clones of EAV-HP from a line N chicken genomic DNA yeast artificial chromosome library and from a meat-type chicken line 21 lambda library. The EAV-HP sequences from the two lines were 98% identical and had a typical provirus structure. The two EAV-HP clones showed identical large deletions spanning part of the
gag
, the entire
pol
, and part of the
env
genes. The
env
region of the EAV-HP clones was 97% identical to the
env
sequence of HPRS-103, the prototype subgroup J ALV. The 5′ region of EAV-HP comprising the R and U5 regions of the long terminal repeat (LTR), the untranslated leader, and the 5′ end of the putative
gag
region were 97% identical to the avian retrotransposon sequence, ART-CH. The remaining
gag
sequence shared less than 60% identity with other ALV sequences. The U3 region of the LTR was distinct from those of other retroviruses but contained some of the conserved motifs required for functioning as a promoter. To examine the ability of this endogenous retroviral LTR to function as a transcriptional promoter, the EAV-HP and HPRS-103 LTR U3 regions were compared in a luciferase reporter gene assay. The low luciferase activity detected with the EAV-HP LTR U3 constructs, at levels close to those observed for a control vector lacking the promoter or enhancer elements, suggested that these elements function as a weak promoter, possibly accounting for their low expression levels in chicken embryos.
The RNA Helicase and Nucleotide Triphosphatase Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential for Viral Replication
Authors: Gu Baohua , Liu Changbao , Lin-Goerke Juili , Maley Derrick R. , Gutshall Lester L. , Feltenberger Cynthia A. , Del Vecchio Alfred M.
Published: July 2002

Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the
Flaviviridae
family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEY
H
to DEY
A
) or both NTPase and helicase activity (motif I, G
K
T to G
A
T and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.
JC Virus Enters Human Glial Cells by Clathrin-Dependent Receptor-Mediated Endocytosis
Authors: Pho M. T. , Ashok A. , Atwood Walter J.
Published: July 2002

The human polyomavirus JC virus (JCV) is the etiologic agent of a fatal central nervous system (CNS) demyelinating disease known as progressive multifocal leukoencephalopathy (PML). PML occurs predominantly in immunosuppressed patients and has increased dramatically as a result of the AIDS pandemic. The major target cell of JCV infection and lytic replication in the CNS is the oligodendrocyte. The mechanisms by which JCV initiates and establishes infection of these glial cells are not understood. The initial interaction between JCV and glial cells involves virus binding to N-linked glycoproteins containing terminal α(2-6)-linked sialic acids. The subsequent steps of entry and targeting of the viral genome to the nucleus have not been described. In this report, we compare the kinetics and mechanisms of infectious entry of JCV into human glial cells with that of the related polyomavirus, simian virus 40 (SV40). We demonstrate that JCV, unlike SV40, enters glial cells by receptor-mediated clathrin-dependent endocytosis.
Inhibition of CD3/CD28-Mediated Activation of the MEK/ERK Signaling Pathway Represses Replication of X4 but Not R5 Human Immunodeficiency Virus Type 1 in Peripheral Blood CD4+T Lymphocytes
Authors: Popik Waldemar , Pitha Paula M.
Published: July 2002

Binding of human immunodeficiency virus type 1 (HIV-1) to CD4 receptors induces multiple cellular signaling pathways, including the MEK/ERK cascade. While the interaction of X4 HIV-1 with CXCR4 does not seem to activate this pathway, viruses using CCR5 for entry efficiently activate MEK/ERK kinases (W. Popik, J. E. Hesselgesser, and P. M. Pitha, J. Virol. 72:6406–6413, 1998; W. Popik and P. M. Pitha, Virology 252:210–217, 1998). Since the importance of MEK/ERK in the initial steps of viral replication is poorly understood, we have examined the role of MEK/ERK signaling in the CD3- and CD28 (CD3/CD28)-mediated activation of HIV-1 replication in resting peripheral blood CD4+T lymphocytes infected with X4 or R5 HIV-1. We have found that the MEK/ERK inhibitor U0126 selectively inhibited CD3/CD28-stimulated replication of X4 HIV-1, while it did not affect the replication of R5 HIV-1. Inhibition of the CD3/CD28-stimulated MEK/ERK pathway did not affect the formation of the early proviral transcripts in cells infected with either X4 or R5 HIV-1, indicating that virus reverse transcription is not affected in the absence of MEK/ERK signaling. In contrast, the levels of nuclear provirus in cells infected with X4 HIV-1, detected by the formation of circular proviral DNA, was significantly lower in cells stimulated in the presence of MEK/ERK inhibitor than in the absence of the inhibitor. However, in cells infected with R5 HIV-1, the inhibition of the MEK/ERK pathway did not affect nuclear localization of the proviral DNA. These data suggest that the nuclear import of X4, but not R5, HIV-1 is dependent on a CD3/CD28-stimulated MEK/ERK pathway.
Herpesvirus mRNAs Are Sorted for Export via Crm1-Dependent and -Independent Pathways
Authors: Soliman Tarik M. , Silverstein Saul J.
Published: July 2002

Cellular pre-mRNA splicing is inhibited by ICP27, a herpes simplex virus regulatory protein, resulting in the shutoff of host protein synthesis. Here we reveal that ICP27 also mediates the export of some virus RNAs via a Crm1-dependent pathway and present evidence that independent domains are required for these functions. Sorting of some viral mRNAs for nuclear export requires Crm1, while other virus mRNAs are exported via another pathway.
Evolutionarily Related Sindbis-Like Plant Viruses Maintain Different Levels of Population Diversity in a Common Host
Authors: Schneider William L. , Roossinck Marilyn J.
Published: July 2002

The levels of population diversity of three related Sindbis-like plant viruses,
Tobacco mosaic virus
(TMV),
Cucumber mosaic virus
(CMV), and
Cowpea chlorotic mottle virus
(CCMV), in infections of a common host,
Nicotiana benthamiana
, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in
N. benthamiana
. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.
CD4+T-Cell-Mediated Antiviral Protection of the Upper Respiratory Tract in BALB/c Mice following Parenteral Immunization with a Recombinant Respiratory Syncytial Virus G Protein Fragment
Authors: Plotnicky-Gilquin Hélène , Robert Alain , Chevalet Laurent , Haeuw Jean-Francois , Beck Alain , Bonnefoy Jean-Yves , Brandt Christian , Siegrist Claire-Anne , Nguyen Thien Ngoc , Power Ultan F.
Published: July 2002

We analyzed the protective mechanisms induced against respiratory syncytial virus subgroup A (RSV-A) infection in the lower and upper respiratory tracts (LRT and URT) of BALB/c mice after intraperitoneal immunization with a recombinant fusion protein incorporating residues 130 to 230 of RSV-A G protein (BBG2Na). Mother-to-offspring antibody (Ab) transfer and adoptive transfer of BBG2Na-primed B cells into SCID mice demonstrated that Abs are important for LRT protection but have no effect on URT infection. In contrast, RSV-A clearance in the URT was achieved in a dose-dependent fashion after adoptive transfer of BBG2Na-primed T cells, while it was abolished in BBG2Na-immunized mice upon in vivo depletion of CD4+, but not CD8+, T cells. Furthermore, the conserved RSV-A G protein cysteines and residues 193 and 194, overlapping the recently identified T helper cell epitope on the G protein (P. W. Tebbey et al., J. Exp. Med. 188:1967–1972, 1998), were found to be essential for URT but not LRT protection. Taken together, these results demonstrate for the first time that CD4+T cells induced upon parenteral immunization with an RSV G protein fragment play a critical role in URT protection of normal mice against RSV infection.
Molecular Characterization of a Bacteriophage (Chp2) from
Chlamydia psittaci
Authors: Liu B. L. , Everson J. S. , Fane B. , Giannikopoulou P. , Vretou E. , Lambden P. R. , Clarke I. N.
Published: July 2002

Comparisons of the proteome of abortifacient
Chlamydia psittaci
isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine
C. psittaci
RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the φX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.
Polyomavirus-Infected Dendritic Cells Induce Antiviral CD8+T Lymphocytes
Authors: Drake Donald R. , Moser Janice M. , Hadley Annette , Altman John D. , Maliszewski Charles , Butz Eric , Lukacher Aron E.
Published: July 2002

CD8+T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8+T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8+T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8+T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8+T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8+T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8+T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8+T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8+T cell surveillance against polyomavirus-induced tumors.
Consequences of Fas-Mediated Human Dendritic Cell Apoptosis Induced by Measles Virus
Authors: Servet-Delprat Christine , Vidalain Pierre-Olivier , Azocar Olga , Le Deist Françoise , Fischer Alain , Rabourdin-Combe Chantal
Published: July 2002

Mortality from measles virus (MV) infection is caused mostly by secondary infections associated with a pronounced immunosuppression. Dendritic cells (DCs) represent a major target of MV and could be involved in immunosuppression. In this study, human monocyte-derived DCs were used to demonstrate that DC apoptosis in MV-infected DC–T-cell cocultures is Fas mediated, whereas apoptotic T cells could not be rescued by blocking the Fas pathway. Two novel consequences of DC apoptosis after MV infection were demonstrated. (i) Fas-mediated apoptosis of DCs facilitates MV release, while CD40 activation enhances MV replication in DCs. Indeed, detailed studies of infectious MV release and intracellular MV nucleoprotein (NP) showed that inhibition of CD40-CD40L ligand interaction blocks NP synthesis. We conclude that the CD40 ligand expressed by activated T cells first enhances MV replication in DCs, and then Fas ligand produced by activated T cells induces Fas-mediated apoptosis of DCs, thus facilitating MV release. (ii) Not only MV-infected DCs but also bystander uninfected DCs undergo a maturation process confirmed by CD1a, CD40, CD80, CD86, CD83, and major histocompatibility complex type II labeling. The bystander maturation effect results from contact and/or engulfment of MV-induced apoptotic DCs by uninfected DCs. A model is proposed to explain how both a specific immune response and immunosuppression can simultaneously occur after MV infection through Fas-mediated apoptosis and CD40 activation of DCs.
Vaccinia Virus Vectors with an Inactivated Gamma Interferon Receptor Homolog Gene (B8R) Are Attenuated In Vivo without a Concomitant Reduction in Immunogenicity
Authors: Verardi Paulo H. , Jones Leslie A. , Aziz Fatema H. , Ahmad Shabbir , Yilma Tilahun D.
Published: July 2002

The vaccinia virus (VV) B8R gene encodes a secreted protein with homology to the gamma interferon (IFN-γ) receptor. In vitro, the B8R protein binds to and neutralizes the antiviral activity of several species of IFN-γ, including human and rat IFN-γ; it does not, however, bind significantly to murine IFN-γ. Here we report on the construction and characterization of recombinant VVs (rVVs) lacking the B8R gene. While the deletion of this gene had no effect on virus replication in vitro, rVVs lacking the B8R gene were attenuated for mice. There was a significant decrease in weight loss and mortality in normal mice, and nude mice survived significantly longer than did controls inoculated with parental virus. This is a surprising result considering the minimal binding of the B8R protein to murine IFN-γ and its failure to block the antiviral activity of this cytokine in vitro. Such reduction in virulence could not be determined in rats, since they are considerably more resistant to VV infection than are mice. Finally, deletion of the B8R gene had no detectable effects on humoral immune responses. Mice and rats vaccinated with the rVVs showed identical humoral responses to both homologous and heterologous genes expressed by VV. This study demonstrates that the deletion of the VV B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.
A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos
Authors: Mebatsion Teshome , Verstegen Stefan , De Vaan Leonarda T. C. , Römer-Oberdörfer Angela , Schrier Carla C.
Published: July 2002

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3′-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.
CD150 (SLAM) Is a Receptor for Measles Virus but Is Not Involved in Viral Contact-Mediated Proliferation Inhibition
Authors: Erlenhoefer Christian , Wurzer Walter J. , Löffler Sieglinde , Schneider-Schaulies Sibylle , ter Meulen Volker , Schneider-Schaulies Jürgen
Published: July 2002

Measles virus (MV) interacts with cellular receptors on the surface of peripheral blood lymphocytes (PBL) which mediate virus binding and uptake. Simultaneously, the direct contact of the viral glycoproteins with the cell surface induces a negative signal blocking progression to the S phase of the cell cycle, resulting in a pronounced proliferation inhibition. We selected a monoclonal antibody (MAb 5C6) directed to the surface of highly MV-susceptible B cells (B95a), which inhibits binding to and infection of cells with MV wild-type and vaccine strains. By screening a retroviral cDNA library from human splenocytes (ViraPort; Stratagene) with this antibody, we cloned and identified the recognized molecule as signaling lymphocytic activation molecule (SLAM; CD150), which is identical to the MV receptor recently found by H. Tatsuo et al. (Nature 406:893–897, 2000). After infection of cells, and after surface contact with MV envelope proteins, SLAM is downregulated from the cell surface of activated PBL and cell lines. Although anti-SLAM and/or anti-CD46 antibodies block virus binding, they do not interfere with the contact-mediated proliferation inhibition. In addition, the cell-type-specific expression of SLAM does not correlate with the sensitivity of cells for proliferation inhibition. The data indicate that proliferation inhibition induced by MV contact is independent of the presence or absence of the virus-binding receptors SLAM and CD46.
Identification of Critical Elements in the tRNA Acceptor Stem and TΨC Loop Necessary for Human Immunodeficiency Virus Type 1 Infectivity
Authors: Yu Qin , Morrow Casey D.
Published: July 2002

A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA
Phe
(psHIV-Phe), which relies on exogenous yeast tRNA
Phe
as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and TΨC stem-loop required for the tRNA primer selection and use in HIV-1 replication. tRNA
Phe
mutants with two- or four-nucleotide deletions in the 3′ end retained the capacity to complement replication of psHIV-Phe. tRNA
Phe
mutants with an extended 5′ end had reduced capacity for complementation, which could be restored by extension of the 3′ end of these tRNA
Phe
mutants with sequences complementary to the HIV-1 U5 region. Further analysis of mutations in the acceptor stem of tRNA
Phe
suggested that an intact acceptor stem RNA structure is important for complementation. Analysis of single-nucleotide changes in the TΨC stem-loop of tRNA
Phe
revealed an unexpected, essential role of this region for rescue of psHIV-Phe.
Human Immunodeficiency Virus Type 1-Specific Immunity after Genetic Immunization Is Enhanced by Modification of Gag and Pol Expression
Authors: Huang Yue , Kong Wing-pui , Nabel Gary J.
Published: July 2002

Immunity to human immunodeficiency virus virion-like structures or a polyprotein has been examined after DNA immunization with Rev-independent expression vectors. A Gag-Pol fusion protein stimulated cytotoxic T lymphocyte and antibody responses to Gag and Pol, while a Gag-Pol pseudoparticle did not elicit substantial Pol responses. This fusion protein may be useful for AIDS vaccines.
Membrane-Fusing Capacity of the Human Immunodeficiency Virus Envelope Proteins Determines the Efficiency of CD4+T-Cell Depletion in Macaques Infected by a Simian-Human Immunodeficiency Virus
Authors: Etemad-Moghadam Bijan , Rhone Daniela , Steenbeke Tavis , Sun Ying , Manola Judith , Gelman Rebecca , Fanton John W. , Racz Paul , Tenner-Racz Klara , Axthelm Michael K. , Letvin Norman L. , Sodroski Joseph
Published: July 2002

The mechanism of the progressive loss of CD4+T lymphocytes, which underlies the development of AIDS in human immunodeficiency virus (HIV-1)-infected individuals, is unknown. Animal models, such as the infection of Old World monkeys by simian-human immunodeficiency virus (SHIV) chimerae, can assist studies of HIV-1 pathogenesis. Serial in vivo passage of the nonpathogenic SHIV-89.6 generated a virus, SHIV-89.6P, that causes rapid depletion of CD4+T lymphocytes and AIDS-like illness in monkeys. SHIV-KB9, a molecularly cloned virus derived from SHIV-89.6P, also caused CD4+T-cell decline and AIDS in inoculated monkeys. It has been demonstrated that changes in the envelope glycoproteins of SHIV-89.6 and SHIV-KB9 determine the degree of CD4+T-cell loss that accompanies a given level of virus replication in the host animals (G. B. Karlsson et. al., J. Exp. Med. 188:1159–1171, 1998). The envelope glycoproteins of the pathogenic SHIV mediated membrane fusion more efficiently than those of the parental, nonpathogenic virus. Here we show that the minimal envelope glycoprotein region that specifies this increase in membrane-fusing capacity is sufficient to convert SHIV-89.6 into a virus that causes profound CD4+T-lymphocyte depletion in monkeys. We also studied two single amino acid changes that decrease the membrane-fusing ability of the SHIV-KB9 envelope glycoproteins by different mechanisms. Each of these changes attenuated the CD4+T-cell destruction that accompanied a given level of virus replication in SHIV-infected monkeys. Thus, the ability of the HIV-1 envelope glycoproteins to fuse membranes, which has been implicated in the induction of viral cytopathic effects in vitro, contributes to the capacity of the pathogenic SHIV to deplete CD4+T lymphocytes in vivo.
A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity
Authors: Lindemann Dirk , Pietschmann Thomas , Picard-Maureau Marcus , Berg Angelika , Heinkelein Martin , Thurow Jana , Knaus Petra , Zentgraf Hanswalter , Rethwilm Axel
Published: July 2002

Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes.
Second-Site Suppressors of Rous Sarcoma Virus CA Mutations: Evidence for Interdomain Interactions
Authors: Bowzard J. Bradford , Wills John W. , Craven Rebecca C.
Published: July 2002

The capsid (CA) protein, the major structural component of retroviruses, forms a shell that encases the ribonucleoprotein complex in the virion core. The most conserved region of CA, ∼20 amino acids of the major homology region (MHR), lies within the carboxy-terminal domain of the protein. Structural and sequence similarities among CA proteins of retroviruses and the CA-like proteins of hepatitis B virus and various retrotransposons suggest that the MHR is involved in an aspect of replication common to these reverse-transcribing elements. Conservative substitutions in this region of the Rous sarcoma virus protein were lethal due to a severe deficiency in reverse transcription, in spite of the presence of an intact genome and active reverse transcriptase in the particles. This finding suggests that the mutations interfered with normal interactions among these constituents. A total of four genetic suppressors of three lethal MHR mutations have now been identified. All four map to the sequence encoding the CA-spacer peptide (SP) region of Gag. The F167Y mutation in the MHR was fully suppressed by a single amino acid change in the alpha helix immediately downstream of the MHR, a region that forms the major dimer interface in human immunodeficiency virus CA. This finding suggests that the F167Y mutation indirectly interfered with dimerization. The F167Y defect could also be repaired by a second, independent suppressor in the C-terminal SP that was removed from CA during maturation. This single residue change, which increased the rate of SP cleavage, apparently corrected the F167Y defect by modifying the maturation pathway. More surprising was the isolation of suppressors of the R170Q and L171V MHR mutations, which mapped to the N-terminal domain of the CA protein. This finding suggests that the two domains, which in the monomeric protein are separated by a flexible linker, must communicate with each other at some unidentified point in the viral replication cycle.
Phylogenetic and Evolutionary Relationships among Yellow Fever Virus Isolates in Africa
Authors: Mutebi John-Paul , Wang Heiman , Li Li , Bryant Juliet E. , Barrett Alan D. T.
Published: July 2002

Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.
Reactivation of Latent Human Cytomegalovirus in CD14+Monocytes Is Differentiation Dependent
Authors: Söderberg-Nauclér Cecilia , Streblow Daniel N. , Fish Kenneth N. , Allan-Yorke Justine , Smith Patricia P. , Nelson Jay A.
Published: July 2002

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4+and CD8+T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14+monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-γ) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-γ was crucial for reactivation of latent HCMV, addition of IFN-γ to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-γ in the reactivation of HCMV.
Attenuation of Murray Valley Encephalitis Virus by Site-Directed Mutagenesis of the Hinge and Putative Receptor-Binding Regions of the Envelope Protein
Authors: Hurrelbrink Robert J. , McMinn Peter C.
Published: July 2002

Molecular determinants of virulence in flaviviruses cluster in two regions on the three-dimensional structure of the envelope (E) protein; the base of domain II, believed to serve as a hinge during pH-dependent conformational change in the endosome, and the lateral face of domain III, which contains an integrin-binding motif Arg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to form the receptor-binding site of the protein. In an effort to better understand the nature of attenuation caused by mutations in these two regions, a full-length infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was employed to produce a panel of site-directed mutants with substitutions at amino acid positions 277 (E-277; hinge region) or 390 (E-390; RGD motif). Viruses with mutations at E-277 (Ser→Ile, Ser→Asn, Ser→Val, and Ser→Pro) showed various levels of in vitro and in vivo attenuation dependent on the level of hydrophobicity of the substituted amino acid. Altered hemagglutination activity observed for these viruses suggests that mutations in the hinge region may indirectly disrupt the receptor-ligand interaction, possibly by causing premature release of the virion from the endosomal membrane prior to fusion. Similarly, viruses with mutations at E-390 (Asp→Asn, Asp→Glu, and Asp→Tyr) were also attenuated in vitro and in vivo; however, the absorption and penetration rates of these viruses were similar to those of wild-type virus. This, coupled with the fact that E-390 mutant viruses were only moderately inhibited by soluble heparin, suggests that RGD-dependent integrin binding is not essential for entry of MVE and that multiple and/or alternate receptors may be involved in cell entry.
Effects of Mutations within the Herpes Simplex Virus Type 1 DNA Encapsidation Signal on Packaging Efficiency
Authors: Hodge Paul D. , Stow Nigel D.
Published: July 2002

The
cis
-acting signals required for cleavage and encapsidation of the herpes simplex virus type 1 genome lie within the terminally redundant region or
a
sequence. The
a
sequence is flanked by short direct repeats (DR1) containing the site of cleavage, and quasi-unique regions, Uc and Ub, occupy positions adjacent to the genomic L and S termini, respectively, such that a novel fragment, Uc-DR1-Ub, is generated upon ligation of the genomic ends. The Uc-DR1-Ub fragment can function as a minimal packaging signal, and motifs have been identified within Uc and Ub that are conserved near the ends of other herpesvirus genomes (
pac
2 and
pac
1, respectively). We have introduced deletion and substitution mutations within the
pac
regions of the Uc-DR1-Ub fragment and assessed their effects on DNA packaging in an amplicon-based transient transfection assay. Within
pac
2, mutations affecting the T tract had the greatest inhibitory effect, but deletion of sequences on either side of this element also reduced packaging, suggesting that its position relative to other sequences within the Uc-DR1-Ub fragment is likely to be important. No single region essential for DNA packaging was detected within
pac
1. However, mutants lacking the G tracts on either side of the
pac
1 T-rich motif exhibited a reduced efficiency of serial propagation, and alteration of the sequences between DR1 and the
pac
1 T element also resulted in defective generation of Ub-containing terminal fragments. The data are consistent with a model in which initiation and termination of packaging are specified by sequences within Uc and Ub, respectively.
Early Spread of Scrapie from the Gastrointestinal Tract to the Central Nervous System Involves Autonomic Fibers of the Splanchnic and Vagus Nerves
Authors: McBride Patricia A. , Schulz-Schaeffer Walter J. , Donaldson Maura , Bruce Moira , Diringer H. , Kretzschmar Hans A. , Beekes Michael
Published: July 2002

Although the ultimate target of infection is the central nervous system (CNS), there is evidence that the enteric nervous system (ENS) and the peripheral nervous system (PNS) are involved in the pathogenesis of orally communicated transmissible spongiform encephalopathies. In several peripherally challenged rodent models of scrapie, spread of infectious agent to the brain and spinal cord shows a pattern consistent with propagation along nerves supplying the viscera. We used immunocytochemistry (ICC) and paraffin-embedded tissue (PET) blotting to identify the location and temporal sequence of pathological accumulation of a host protein, PrP, in the CNS, PNS, and ENS of hamsters orally infected with the 263K scrapie strain. Enteric ganglia and components of splanchnic and vagus nerve circuitry were examined along with the brain and spinal cord. Bioassays were carried out with selected PNS constituents. Deposition of pathological PrP detected by ICC was consistent with immunostaining of a partially protease-resistant form of PrP (PrPSc) in PET blots. PrPSccould be observed from approximately one-third of the way through the incubation period in enteric ganglia and autonomic ganglia of splanchnic or vagus circuitry prior to sensory ganglia. PrPScaccumulated, in a defined temporal sequence, in sites that accurately reflected known autonomic and sensory relays. Scrapie agent infectivity was present in the PNS at low or moderate levels. The data suggest that, in this scrapie model, the infectious agent primarily uses synaptically linked autonomic ganglia and efferent fibers of the vagus and splanchnic nerves to invade initial target sites in the brain and spinal cord.
The Latency-Associated Nuclear Antigen Encoded by Kaposi's Sarcoma-Associated Herpesvirus Activates Two Major Essential Epstein-Barr Virus Latent Promoters
Authors: Groves Angela K. , Cotter Murray A. , Subramanian Chitra , Robertson Erle S.
Published: July 2002

The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of
LMP1
and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the −204 to +40 region had increased levels of activation compared with a larger region, −512 to +40, which contains two recombination signal-binding protein Jκ binding sites. The smaller, −204 to +40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the −204 to +40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.
Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases
Authors: Beck Zachary Q. , Lin Ying-Chuan , Elder John H.
Published: July 2002

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH
2
(arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH
2
NH)VVNGL-NH
2.
This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.
Mutagenesis of the
Dengue Virus Type 2
NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication
Authors: Matusan Anita E. , Pryor Melinda J. , Davidson Andrew D. , Wright Peter J.
Published: July 2002

The protein NS3 of
Dengue virus type 2
(DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family
Flaviviridae
. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione
S-
transferase in
Escherichia coli.
The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R
376
KNGK
380
, abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R
184
KR
186
and D
436
GEE
439
, we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D
334
EE
336
and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.