bluuebunny/crossref_metadata_2025 huggingface dataset:
Biofilm Formation by
Candida dubliniensis
Authors: Ramage Gordon , Vande Walle Kacy , Wickes Brian L. , López-Ribot José L.
Published: July 2002

Candida dubliniensis
is an opportunistic yeast closely related to
Candida albicans
that has been recently implicated in oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Most manifestations of candidiasis are associated with biofilm formation, with cells in biofilms displaying properties dramatically different from free-living cells grown under normal laboratory conditions. Here, we report on the development of in vitro models of
C. dubliniensis
biofilms on the surfaces of biomaterials (polystyrene and acrylic) and on the characteristics associated with biofilm formation by this newly described species. Time course analysis using a formazan salt reduction assay to monitor metabolic activities of cells within the biofilm, together with microscopy studies, revealed that biofilm formation by
C. dubliniensis
occurred after initial focal adherence, followed by growth, proliferation, and maturation over 24 to 48 h. Serum and saliva preconditioning films enhanced the initial attachment of
C. dubliniensis
and subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to further characterize
C. dubliniensis
biofilms. Mature
C. dubliniensis
biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material.
C. dubliniensis
biofilms displayed spatial heterogeneity and an architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile
C. dubliniensis
cells, including the type strain and eight different clinical isolates, against fluconazole and amphotericin B compared to their planktonic counterparts.
C. dubliniensis
biofilm formation may allow this species to maintain its ecological niche as a commensal and during infection with important clinical repercussions.
Application of PCR to Distinguish Common Species of Dermatophytes
Authors: Faggi Elisabetta , Pini Gabriella , Campisi Enza , Bertellini Chiara , Difonzo Elisa , Mancianti Francesca
Published: July 2002

This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)
4
. The primer was able to amplify all the strains, producing species-specific profiles for
Microsporum canis
,
Microsporum gypseum
,
Trichophyton rubrum
,
Trichophyton ajelloi
, and
Epidermophyton floccosum.
Intraspecific variability was not observed for these species. Instead, three different profiles were observed in the
Trichophyton mentagrophytes
group.
The
ica
Operon and Biofilm Production in Coagulase-Negative Staphylococci Associated with Carriage and Disease in a Neonatal Intensive Care Unit
Authors: de Silva G. D. I. , Kantzanou M. , Justice A. , Massey R. C. , Wilkinson A. R. , Day N. P. J. , Peacock S. J.
Published: July 2002

Coagulase-negative staphylococci (CoNS) are a major cause of sepsis in the neonatal intensive care unit (NICU). We evaluated the hypothesis that the
ica
operon and biofilm production are associated with CoNS disease in this setting. CoNS associated with bacteremia or blood culture contamination and from the skin of infants with CoNS bacteremia or healthy controls were obtained during a prospective case-control study on a busy NICU. A total of 180 strains were identified, of which 122 (68%) were
Staphylococcus epidermidis
and the remainder were
S. capitis
(
n
= 29),
S. haemolyticus
(
n
= 11),
S. hominis
(
n
= 9),
S. warneri
(
n
= 8), and
S. auricularis
(
n
= 1). The presence of the genes
icaA, icaB, icaC
, and
icaD
was determined by PCR, and biofilm production was examined using qualitative (Congo red agar [CRA]) and quantitative (microtiter plate) techniques. There were no significant differences in the presence of the
ica
operon or CRA positivity among the four groups of strains. However, quantitative biofilm production was significantly greater in strains isolated from either the blood or the skin of neonates with
S. epidermidis
bacteremia. We conclude that the quantity of biofilm produced may be associated with the ability to cause CoNS infection. This conclusion suggests that the regulation of biofilm expression may play a central role in the disease process.
Discrimination of Single-Copy IS6110DNA Fingerprints ofMycobacterium tuberculosisIsolates by High-Resolution Minisatellite-Based Typing
Authors: Lee Ann S. G. , Tang Lynn L. H. , Lim Irene H. K. , Bellamy Richard , Wong Sin-Yew
Published: July 2002

Seven isoniazid-resistant isolates with mutations in the NADH dehydrogenase (ndh) gene were molecularly typed by IS6110-based restriction fragment length polymorphism analysis. All seven isolates with the R268H mutation had identical 1.4-kb IS6110fingerprints. High-resolution minisatellite-based typing discriminated five of these isolates; two isolates were identical.
In Vitro Activities of Broad-Spectrum Cephalosporins against Nonmeningeal Isolates of
Streptococcus pneumoniae
: MIC Interpretation Using NCCLS M100-S12 Recommendations
Authors: Sahm Daniel F. , Thornsberry Clyde , Mayfield David C. , Jones Mark E. , Karlowsky James A.
Published: July 2002

Publication of the NCCLS M100-S12 document in January 2002 introduced ceftriaxone and cefotaxime MIC interpretative breakpoints of ≤1 μg/ml (susceptible), 2 μg/ml (intermediate), and ≥4 μg/ml (resistant) for nonmeningeal isolates of
Streptococcus pneumoniae
. To estimate the effect of these breakpoint changes on clinical laboratory susceptibility testing results, nonmeningeal pneumococcal isolate (blood and respiratory) data from The Surveillance Network Database-USA, an electronic surveillance database, for the years 1996 to 2000 were collated and studied. Of 9,863 nonmeningeal isolates tested against ceftriaxone, 82.7% were susceptible, 13.2% were intermediate, and 4.1% were resistant by the M100-S11 NCCLS breakpoints (2001); by M100-S12 breakpoints, 95.9% of the isolates were susceptible, 3.1% were intermediate, and 1.0% were resistant. Of 10,777 nonmeningeal isolates tested against cefotaxime, 79.2% were susceptible, 14.3% were intermediate, and 6.5% were resistant by M100-S11 breakpoints; by M100-S12 breakpoints, 93.5% were susceptible, 4.2% were intermediate, and 2.3% were resistant. Overall, the new M100-S12 ceftriaxone and cefotaxime interpretative breakpoints for nonmeningeal isolates of
S. pneumoniae
decreased the number of isolates interpreted as intermediate by 10% and as resistant by 3 to 4%.
Pulmonary Colonization by
Chrysosporium zonatum
Associated with Allergic Inflammation in an Immunocompetent Subject
Authors: Hayashi Shinichiro , Naitoh Keiko , Matsubara Shinichi , Nakahara Yoshiaki , Nagasawa Zenzo , Tanabe Ichiro , Kusaba Kohzi , Tadano Jutaro , Nishimura Kazuko , Sigler Lynne
Published: July 2002

We report a case of noninvasive pulmonary disease due to
Chrysosporium zonatum
in an immunocompetent male. The fungus colonized an existing tuberculous cavity and was isolated from transbronchial lavage fluid and from a percutaneous aspiration specimen. The disease was accompanied by the unusual feature of an allergic reaction. The fungus ball was successfully treated by intracavitary administration of amphotericin B.
C. zonatum
is the anamorph of the heterothallic ascomycete
Uncinocarpus orissi
, and the identity of the case isolate was verified by formation of ascospores in mating tests with reference isolates.
Antigenic Diversity and Distribution of Rabies Virus in Mexico
Authors: Velasco-Villa Andrés , Gómez-Sierra Mauricio , Hernández-Rodríguez Gustavo , Juárez-Islas Victor , Meléndez-Félix Alejandra , Vargas-Pino Fernando , Velázquez-Monroy Oscar , Flisser Ana
Published: July 2002

Rabies remains a public health problem in the Americas because of the great diversity of wild reservoirs that maintain the virus in nature. Here we report the antigenic characterization of 254 rabies viruses isolated from 148 nonreservoir and 106 reservoir hosts collected in 27 states of Mexico. Nine out of 11 antigenic variants previously reported in the United States were detected in Mexico by using the limited panel of monoclonal antibodies donated by the Centers for Disease Control and Prevention. Some rabies virus variants were isolated from their natural reservoirs, which were also taxonomically identified. Terrestrial reservoirs included stray dogs with V1,
Urocyon cineroargenteus
(gray foxes) with V7, and two subspecies of
Spilogale putorius
(spotted skunks) with different viral variants (V8 and V10). Aerial hosts included
Tadarida brasiliensis mexicana
and
Desmodus rotundus
, which harbored V9 and V4 and harbored V11, respectively. All variants, with the exception of V9, were isolated from nonreservoir hosts, while V3, V4, and V5 were not isolated from their natural reservoirs but only from livestock. Rabies virus antigenic typing allowed us to determine rabies reservoirs and their distribution in Mexico, data which will probably improve prevention and control of the illness in humans and in the reservoir hosts.
Enhanced Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus
Authors: Studentsov Yevgeniy Y. , Schiffman Mark , Strickler Howard D. , Ho Gloria Y. F. , Pang Yuk-Ying Susana , Schiller John , Herrero Rolando , Burk Robert D.
Published: July 2002

Measurement of antibodies to human papillomavirus (HPV) is complicated by many factors. Although enzyme-linked immunosorbent assays (ELISAs) that use virus-like particles (VLPs) have proved useful, the assays have, in general, had moderate sensitivities and low signal-to-noise ratios. To enhance the performance of the assay, a systematic investigation was undertaken to examine key variables used in ELISAs for the detection of antibodies to VLPs of HPV. Incorporation of two vinyl polymers, polyvinyl alcohol (molecular weight, 50,000) (PVA-50) and polyvinylpyrrolidone (molecular weight, 360,000) (PVP-360), was found to increase the sensitivity as well as the specificity of the assay for the detection of antibodies to VLPs of HPV. In particular, the addition of PVA-50 to the blocking solution reduced the amount of nonspecific binding of antibodies to VLPs and the microplate surface, whereas the addition of PVP-360 increased the sensitivity of antibody detection. The new ELISA demonstrated increased sensitivity and specificity for the detection of cervical HPV type 16 infection compared to those of a prototype assay with coded clinical serum samples from women with known cervicovaginal HPV infection status. It is anticipated that the enhanced ELISA conditions will have wide application to a large number of clinical diagnostic assays.
Microsatellite Characterization of
Plasmodium falciparum
from Cerebral and Uncomplicated Malaria Patients in Southern Vietnam
Authors: Ferreira Marcelo U. , Nair Shalini , Hyunh Thien Van , Kawamoto Fumihiko , Anderson Timothy J. C.
Published: July 2002

If parasite genotype influences the clinical course of malaria, we expect that isolates from patients with similar pathology would be more closely related than would be expected by chance. To explore this prediction, we typed nine microsatellite markers in sympatric
Plasmodium falciparum
isolates from cerebral and uncomplicated malaria patients from Vietnam. Temporal structure and linkage disequilibrium were also examined in this data set.
Development of Variable-Number Tandem Repeat Typing ofMycobacterium bovis: Comparison of Results with Those Obtained by Using Existing Exact Tandem Repeats and Spoligotyping
Authors: Roring Solvig , Scott Alistair , Brittain David , Walker Ian , Hewinson Glyn , Neill Sydney , Skuce Robin
Published: July 2002

Various genetic markers have been exploited for fingerprinting theMycobacterium tuberculosiscomplex (MTBC) in molecular epidemiological studies, mainly through identifying restriction fragment length polymorphisms (RFLP). In large-scale studies, RFLP typing has practical processing and analysis limitations; therefore, attempts have been made to move towards PCR-based typing techniques. Spoligotyping (spacer oligotyping) and, more recently, variable-number tandem repeat (VNTR) typing have provided PCR-derived typing techniques. This study describes the identification and characterization of novel VNTR loci, consisting of tandem repeats in the size range of 53 to 59 bp in the MTBC, and their assessment as typing tools in 47Mycobacterium bovisfield isolates and nine MTBC strains. Spoligotyping and the previously described set of exact tandem repeats (ETRs) (R. Frothingham and W. A. Meeker-O'Connell, Microbiology144:1189-1196, 1998) were also applied to the same panel of isolates. The allelic diversity of the individual VNTR loci was calculated, and a comparison of the novel VNTRs was made against the results obtained by spoligotyping and the existing set of ETRs. Eleven unique spoligotypes were discriminated in the panel of 47M. bovisisolates. Greater resolution was obtained through the combination of the most-discriminating VNTRs from both sets. Considerable discrimination was achieved, with the 47M. bovisisolates resolved into 14 unique profiles, while all nine MTBC isolates were uniquely differentiated. The novel VNTR markers described increased the discrimination possible in strain typing ofM. bovis, with the added benefit of an intuitive digital nomenclature, with the allele copy number of the individual VNTRs providing a profile. VNTR typing was shown to be a valuable technique with great potential for further development and application to epidemiological tracing of tuberculosis transmissions.
Core Protein Phosphorylation Modulates Pregenomic RNA Encapsidation to Different Extents in Human and Duck Hepatitis B Viruses
Authors: Gazina Elena V. , Fielding James E. , Lin Bo , Anderson David A.
Published: July 2002

To clarify the role of core protein phosphorylation in pregenomic-RNA encapsidation of human and duck hepatitis B viruses (HBV and DHBV, respectively), we have examined the phosphorylation states of different forms of intracellular HBV core protein and the phenotypic effects of mutations in the phosphorylation sites of HBV and DHBV core proteins. We show that HBV core protein is phosphorylated to similar extents in the form of protein dimers and after further assembly in pregenomic RNA-containing capsids. Individual and multiple substitutions of alanine and aspartic acid for serine in the phosphorylation sites of HBV core protein resulted in site-specific and synergistic effects on RNA encapsidation, ranging from 2-fold enhancement to more than 10-fold inhibition. Core protein variants with mutations in all phosphorylation sites exhibited dominant-negative effects on RNA encapsidation by wild-type protein. The results suggest that the presence of phosphoserine at position 162 of HBV core protein is required for pregenomic-RNA encapsidation, whereas phosphoserine at position 170 optimizes the process and serine might be preferable in position 155. Examination of the pregenomic-RNA-encapsidating capacities of DHBV core protein variants, in which four phosphorylation sites were jointly mutated to alanine or aspartic acid, suggests that phosphorylation of DHBV core protein at these sites may optimize pregenomic-RNA encapsidation but that its impact is much less profound than in the case of HBV. The possible mechanisms by which RNA encapsidation may be modulated by core protein phosphorylation are discussed in the context of the observed differences between the two viruses.
The Intracytoplasmic Domain of the Env Transmembrane Protein Is a Locus for Attenuation of Simian Immunodeficiency Virus SIVmac in Rhesus Macaques
Authors: Shacklett Barbara L. , Weber Claudia Jo , Shaw Karen E. S. , Keddie Elise M. , Gardner Murray B. , Sonigo Pierre , Luciw Paul A.
Published: July 2002

The human and simian immunodeficiency virus (HIV-1 and SIVmac) transmembrane proteins contain unusually long intracytoplasmic domains (ICD-TM). These domains are suggested to play a role in envelope fusogenicity, interaction with the viral matrix protein during assembly, viral infectivity, binding of intracellular calmodulin, disruption of membranes, and induction of apoptosis. Here we describe a novel mutant virus, SIVmac-M4, containing multiple mutations in the coding region for the ICD-TM of pathogenic molecular clone SIVmac239. Parental SIVmac239-Nef+ produces high-level persistent viremia and simian AIDS in both juvenile and newborn rhesus macaques. The ICD-TM region of SIVmac-M4 contains three stop codons, a +1 frameshift, and mutation of three highly conserved, charged residues in the conserved C-terminal alpha-helix referred to as lentivirus lytic peptide 1 (LLP-1). Overlapping reading frames fortat,rev, andnefare not affected by these changes. In this study, four juvenile macaques received SIVmac-M4 by intravenous injection. Plasma viremia, as measured by branched-DNA (bDNA) assay, reached a peak at 2 weeks postinoculation but dropped to below detectable levels by 12 weeks. At over 1.5 years postinoculation, all four juvenile macaques remain healthy and asymptomatic. In a subsequent experiment, four neonatal rhesus macaques were given SIVmac-M4 intravenously. These animals exhibited high levels of viremia in the acute phase (2 weeks postinoculation) but are showing a relatively low viral load in the chronic phase of infection, with no clinical signs of disease for 1 year. These findings demonstrated that the intracytoplasmic domain of the transmembrane Env (Env-TM) is a locus for attenuation in rhesus macaques.
The Chicken Anemia Virus-Derived Protein Apoptin Requires Activation of Caspases for Induction of Apoptosis in Human Tumor Cells
Authors: Danen-van Oorschot A. A. A. M. , van der Eb A. J. , Noteborn M. H. M.
Published: July 2002

The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e.g., many chemotherapeutic agents and radiation) require functional p53 and are inhibited by Bcl-2, Apoptin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptotic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis. Although Apoptin activity was not affected by CrmA, p35 did inhibit Apoptin-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. However, in most of these cells, cytochrome
c
is still released and the mitochondria are not stained by CMX-Ros, indicating a drop in mitochondrial membrane potential. These results imply that although the final apoptotic events are blocked by p35, parts of the upstream apoptotic pathway that affect mitochondria are already activated by Apoptin. Taken together, these data show that the viral protein Apoptin employs cellular apoptotic factors for induction of apoptosis. Although activation of upstream caspases is not required, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.
The First Step of Adenovirus Type 2 Disassembly Occurs at the Cell Surface, Independently of Endocytosis and Escape to the Cytosol
Authors: Nakano M. Y. , Boucke K. , Suomalainen M. , Stidwill R. P. , Greber U. F.
Published: July 2002

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.
Semliki Forest Virus Budding: Assay, Mechanisms, and Cholesterol Requirement
Authors: Lu Yanping E. , Kielian Margaret
Published: July 2002

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.
Neuroinvasion by Human Respiratory Coronaviruses
Authors: Arbour Nathalie , Day Robert , Newcombe Jia , Talbot Pierre J.
Published: July 2002

Human coronaviruses (HCoV) cause common colds but can also infect neural cell cultures. To provide definitive experimental evidence for the neurotropism and neuroinvasion of HCoV and its possible association with multiple sclerosis (MS), we have performed an extensive search and characterization of HCoV RNA in a large panel of human brain autopsy samples. Very stringent reverse transcription-PCR with two primer pairs for both viral strains (229E and OC43), combined with Southern hybridization, was performed on samples from 90 coded donors with various neurological diseases (39 with MS and 26 with other neurological diseases) or normal controls (25 patients). We report that 44% (40 of 90) of donors were positive for 229E and that 23% (21 of 90) were positive for OC43. A statistically significant higher prevalence of OC43 in MS patients (35.9%; 14 of 39) than in controls (13.7%; 7 of 51) was observed. Sequencing of nucleocapsid protein (N) gene amplicons revealed point mutations in OC43, some consistently found in three MS patient brains and one normal control but never observed in laboratory viruses. In situ hybridization confirmed the presence of viral RNA in brain parenchyma, outside blood vessels. The presence of HCoV in human brains is consistent with neuroinvasion by these respiratory pathogens. Further studies are needed to distinguish between opportunistic and disease-associated viral presence in human brains.
Induction of the Chemokines Interleukin-8 and IP-10 by Human Immunodeficiency Virus Type 1 Tat in Astrocytes
Authors: Kutsch O. , Oh J.-W. , Nath A. , Benveniste E. N.
Published: July 2002

A finding commonly observed in human immunodeficiency virus type 1 (HIV-1)-infected patients is invasion of the brain by activated T cells and infected macrophages, eventually leading to the development of neurological disorders and HIV-1-associated dementia. The recruitment of T cells and macrophages into the brain is likely the result of chemokine expression. Indeed, earlier studies revealed that levels of different chemokines were increased in the cerebrospinal fluid of HIV-1-infected patients whereas possible triggers and cellular sources for chemokine expression in the brain remain widely undefined. As previous studies indicated that HIV-1 Tat, the retroviral transactivator, is capable of inducing a variety of cellular genes, we investigated its capacity to induce production of chemokines in astrocytes. Herein, we demonstrate that HIV-1 Tat72aais a potent inducer of MCP-1, interleukin-8 (IL-8), and IP-10 expression in astrocytes. Levels of induced IP-10 protein were sufficiently high to induce chemotaxis of peripheral blood lymphocytes. In addition, Tat72aainduced IL-8 expression in astrocytes. IL-8 mRNA induction was seen less then 1 h after Tat72aastimulation, and levels remained elevated for up to 24 h, leading to IL-8 protein production. Tat72aa-mediated MCP-1 and IL-8 mRNA induction was susceptible to inhibition by the MEK1/2 inhibitor UO126 but was only modestly decreased by the inclusion of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190. In contrast, Tat-mediated IP-10 mRNA induction was suppressed by SB202190 but not by the MEK1/2 inhibitor UO126. These findings indicate that MAPKs play a major role in Tat72aa-mediated chemokine induction in astrocytes.
RNA Sequences Involved in Transcriptional Termination of Respiratory Syncytial Virus
Authors: Harmon Shawn B. , Megaw A. George , Wertz Gail W.
Published: July 2002

RNA signals at the ends of the genes of respiratory syncytial (RS) virus direct polyadenylation and termination of viral transcription. These gene ends contain two conserved regions, a pentanucleotide and a tract of uridylate (U) residues, separated by an A/U-rich central region that is less well conserved. The U tract is thought to be the template for polyadenylation of viral mRNAs by reiterative transcription. The
cis
-acting requirements for termination were investigated by mutagenesis of the matrix (M) gene end (3′-UCAAUUAUUUUUU-5′) in a dicistronic RNA replicon. Termination efficiencies were quantitated by intracellular metabolic labeling of monocistronic mRNAs and the dicistronic readthrough RNAs that result when termination fails to occur. All three regions of the gene end were necessary for termination. Mutation of each of the first 8 nucleotides of the M gene end to all other nucleotides showed that nucleotides 2 to 6 were important for termination and intolerant of change, whereas nucleotides 1 and 7 were tolerant of change. At position 8, A or U allowed termination, but G or C did not. Both the length and the position of the U tract were important for termination. U residues at positions 9 to 12 were necessary, while additional U residues at position 8, and especially position 13, enhanced termination efficiency. Altering the length of the central region abolished termination, suggesting that the position of the U tract with respect to the 3′-UCAAU-5′ sequence was critical. The termination efficiencies of each of the 10 genes of RS virus are different. Since transcription is obligatorily sequential and termination of each gene is required for transcription of the next gene downstream, these differences may contribute to gene regulation. In agreement with our data, the naturally occurring gene ends of RS virus that terminate inefficiently have short U tracts or other sequence features that correlated with decreased termination when similar mutations were analyzed in RNA replicons.
CD4+T Cells and Gamma Interferon in the Long-Term Control of Persistent Friend Retrovirus Infection
Authors: Iwashiro Michihiro , Peterson Karin , Messer Ronald J. , Stromnes Ingunn M. , Hasenkrug Kim J.
Published: July 2002

We have used the Friend virus model to determine the basic mechanisms by which the immune system can control persistent retroviral infections. Previously we showed that CD4+T cells play an essential role in keeping persistent retrovirus in check. The present in vitro experiments with a Friend virus-specific CD4+T-cell clone revealed that these cells produce gamma interferon (IFN-γ), which acts with two distinct mechanisms of antiviral activity. First, IFN-γ had a direct inhibitory effect on virus production. This inhibitory effect was noncytolytic and, interestingly, was not associated with decreased cell surface expression of viral antigens. The second mechanism of IFN-γ-mediated antiviral activity was an enhancement of CD4+T-cell-mediated cytolytic activity. We also found an in vivo role for IFN-γ in the control of persistent Friend virus infections. Neutralization of IFN-γ in persistently infected mice resulted in significantly increased levels of virus in the spleen, and a significant percentage of IFN-γ-deficient mice were unable to maintain long-term control over Friend virus infections.
Ebola Virus VP40-Induced Particle Formation and Association with the Lipid Bilayer
Authors: Jasenosky Luke D. , Neumann Gabriele , Lukashevich Igor , Kawaoka Yoshihiro
Published: July 2002

Viral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.
Relationships between CD4 Independence, Neutralization Sensitivity, and Exposure of a CD4-Induced Epitope in a Human Immunodeficiency Virus Type 1 Envelope Protein
Authors: Edwards Terri G. , Hoffman Trevor L. , Baribaud Frédéric , Wyss Stéphanie , LaBranche Celia C. , Romano Josephine , Adkinson Joshua , Sharron Matthew , Hoxie James A. , Doms Robert W.
Published: July 2002

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HIV-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-negative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD4 due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization. To study the relationships between CD4 independence, neutralization sensitivity, and exposure of CD4-induced epitopes associated with the coreceptor binding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2. We found that a frameshift mutation just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the coreceptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regions imparted CD4 independence, with other changes playing a modulatory role. The N386K mutation resulted in loss of an N-linked glycosylation site, but additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate structures shield important neutralization determinants. Exposure of the CD4-induced epitope recognized by monoclonal antibody 17b and which overlaps the coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 independence. Finally, while not all neutralization-sensitive Envs were CD4 independent, all CD4-independent Envs exhibited enhanced sensitivity to neutralization by HIV-1-positive human sera, indicating that the humoral immune response can exert strong selective pressure against the CD4-independent phenotype in vivo. Whether this can be used to advantage in designing more effective immunogens remains to be seen.
Induction of Neutralizing Antibodies and Gag-Specific Cellular Immune Responses to an R5 Primary Isolate of Human Immunodeficiency Virus Type 1 in Rhesus Macaques
Authors: Montefiori David C. , Safrit Jeffrey T. , Lydy Shari L. , Barry Ashley P. , Bilska Miroslawa , Vo Ha T. T. , Klein Michel , Tartaglia James , Robinson Harriet L. , Rovinski Benjamin
Published: July 2002

The ability to generate antibodies that cross-neutralize diverse primary isolates is an important goal for human immunodeficiency virus type 1 (HIV-1) vaccine development. Most of the candidate HIV-1 vaccines tested in humans and nonhuman primates have failed in this regard. Past efforts have focused almost entirely on the envelope glycoproteins of a small number of T-cell line-adapted strains of the virus as immunogens. Here we assessed the immunogenicity of noninfectious virus-like particles (VLP) consisting of Gag, Pro (protease), and Env from R5 primary isolate HIV-1Bx08. Immunogens were delivered to rhesus macaques in the form of either purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1Bx08were detected in animals that received (i) coinoculations with DNABx08and VLPBx08, (ii) DNABx08followed by ALVACBx08boosting, and (iii) VLPBx08alone. The neutralizing antibodies were highly strain specific despite the fact that they did not appear to be directed to linear epitopes in the V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon (IFN-γ)-producing cells. These cellular immune responses required the inclusion of DNABx08in the immunization modality, since few or no IFN-γ-producing cells were detected in animals that received either VLPBx08or ALVACBx08alone. The results demonstrate the feasibility of generating neutralizing antibodies and cellular immune responses that target an R5 primary HIV-1 isolate by vaccination in primates.
Nonenveloped Nucleocapsids of Hepatitis C Virus in the Serum of Infected Patients
Authors: Maillard P. , Krawczynski K. , Nitkiewicz J. , Bronnert C. , Sidorkiewicz M. , Gounon P. , Dubuisson J. , Faure G. , Crainic R. , Budkowska A.
Published: July 2002

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.
Antibody Protects Macaques against Vaginal Challenge with a Pathogenic R5 Simian/Human Immunodeficiency Virus at Serum Levels Giving Complete Neutralization In Vitro
Authors: Parren Paul W. H. I. , Marx Preston A. , Hessell Ann J. , Luckay Amara , Harouse Janet , Cheng-Mayer Cecilia , Moore John P. , Burton Dennis R.
Published: July 2002

A major unknown in human immunodeficiency virus (HIV-1) vaccine design is the efficacy of antibodies in preventing mucosal transmission of R5 viruses. These viruses, which use CCR5 as a coreceptor, appear to have a selective advantage in transmission of HIV-1 in humans. Hence R5 viruses predominate during primary infection and persist throughout the course of disease in most infected people. Vaginal challenge of macaques with chimeric simian/human immunodeficiency viruses (SHIV) is perhaps one of the best available animal models for human HIV-1 infection. Passive transfer studies are widely used to establish the conditions for antibody protection against viral challenge. Here we show that passive intravenous transfer of the human neutralizing monoclonal antibody b12 provides dose-dependent protection to macaques vaginally challenged with the R5 virus SHIV
162P4
. Four of four monkeys given 25 mg of b12 per kg of body weight 6 h prior to challenge showed no evidence of viral infection (sterile protection). Two of four monkeys given 5 mg of b12/kg were similarly protected, whereas the other two showed significantly reduced and delayed plasma viremia compared to control animals. In contrast, all four monkeys treated with a dose of 1 mg/kg became infected with viremia levels close to those for control animals. Antibody b12 serum concentrations at the time of virus challenge corresponded to approximately 400 (25 mg/kg), 80 (5 mg/kg), and 16 (1 mg/kg) times the in vitro (90%) neutralization titers. Therefore, complete protection against mucosal challenge with an R5 SHIV required essentially complete neutralization of the infecting virus. This suggests that a vaccine based on antibody alone would need to sustain serum neutralizing antibody titers (90%) of the order of 1:400 to achieve sterile protection but that lower titers, around 1:100, could provide a significant benefit. The significance of such substerilizing neutralizing antibody titers in the context of a potent cellular immune response is an important area for further study.
The Pathogenicity of Human Immunodeficiency Virus (HIV) Type 1 Nef in CD4C/HIV Transgenic Mice Is Abolished by Mutation of Its SH3-Binding Domain, and Disease Development Is Delayed in the Absence of Hck
Authors: Hanna Zaher , Weng Xiaoduan , Kay Denis G. , Poudrier Johanne , Lowell Clifford , Jolicoeur Paul
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P
72
XXP
75
XXP
78
) was mutated to A
72
XXA
75
XXQ
78
. This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an
hck
−/−
(knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.
African Swine Fever Virus Protein p54 Interacts with the Microtubular Motor Complex through Direct Binding to Light-Chain Dynein
Authors: Alonso Covadonga , Miskin James , Hernáez Bruno , Fernandez-Zapatero Patricia , Soto Lourdes , Cantó Carmen , Rodrı́guez-Crespo Ignacio , Dixon Linda , Escribano José M.
Published: July 2002

Dynein is a minus-end-directed microtubule-associated motor protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus protein p54 to the light chain of cytoplasmic dynein (LC8). Interaction of p54 and LC8 occurs both in vitro and in cells, and the two proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of p54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.
DC-SIGN Interactions with Human Immunodeficiency Virus: Virus Binding and Transfer Are Dissociable Functions
Authors: Pöhlmann Stefan , Leslie George J. , Edwards Terri G. , Macfarlan Todd , Reeves Jacqueline D. , Hiebenthal-Millow Kirsten , Kirchhoff Frank , Baribaud Frédéric , Doms Robert W.
Published: July 2002

The C-type lectins DC-SIGN and DC-SIGNR capture and transfer human immunodeficiency virus (HIV) to susceptible cells, although the underlying mechanism is unclear. Here we show that DC-SIGN/DC-SIGNR-mediated HIV transmission involves dissociable binding and transfer steps, indicating that efficient virus transmission is not simply due to tethering of virus to the cell surface.
Requirement of Cysteines and Length of the Human Respiratory Syncytial Virus M2-1 Protein for Protein Function and Virus Viability
Authors: Tang Roderick S. , Nguyen Nick , Cheng Xing , Jin Hong
Published: July 2002

The M2-1 protein of
human respiratory syncytial virus
(hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys
3
-His
1
motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using β-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys
3
-His
1
motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys
3
-His
1
motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys
3
-His
1
motif. This indicates that the Cys
3
-His
1
motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length β-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.
Stabilization but Not the Transcriptional Activity of Herpes Simplex Virus VP16-Induced Complexes Is Evolutionarily Conserved among HCF Family Members
Authors: Lee Soyoung , Herr Winship
Published: July 2002

The human herpes simplex virus (HSV) protein VP16 induces formation of a transcriptional regulatory complex with two cellular factors—the POU homeodomain transcription factor Oct-1 and the cell proliferation factor HCF-1—to activate viral immediate-early-gene transcription. Although the cellular role of Oct-1 in transcription is relatively well understood, the cellular role of HCF-1 in cell proliferation is enigmatic. HCF-1 and the related protein HCF-2 form an HCF protein family in humans that is related to a
Caenorhabditis elegans
homolog called CeHCF. In this study, we show that all three proteins can promote VP16-induced-complex formation, indicating that VP16 targets a highly conserved function of HCF proteins. The resulting VP16-induced complexes, however, display different transcriptional activities. In contrast to HCF-1 and CeHCF, HCF-2 fails to support VP16 activation of transcription effectively. These results suggest that, along with HCF-1, HCF-2 could have a role, albeit probably a different role, in HSV infection. CeHCF can mimic HCF-1 for both association with viral and cellular proteins and transcriptional activation, suggesting that the function(s) of HCF-1 targeted by VP16 has been highly conserved throughout metazoan evolution.