bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and Evaluation of Real-Time PCR-Based Fluorescence Assays for Detection of
Chlamydia
pneumoniae
Authors: Tondella Maria Lucia C. , Talkington Deborah F. , Holloway Brian P. , Dowell Scott F. , Cowley Karyn , Soriano-Gabarro Montse , Elkind Mitchell S. , Fields Barry S.
Published: July 2002

Chlamydia
pneumoniae
is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of
C. pneumoniae
is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for
C. pneumoniae
were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the
ompA
gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine
C. pneumoniae
isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the
C. trachomatis
serovars,
C. psittaci
strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and
ompA
genes. The assays were compared by testing
C. pneumoniae
purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected
C. pneumoniae
in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of
C. pneumoniae
in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of
C. pneumoniae
in clinical specimens.
PCR-Based Diagnosis of Acute and Chronic Cutaneous Leishmaniasis Caused by
Leishmania
(
Viannia
)
Authors: Weigle Kristen A. , Labrada Luz Angela , Lozano Caterin , Santrich Cecilia , Barker Douglas C.
Published: July 2002

We evaluated PCR methods for diagnosis of acute and chronic cutaneous leishmaniasis (CL) in an area of Colombia where
Leishmania
(
Viannia
) is endemic. The PCR method specifically amplified whole linearized minicircle kinetoplast DNA (kDNA) of the
Leishmania
subgenus
Viannia
from biopsy lysates. PCR products were detected in agarose gels. For 255 acute cases, this PCR method had greater sensitivity (75.7%) than each conventional method, i.e., microscopic examination of Giemsa-stained lesion scraping (46.7%), biopsy culture (55.3%), aspirate culture (46.3%), and the conventional methods combined (70.2%). Among 44 cases of chronic CL, amplification of biopsy DNA was more sensitive (45.5%) than the individual (4.5 to 27.7%) and combined (27.3%) conventional methods. The detection of kDNA in biopsies from chronic lesions was enhanced by a chemiluminescent dot blot hybridization, which produced a sensitivity of 65.8% when alone and 90.9% when in combination with DNA extraction of biopsy lysates (
P
< 0.001). Three biopsies from 84 skin lesions of other etiologies were falsely positive by PCR (specificity, 96.4%). PCR detected kDNA more frequently in biopsies (detection level, 83.9%) than in aspirates (74.7%) from 103 cases of acute CL. Among aspirates from 53 chronic cases of CL, the alternative methods, DNA extraction and hybridization, increased sensitivity from 41.5 to 56.6% (
P
> 0.05). This enhanced PCR method in chronic biopsies was so much more sensitive than conventional methods that it should be considered the preferred diagnostic method for chronic CL. These findings support the appropriate incorporation of PCR into diagnostic strategies for cutaneous leishmaniasis.
Septicemia Due to
Pasteurella pneumotropica
: 16S rRNA Sequencing for Diagnosis Confirmation
Authors: Frebourg Noëlle Barbier , Berthelot Gilles , Hocq Raymond , Chibani Ahmed , Lemeland Jean-François
Published: July 2002

Bacteremia due to
Pasteurella pneumotropica
occurs infrequently. We report a case of septicemia in a 72-year-old woman who had no underlying illness. The microorganism was isolated from 10 blood cultures and identified by conventional and molecular methods. This is the first reported case of
P. pneumotropica
septicemia in an immunocompetent patient. The history of
P. pneumotropica
diseases in animals and humans and their varied clinical features are reviewed.
Species-Level Identification of Orthopoxviruses with an Oligonucleotide Microchip
Authors: Lapa Sergey , Mikheev Maxim , Shchelkunov Sergei , Mikhailovich Vladimir , Sobolev Alexander , Blinov Vladimir , Babkin Igor , Guskov Alexander , Sokunova Elena , Zasedatelev Alexander , Sandakhchiev Lev , Mirzabekov Andrei
Published: July 2002

A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within thecrmBgene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used).
Pneumocystis carinii
f. sp.
hominis
Is Not Infectious for SCID mice
Authors: Durand-Joly Isabelle , Aliouat El Moukhtar , Recourt Céline , Guyot Karine , François Nadine , Wauquier Michèle , Camus Daniel , Dei-Cas Eduardo
Published: July 2002

The infectious power of
Pneumocystis carinii
f. sp.
hominis
was explored by inoculating SCID mice intranasally with either
P. carinii
f. sp.
hominis
or
P. carinii
f. sp.
muris
isolates. Only mice inoculated with mouse parasites developed
Pneumocystis
pneumonia, as assessed by microscopy and PCR. These results suggest that humans do not contract pneumocystosis from animals.
Recombinant Chimeric Yellow Fever-Dengue Type 2 Virus Is Immunogenic and Protective in Nonhuman Primates
Authors: Guirakhoo F. , Weltzin R. , Chambers T. J. , Zhang Z.-X. , Soike K. , Ratterree M. , Arroyo J. , Georgakopoulos K. , Catalan J. , Monath T. P.
Published: July 2002

A chimeric yellow fever (YF)-dengue type 2 (dengue-2) virus (ChimeriVax-D2) was constructed using a recombinant cDNA infectious clone of a YF vaccine strain (YF 17D) as a backbone into which we inserted the premembrane (prM) and envelope (E) genes of dengue-2 virus (strain PUO-218 from a case of dengue fever in Bangkok, Thailand). The chimeric virus was recovered from the supernatant of Vero cells transfected with RNA transcripts and amplified once in these cells to yield a titer of 6.3 log10PFU/ml. The ChimeriVax-D2 was not neurovirulent for 4-week-old outbred mice inoculated intracerebrally. This virus was evaluated in rhesus monkeys for its safety (induction of viremia) and protective efficacy (induction of anti-dengue-2 neutralizing antibodies and protection against challenge). In one experiment, groups of non-YF-immune monkeys received graded doses of ChimeriVax-D2; a control group received only the vaccine diluents. All monkeys (except the control group) developed a brief viremia and showed no signs of illness. Sixty-two days postimmunization, animals were challenged with 5.0 log10focus forming units (FFU) of a wild-type dengue-2 virus. No viremia (<1.7 log10FFU/ml) was detected in any vaccinated group, whereas all animals in the placebo control group developed viremia. All vaccinated monkeys developed neutralizing antibodies in a dose-dependent response. In another experiment, viremia and production of neutralizing antibodies were determined in YF-immune monkeys that received either ChimeriVax-D2 or a wild-type dengue-2 virus. Low viremia was detected in ChimeriVax-D2-inoculated monkeys, whereas all dengue-2-immunized animals became viremic. All of these animals were protected against challenge with a wild-type dengue-2 virus, whereas all YF-immune monkeys and nonimmune controls became viremic upon challenge. Genetic stability of ChimeriVax-D2 was assessed by continuous in vitro passage in VeroPM cells. The titer of ChimeriVax-D2, the attenuated phenotype for 4-week-old mice, and the sequence of the inserted prME genes were unchanged after 18 passages in Vero cells. The high replication efficiency, attenuation phenotype in mice and monkeys, immunogenicity and protective efficacy, and genomic stability of ChimeriVax-D2 justify it as a novel vaccine candidate to be evaluated in humans.
Additive Effect of Neutralizing Antibody and Antiviral Drug Treatment in Preventing Virus Escape and Persistence
Authors: Seiler Peter , Senn Beatrice M. , Klenerman Paul , Kalinke Ulrich , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Poorly cytopathic or noncytopathic viruses can escape immune surveillance and establish a chronic infection. Here we exploited the strategy of combining antiviral drug treatment with the induction of a neutralizing antibody response to avoid the appearance of neutralization-resistant virus variants. Despite the fact that H25 immunoglobulin transgenic mice infected with lymphocytic choriomeningitis virus mounted an early neutralizing antibody response, the virus escaped from neutralization and persisted. After ribavirin treatment of H25 transgenic mice, the appearance of neutralization-resistant virus was prevented and virus was cleared. Thus, the combination of virus-neutralizing antibodies and chemotherapy efficiently controlled the infection, whereas each defense line alone did not. Similar additive effects may be unexpectedly efficient and beneficial in humans after infections with persistent viruses such as hepatitis C virus and hepatitis B virus and possibly human immunodeficiency virus.
Optimized Viral Dose and Transient Immunosuppression Enable Herpes Simplex Virus ICP0-Null Mutants To Establish Wild-Type Levels of Latency In Vivo
Authors: Halford William P. , Schaffer Priscilla A.
Published: July 2002

The reduced efficiency with which herpes simplex virus type 1 (HSV-1) mutants establish latent infections in vivo has been a fundamental obstacle in efforts to determine the roles of individual viral genes in HSV-1 reactivation. For example, in the absence of the “nonessential” viral immediate-early protein, ICP0, HSV-1 is severely impaired in its ability to (i) replicate at the site of inoculation and (ii) establish latency in neurons of the peripheral nervous system. The mouse ocular model of HSV latency was used in the present study to determine if the conditions of infection can be manipulated such that replication-impaired, ICP0-null mutants establish wild-type levels of latency, as measured by viral genome loads in latently infected trigeminal ganglia (TG). To this end, the effects of inoculum size and transient immunosuppression on the levels of acute replication in mouse eyes and of viral DNA in latently infected TG were examined. Following inoculation of mice with 2 × 103, 2 × 104, 2 × 105, or 2 × 106PFU/eye, wild-type virus replicated in mouse eyes and established latency in TG with similar efficiencies at all four doses. In contrast, increasing the inoculum size of the ICP0-null mutants n212 and 7134 from 2 × 105to 2 × 106PFU/eye significantly decreased the levels of infectious virus detected in the tear films of mice from days 4 to 9 postinfection. In an attempt to establish the biological basis for this finding, the effect of viral dose on the induction of the host proinflammatory response was examined. Quantitative reverse transcription-PCR demonstrated that increasing the inoculum of 7134 from 2 × 104to 2 × 106PFU/eye significantly increased the expression of proinflammatory (interleukin 6), cell adhesion (intercellular adhesion molecule 1), and phagocyte-associated (CD11b) genes in mouse eyes 24 h postinfection. Furthermore, transient immunosuppression of mice with cyclophosphamide, but not cyclosporin A, significantly enhanced both the levels of acute n212 and 7134 replication in the eye and the levels of mutant viral genomes present in latently infected TG in a dose-dependent manner. Thus, the results of this study demonstrate that acute replication in the eye and the number of ICP0-null mutant genomes in latently infected TG can be increased to wild-type levels for both n212 and 7134 by (i) optimization of inoculum size and (ii) transient immunosuppression with cyclophosphamide.
Binding of Tat to TAR and Recruitment of Positive Transcription Elongation Factor b Occur Independently in Bovine Immunodeficiency Virus
Authors: Barboric Matjaz , Taube Ran , Nekrep Nada , Fujinaga Koh , Peterlin B. Matija
Published: July 2002

Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5′ bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV.
Functional Significance of the Interaction of Hepatitis A Virus RNA with Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH): Opposing Effects of GAPDH and Polypyrimidine Tract Binding Protein on Internal Ribosome Entry Site Function
Authors: Yi MinKyung , Schultz Derk E. , Lemon Stanley M.
Published: July 2002

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a cellular enzyme involved in glycolysis, binds specifically to several viral RNAs, but the functional significance of this interaction is uncertain. Both GAPDH and polypyrimidine tract binding protein (PTB) bind to overlapping sites in stem-loop IIIa of the internal ribosome entry site (IRES) of
Hepatitis A virus
(HAV), a picornavirus. Since the binding of GAPDH destabilizes the RNA secondary structure, we reasoned that GAPDH may suppress the ability of the IRES to direct cap-independent translation, making its effects antagonistic to the translation-enhancing activity of PTB (D. E. Schultz, C. C. Hardin, and S. M. Lemon, J. Biol. Chem. 271:14134–14142, 1996). To test this hypothesis, we constructed plasmids containing a dicistronic transcriptional unit in which the HAV IRES was placed between an upstream GAPDH-coding sequence and a downstream
Renilla
luciferase (RLuc) sequence. Transfection with this plasmid results in overexpression of GAPDH and in RLuc production as a measure of IRES activity. RLuc activity was compared with that from a control, null-expression plasmid that was identical except for a frameshift mutation within the 5′ GAPDH coding sequence. In transfection experiments, GAPDH overexpression significantly suppressed HAV IRES activity in BSC-1 and FRhK-4 cells but not in Huh-7 cells, which have a significantly greater cytoplasmic abundance of PTB. GAPDH suppression of HAV translation was greater with the wild-type HAV IRES than with the IRES from a cell culture-adapted virus (HM175/P16) that has reproducibly higher basal translational activity in BSC-1 cells. Stem-loop IIIa RNA from the latter IRES had significantly lower affinity for GAPDH in filter binding experiments. Thus, the binding of GAPDH to the IRES of HAV suppresses cap-independent viral translation in vivo in African green monkey kidney cells. The enhanced replication capacity of cell culture-adapted HAV in such cells may be due in part to reduced affinity of the viral IRES for GAPDH.
Formation of the Poliovirus Replication Complex Requires Coupled Viral Translation, Vesicle Production, and Viral RNA Synthesis
Authors: Egger Denise , Teterina Natalya , Ehrenfeld Ellie , Bienz Kurt
Published: July 2002

Poliovirus (PV) infection induces the rearrangement of intracellular membranes into characteristic vesicles which assemble into an RNA replication complex. To investigate this transformation, endoplasmic reticulum (ER) membranes in HeLa cells were modified by the expression of different cellular or viral membrane-binding proteins. The membrane-binding proteins induced two types of membrane alterations, i.e., extended membrane sheets and vesicles similar to those found during a PV infection. Cells expressing membrane-binding proteins were superinfected with PV and then analyzed for virus replication, location of membranes, viral protein, and RNA by immunofluorescence and fluorescent in situ hybridization. Cultures expressing cellular or viral membrane-binding proteins, but not those expressing soluble proteins, showed a markedly reduced ability to support PV replication as a consequence of the modification of ER membranes. The altered membranes, regardless of their morphology, were not used for the formation of viral replication complexes during a subsequent PV infection. Specifically, membrane sheets were not substrates for PV-induced vesicle formation, and, surprisingly, vesicles induced by and carrying one or all of the PV replication proteins did not contribute to replication complexes formed by the superinfecting PV. The formation of replication complexes required active viral RNA replication. The extensive alterations induced by membrane-binding proteins in the ER resulted in reduced viral protein synthesis, thus affecting the number of cells supporting PV multiplication. Our data suggest that a functional replication complex is formed in
cis
, in a coupled process involving viral translation, membrane modification and vesicle budding, and viral RNA synthesis.
The Human Papillomavirus Type 16 E7 Oncogene Is Required for the Productive Stage of the Viral Life Cycle
Authors: Flores Elsa R. , Allen-Hoffmann B. Lynn , Lee Denis , Lambert Paul F.
Published: July 2002

The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.
Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion
Authors: Klupp Barbara G. , Nixdorf Ralf , Mettenleiter Thomas C.
Published: July 2002

A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.
Cathepsin G, a Neutrophil-Derived Serine Protease, Increases Susceptibility of Macrophages to Acute Human Immunodeficiency Virus Type 1 Infection
Authors: Moriuchi Hiroyuki , Moriuchi Masako , Fauci Anthony S.
Published: July 2002

Neutrophils dominate acute inflammatory responses that generally evolve into chronic inflammatory reactions mediated by monocyte/macrophages and lymphocytes. The latter cell types also serve as major targets for human immunodeficiency virus type 1 (HIV-1). In this study we have investigated the role of neutrophil products, particularly cathepsin G, in HIV infection. Cathepsin G induced chemotaxis and production of proinflammatory cytokines by macrophages but not CD4+T cells. Pretreatment with cathepsin G markedly increased susceptibility of macrophages but not CD4+T cells to acute HIV-1 infection. When macrophages were exposed to pertussis toxin prior to cathepsin G treatment, the cathepsin G-mediated effect was almost abrogated, suggesting that enhancement of HIV-1 replication by cathepsin G requires Gi protein-mediated signal transduction. Although prolonged exposure to cathepsin G suppressed HIV infection of macrophages, serine protease inhibitors, which are exuded from the bloodstream later during inflammatory processes, neutralized the inhibitory effect. Neutrophil extracts or supernatants from neutrophil cultures, which contain cathepsin G, had effects similar to purified cathepsin G. Thus, cathepsin G, and possibly other neutrophil-derived serine proteases, may have multiple activities in HIV-1 infection of macrophages, including chemoattraction of monocyte/macrophages (HIV-1 targets) to inflamed tissue, activation of target cells, and increase in their susceptibility to acute HIV-1 infection.
Complete DNA Sequence of the Rat Cytomegalovirus Genome
Authors: Vink Cornelis , Beuken Erik , Bruggeman Cathrien A.
Published: July 2002

We have determined the complete genome sequence of the Maastricht strain of rat cytomegalovirus (RCMV). The RCMV genome has a length of 229,896 bp and is arranged as a single unique sequence flanked by 504-bp terminal direct repeats. RCMV was found to have counterparts of all but one of the open reading frames (ORFs) that are conserved between murine CMV (MCMV) and human CMV (HCMV). Like HCMV, RCMV lacks homologs of the genes belonging to the MCMV m02 glycoprotein gene family. However, RCMV contains 15 ORFs with homology to members of the MCMV m145 glycoprotein gene family. Four ORFs are predicted to encode homologs of host proteins; R33 and R78 both putatively encode G protein-coupled receptors, whereas r144 and r131 encode homologs of major histocompatibility class I heavy chains and CC chemokines, respectively. An intriguing feature of the RCMV genome is the presence of an ORF, r127, with similarity to the
rep
gene of parvoviruses as well as ORF U94 of human herpesvirus 6A (HHV-6A) and HHV-6B. Counterparts of these ORFs have not been found in the other sequenced herpesviruses.
Mink Cell Focus-Forming Murine Leukemia Virus Infection Induces Apoptosis of Thymic Lymphocytes
Authors: Yoshimura Fayth K. , Wang Tao , Yu Fei , Kim Hyeong-Reh C. , Turner Jerrold R.
Published: July 2002

In a previous study we identified the subpopulations of thymus cells that were infected by the lymphomagenic MCF13 murine leukemia virus (MLV) (F. K. Yoshimura, T. Wang, and M. Cankovic, J. Virol. 73:4890–4898, 1999) and observed an effect on thymus size by virus infection. In this report we describe our results which demonstrate that MCF13 MLV infection of thymuses reduced the number of T lymphocytes in this organ. Histological examination showed diffuse lymphocyte depletion, which was most striking in the CD4+CD8+lymphocyte-enriched cortical zone. Consistent with this, flow cytometric analysis showed that the lymphocytes which were depleted were predominantly the immature CD3−CD4+CD8+and CD3+CD4+CD8+cells. A comparison of the percentages of live, apoptotic, and dead cells of the gp70+and gp70−thymic lymphocytes suggested that this effect on thymus cellularity is a result of virus infection. Studies of the survival of thymic T lymphocytes in culture showed that cells from MCF13 MLV-inoculated mice underwent greater apoptosis and death than cells from control animals. Assays for apoptosis included 7-amino-actinomycin D staining, DNA fragmentation, and cleavage of caspase-3 and poly(ADP-ribose) polymerase proenzymes. Our results suggest that apoptosis of thymic lymphocytes by virus infection is an important step in the early stages of MCF13 MLV tumorigenesis.
Study of the Assembly of Vesicular Stomatitis Virus N Protein: Role of the P Protein
Authors: Green Todd J. , Macpherson Silvia , Qiu Shihong , Lebowitz Jacob , Wertz Gail W. , Luo Ming
Published: July 2002

To derive structural information about the vesicular stomatitis virus (VSV) nucleocapsid (N) protein, the N protein and the VSV phosphoprotein (P protein) were expressed together in
Escherichia coli
. The N and P proteins formed soluble protein complexes of various molar ratios when coexpressed. The major N/P protein complex was composed of 10 molecules of the N protein, 5 molecules of the P protein, and an RNA. A soluble N protein-RNA oligomer free of the P protein was isolated from the N/P protein-RNA complex using conditions of lowered pH. The molecular weight of the N protein-RNA oligomer, 513,879, as determined by analytical ultracentrifugation, showed that it was composed of 10 molecules of the N protein and an RNA of approximately 90 nucleotides. The N protein-RNA oligomer had the appearance of a disk with outer diameter, inner diameter, and thickness of 148 ± 10 Å, 78 ± 9 Å, and 83 ± 8 Å, respectively, as determined by electron microscopy. RNA in the complexes was protected from RNase digestion and was stable at pH 11. This verified that N/P protein complexes expressed in
E. coli
were competent for encapsidation. In addition to coexpression with the full-length P protein, the N protein was expressed with the C-terminal 72 amino acids of the P protein. This portion of the P protein was sufficient for binding to the N protein, maintaining it in a soluble state, and for assembly of N protein-RNA oligomers. With the results provided in this report, we propose a model for the assembly of an N/P protein-RNA oligomer.
Multiple Blocks to Human Immunodeficiency Virus Type 1 Replication in Rodent Cells
Authors: Bieniasz Paul D. , Cullen Bryan R.
Published: July 2002

The recent identification of human gene products that are required for early steps in the human immunodeficiency virus type 1 (HIV-1) life cycle has raised the possibility that rodents might be engineered to support HIV-1 infection. Therefore, we have examined the ability of modified mouse, rat, and hamster cell lines to support productive HIV-1 replication. Rodent cells, engineered to support Tat function by stable expression of a permissive cyclin T1 protein, proved to be able to support reverse transcription, integration, and early gene expression at levels comparable to those observed in human cell lines. Surprisingly, however, levels of CD4- and coreceptor-dependent virus entry were reduced to a variable but significant extent in both mouse and rat fibroblast cell lines. Additional posttranscriptional defects were observed, including a reduced level of unspliced HIV-1 genomic RNA and reduced structural gene expression. Furthermore, the HIV-1 Gag precursor is generally inefficiently processed and is poorly secreted from mouse and rat cells in a largely noninfectious form. These posttranscriptional defects, together, resulted in a dramatically reduced yield of infectious virus (up to 10,000-fold) over a single cycle of HIV-1 replication, as compared to human cells. Interestingly, these defects were less pronounced in one hamster cell line, CHO, which not only was able to produce infectious HIV-1 particles at a level close to that observed in human cells, but also could support transient, low-level HIV-1 replication. Importantly, the blocks to infectious virus production in mouse and rat cells are recessive, since they can be substantially suppressed by fusion with uninfected human cells. These studies imply the existence of one or more human gene products, either lacking or nonfunctional in most rodent cells that are critical for infectious HIV-1 virion morphogenesis.
Measles Virus Assembly within Membrane Rafts
Authors: Vincent Séverine , Gerlier Denis , Manié Serge N.
Published: July 2002

During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305–311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.
Glycosaminoglycan Sulfation Requirements for Respiratory Syncytial Virus Infection
Authors: Hallak Louay K. , Spillmann Dorothe , Collins Peter L. , Peeples Mark E.
Published: July 2002

Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant green fluorescent protein-expressing RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in
N
-sulfation was three times less efficient than infection of the parental CHO cell line, indicating that
N
-sulfation is important. In contrast, infection of a cell line deficient in 2-
O
-sulfation was as efficient as infection of the parental cell line, indicating that 2-
O
-sulfation is not required for RSV infection. Incubating RSV with the purified soluble heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified heparin chains that lack their
N
-, C6-
O
-, or C2-
O
-sulfate groups. Only heparin chains lacking the
N
-sulfate group lost the ability to neutralize infection, confirming that
N
-sulfation, but not C6-
O
- or C2-
O
-sulfation, is important for RSV infection. Analysis of heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required. This specificity indicates that the role of cell surface GAGs in RSV infection is not based on a simple charge interaction between the virus and sulfate groups but instead involves a specific GAG structural configuration that includes
N
-sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264–275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.
Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity
Authors: Li Peggy P. , Nakanishi Akira , Tran Mary A. , Salazar Adler M. , Liddington Robert C. , Kasamatsu Harumi
Published: July 2002

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants—C9A, C49A, C87A, C104A, C207S, C254A, and C267L—retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca
2+
-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca
2+
ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.
DNA-Directed Expression of Functional Flock House Virus RNA1 Derivatives inSaccharomyces cerevisiae, Heterologous Gene Expression, and Selective Effects on Subgenomic mRNA Synthesis
Authors: Price B. Duane , Roeder Mark , Ahlquist Paul
Published: July 2002

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication inSaccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3′-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in eithercisortrans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.
Glycoprotein D or J Delivered intransBlocks Apoptosis in SK-N-SH Cells Induced by a Herpes Simplex Virus 1 Mutant Lacking Intact Genes Expressing Both Glycoproteins
Authors: Zhou Guoying , Galvan Veronica , Campadelli-Fiume Gabriella , Roizman Bernard
Published: July 2002

We have made two stocks of a herpes simplex virus 1 mutant lacking intact US5 and US6 open reading frames encoding glycoproteins J (gJ) and D (gD), respectively. The stock designated gD−/+, made in cells carrying US6 and expressing gD, was capable of productively infecting cells, whereas the stock designated gD−/−, made in cells lacking viral DNA sequences, was known to attach but not initiate infection. We report the following. (i) Both stocks of virus induced apoptosis in SK-N-SH cells. Thus, annexin V binding to cell surfaces was detected as early as 8 h after infection. (ii) US5 or US6 cloned into the baculovirus under the human cytomegalovirus immediate-early promoter was expressed in SK-N-SH cells and blocked apoptosis in cells infected with either gD−/+or gD−/−virus, whereas glycoprotein B, infected cell protein 22, or the wild-type baculovirus did not block apoptosis. (iii) In SK-N-SH cells, internalized, partially degraded virus particles were detected at 30 min after exposure to gD−/−virus but not at later intervals. (iv) Concurrent infection of cells with baculoviruses did not alter the failure of gD−/−virus from expressing its genes or, conversely, the expression of viral genes by gD−/+virus. These results underscore the capacity of herpes simplex virus to initiate the apoptotic cascade in the absence of de novo protein synthesis and indicate that both gD and gJ independently, and most likely at different stages in the reproductive cycle, play a key role in blocking the apoptotic cascade leading to cell death.
Infection of Human T Lymphocytes with Varicella-Zoster Virus: an Analysis with Viral Mutants and Clinical Isolates
Authors: Soong Weily , Schultz Julie C. , Patera Andriani C. , Sommer Marvin H. , Cohen Jeffrey I.
Published: July 2002

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.
The Lymphocytic Choriomeningitis Virus RING Protein Z Associates with Eukaryotic Initiation Factor 4E and Selectively Represses Translation in a RING-Dependent Manner
Authors: Campbell Dwyer Elizabeth J. , Lai HuiKang , MacDonald Rhea C. , Salvato Maria S. , Borden Katherine L. B.
Published: July 2002

Only a few host cell proteins that associate with arenaviruses have been identified. To date, the arenavirus Z protein associates with the promyelocytic leukemia protein PML and the ribosomal P proteins. The majority of PML is present in nuclear bodies which are translocated to the cytoplasm by infection with the arenavirus, lymphocytic choriomeningitis virus (LCMV). The Z protein is a small zinc-binding RING protein with an unknown function which is required for the viral life cycle. Here, we demonstrate an association between Z and the host cell translation factor, eukaryotic initiation factor 4E (eIF-4E) in infected and transfected cells. Z's association with both ribosomal proteins and this translation factor led us to investigate whether Z could modulate host cell translation. In cell culture, Z selectively represses protein production in an eIF-4E-dependent manner. Specifically, we see reduction in cyclin D1 protein production with no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells transfected with Z. Previous reports indicate that cyclin D1 is sensitive to eIF-4E levels, whereas GAPDH is not. Consistent with this, we observe preferential downregulation of cyclin D1 during infection and no effect on GAPDH. Further, no changes in RNA levels were observed for cyclin D1 or GAPDH transcripts. The interaction between eIF-4E and Z may provide a mechanism for slower growth observed in infected cells and a viral strategy for establishing chronic infection.
Construction and Analysis of an Infectious Human Immunodeficiency Virus Type 1 Subtype C Molecular Clone
Authors: Ndung'u Thumbi , Renjifo Boris , Essex Max
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) subtype C is now the predominant subtype in the global epidemic. This subtype is encountered in southern Africa and parts of Asia, where the epidemic is rapidly spreading. One possible explanation for these epidemiological observations is that this subtype has genetic characteristics that may contribute to its spread and/or pathogenic potential. In this report, we describe the construction of MJ4, an infectious chimeric molecular clone of HIV-1 subtype C that replicates in donor peripheral blood mononuclear cells and macrophages. We also tested this clone for its ability to use the chemokine receptors CCR1, CCR2b, CCR3, CXCR4, and CCR5 and found that the clone utilizes only CCR5 as the coreceptor for cell entry. The MJ4 clone will be useful in further biological and virological characterization of HIV-1 subtype C and will be an important tool in the continuing efforts to understand what may constitute protective immunity in HIV-1. The clone may also be used in experimental design of vaccine candidates that may be directed against HIV-1 subtype C.
Repression of Vaccinia Virus Holliday Junction Resolvase Inhibits Processing of Viral DNA into Unit-Length Genomes
Authors: Garcia Alonzo D. , Moss Bernard
Published: July 2002

The vaccinia virus A22R gene encodes a protein that is homologous to the bacterial enzyme RuvC and specifically cleaves and resolves four-way DNA Holliday junctions into linear duplex products. To investigate the role of the vaccinia virus Holliday junction resolvase during an infection, we constructed two recombinant viruses: vA22-HA, which has a short C-terminal epitope tag appended to the A22R open reading frame, and vA22i, in which the original A22R gene is deleted and replaced by an inducible copy. Polyacrylamide gel electrophoresis and Western blot analysis of extracts and purified virions from cells infected with vA22-HA revealed that the resolvase was expressed after the onset of DNA replication and incorporated into virion cores. vA22i exhibited a conditional replication defect. In the absence of an inducer, (i) viral protein synthesis was unaffected, (ii) late-stage viral DNA replication was reduced, (iii) most of the newly synthesized viral DNA remained in a branched or concatemeric form that caused it to be trapped at the application site during pulsed-field gel electrophoresis, (iv) cleavage of concatemer junctions was inhibited, and (v) virion morphogenesis was arrested at an immature stage. These data indicated multiple roles for the vaccinia virus Holliday junction resolvase in the replication and processing of viral DNA into unit-length genomes.