bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a Universal Group B
Streptococcus
Vaccine by Multiple Genome Screen
Authors: Maione Domenico , Margarit Immaculada , Rinaudo Cira D. , Masignani Vega , Mora Marirosa , Scarselli Maria , Tettelin Hervé , Brettoni Cecilia , Iacobini Emilia T. , Rosini Roberto , D'Agostino Nunzio , Miorin Lisa , Buccato Scilla , Mariani Massimo , Galli Giuliano , Nogarotto Renzo , Nardi-Dei Vincenzo , Vegni Filipo , Fraser Claire , Mancuso Giuseppe , Teti Giuseppe , Madoff Lawrence C. , Paoletti Lawrence C. , Rappuoli Rino , Kasper Dennis L. , Telford John L. , Grandi Guido
Published: July 2005

Group B
Streptococcus
(GBS) is a multiserotype bacterial pathogen representing a major cause of life-threatening infections in newborns. To develop a broadly protective vaccine, we analyzed the genome sequences of eight GBS isolates and cloned and tested 312 surface proteins as vaccines. Four proteins elicited protection in mice, and their combination proved highly protective against a large panel of strains, including all circulating serotypes. Protection also correlated with antigen accessibility on the bacterial surface and with the induction of opsonophagocytic antibodies. Multigenome analysis and screening described here represent a powerful strategy for identifying potential vaccine candidates against highly variable pathogens.
Genome Sequence of
Theileria parva
, a Bovine Pathogen That Transforms Lymphocytes
Authors: Gardner Malcolm J. , Bishop Richard , Shah Trushar , de Villiers Etienne P. , Carlton Jane M. , Hall Neil , Ren Qinghu , Paulsen Ian T. , Pain Arnab , Berriman Matthew , Wilson Robert J. M. , Sato Shigeharu , Ralph Stuart A. , Mann David J. , Xiong Zikai , Shallom Shamira J. , Weidman Janice , Jiang Lingxia , Lynn Jeffery , Weaver Bruce , Shoaibi Azadeh , Domingo Alexander R. , Wasawo Delia , Crabtree Jonathan , Wortman Jennifer R. , Haas Brian , Angiuoli Samuel V. , Creasy Todd H. , Lu Charles , Suh Bernard , Silva Joana C. , Utterback Teresa R. , Feldblyum Tamara V. , Pertea Mihaela , Allen Jonathan , Nierman William C. , Taracha Evans L. N. , Salzberg Steven L. , White Owen R. , Fitzhugh Henry A. , Morzaria Subhash , Venter J. Craig , Fraser Claire M. , Nene Vishvanath
Published: July 2005

We report the genome sequence of
Theileria parva
, an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with
Plasmodium falciparum
, and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.
Effects of Landscape Corridors on Seed Dispersal by Birds
Authors: Levey Douglas J. , Bolker Benjamin M. , Tewksbury Joshua J. , Sargent Sarah , Haddad Nick M.
Published: July 2005

Habitat fragmentation threatens biodiversity by disrupting dispersal. The mechanisms and consequences of this disruption are controversial, primarily because most organisms are difficult to track. We examined the effect of habitat corridors on long-distance dispersal of seeds by birds, and tested whether small-scale (<20 meters) movements of birds could be scaled up to predict dispersal of seeds across hundreds of meters in eight experimentally fragmented landscapes. A simulation model accurately predicted the observed pattern of seed rain and revealed that corridors functioned through edge-following behavior of birds. Our study shows how models based on easily observed behaviors can be scaled up to predict landscape-level processes.
Dancing Einstein
Authors: Bohannon John
Published: July 2005

Constant Speed
. Physics in Motion.
Choreographed by Mark Baldwin
. Rambert Dance Company, London. Sadler's Wells Theatre, London, 24-28 May 2005. For future performances, see
www.rambert.org.uk

This set of six dances inspired by Einstein's 1905 papers was commissioned by the Institute of Physics as part of its Einstein year celebrations.
Castanospermine, a Potent Inhibitor of Dengue Virus Infection In Vitro and In Vivo
Authors: Whitby Kevin , Pierson Theodore C. , Geiss Brian , Lane Kelly , Engle Michael , Zhou Yi , Doms Robert W. , Diamond Michael S.
Published: July 2005

Previous studies have suggested that α-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P≤ 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.
Mycobacterial Codon Optimization Enhances Antigen Expression and Virus-Specific Immune Responses in RecombinantMycobacterium bovisBacille Calmette-Guérin Expressing Human Immunodeficiency Virus Type 1 Gag
Authors: Kanekiyo Masaru , Matsuo Kazuhiro , Hamatake Makiko , Hamano Takaichi , Ohsu Takeaki , Matsumoto Sohkichi , Yamada Takeshi , Yamazaki Shudo , Hasegawa Atsuhiko , Yamamoto Naoki , Honda Mitsuo
Published: July 2005

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes toMycobacterium bovisbacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene ofgag(p24gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.
Detailed Mapping of the Nuclear Export Signal in the Rous Sarcoma Virus Gag Protein
Authors: Scheifele Lisa Z. , Ryan Eileen P. , Parent Leslie J.
Published: July 2005

The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA
99:
3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA
99:
3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.
A Somatic Knockout of CBF1 in a Human B-Cell Line Reveals that Induction of CD21 and CCR7 by EBNA-2 Is Strictly CBF1 Dependent and that Downregulation of Immunoglobulin M Is Partially CBF1 Independent
Authors: Maier Sabine , Santak Maja , Mantik Anja , Grabusic Kristina , Kremmer Elisabeth , Hammerschmidt Wolfgang , Kempkes Bettina
Published: July 2005

CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies.
Interaction of Bovine Papillomavirus E2 Protein with Brd4 Stabilizes Its Association with Chromatin
Authors: McPhillips Maria G. , Ozato Keiko , McBride Alison A.
Published: July 2005

The bovine papillomavirus E2 protein maintains and segregates the viral extrachromosomal genomes by tethering them to cellular mitotic chromosomes. E2 interacts with a cellular bromodomain protein, Brd4, to mediate the segregation of viral genomes into daughter cells. Brd4 binds acetylated histones and has been observed to diffusely coat mitotic chromosomes in several cell types. In this study, we show that in mitotic C127 cells, Brd4 diffusely coated the condensed chromosomes. However, in the presence of the E2 protein, E2 and Brd4 colocalized in punctate dots that were randomly distributed over the chromosomes. A similar pattern of E2 and Brd4 colocalization on mitotic chromosomes was observed in CV-1 cells, whereas only a faint chromosomal coating of Brd4 was detected in the absence of the E2 protein. Therefore, the viral E2 protein relocalizes and/or stabilizes the association of Brd4 with chromosomes in mitotic cells. The colocalization of E2 and Brd4 was also observed in interphase cells, indicating that this protein-protein interaction persists throughout the cell cycle. The interaction of E2 with Brd4 greatly stabilized the association of Brd4 with interphase chromatin. In both mitotic and interphase cells, this stabilization required a transcriptionally competent transactivation domain, but not the DNA binding function of the E2 protein. Thus, the E2 protein modulates the chromatin association of Brd4 during both interphase and mitosis. This study demonstrates that the segregation of papillomavirus genomes is not simply due to the passive hitchhiking of the E2/genome complex with a convenient cellular chromosomal protein.
AIP1/Alix Is a Binding Partner of Sendai Virus C Protein and Facilitates Virus Budding
Authors: Sakaguchi Takemasa , Kato Atsushi , Sugahara Fumihiro , Shimazu Yukie , Inoue Makoto , Kiyotani Katsuhiro , Nagai Yoshiyuki , Yoshida Tetsuya
Published: July 2005

The C protein, an accessory protein of Sendai virus (SeV), has anti-interferon capacity and suppresses viral RNA synthesis. In addition, it is thought that the C protein is involved in virus budding because of the low efficiency of release of progeny virions from C-knockout virus-infected cells and because of the requirement of the C protein for efficient release of virus-like particles. Here, we identified AIP1/Alix, a host protein involved in apoptosis and endosomal membrane trafficking, as an interacting partner of the C protein using a yeast two-hybrid system. The amino terminus of AIP1/Alix and the carboxyl terminus of the C protein are important for the interaction in mammalian cells. Mutant C proteins unable to bind AIP1/Alix failed to accelerate the release of virus-like particles from cells. Furthermore, overexpression of AIP1/Alix enhanced SeV budding from infected cells in a C-protein-dependent manner, while the release of nucleocapsid-free empty virions was also enhanced. Finally, AIP1/Alix depletion by small interfering RNA resulted in suppression of SeV budding. The results of this study suggest that AIP1/Alix plays a role in efficient SeV budding and that the SeV C protein facilitates virus budding through interaction with AIP1/Alix.
Human Tripartite Motif 5α Domains Responsible for Retrovirus Restriction Activity and Specificity
Authors: Perez-Caballero David , Hatziioannou Theodora , Yang Annie , Cowan Simone , Bieniasz Paul D.
Published: July 2005

The tripartite motif 5α protein (TRIM5α) is one of several factors expressed by mammalian cells that inhibit retrovirus replication. Human TRIM5α (huTRIM5α) inhibits infection by N-tropic murine leukemia virus (N-MLV) but is inactive against human immunodeficiency virus type 1 (HIV-1). However, we show that replacement of a small segment in the carboxy-terminal B30.2/SPRY domain of huTRIM5α with its rhesus macaque counterpart (rhTRIM5α) endows it with the ability to potently inhibit HIV-1 infection. The B30.2/SPRY domain and an additional domain in huTRIM5α, comprising the amino-terminal RING and B-box components of the TRIM motif, are required for N-MLV restriction activity, while the intervening coiled-coil domain is necessary and sufficient for huTRIM5α multimerization. Truncated huTRIM5α proteins that lack either or both the N-terminal RING/B-Box or the C-terminal B30.2/SPRY domain form heteromultimers with full-length huTRIM5α and are dominant inhibitors of its N-MLV restricting activity, suggesting that homomultimerization of intact huTRIM5α monomers is necessary for N-MLV restriction. However, localization in large cytoplasmic bodies is not required for inhibition of N-MLV by huTRIM5α or for inhibition of HIV-1 by chimeric or rhTRIM5α.
The Replicative Fitness of Primary Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2 Isolates
Authors: Ariën Kevin K. , Abraha Awet , Quiñones-Mateu Miguel E. , Kestens Luc , Vanham Guido , Arts Eric J.
Published: July 2005

The main (M) group of human immunodeficiency virus type 1 (HIV-1) is responsible for the global AIDS epidemic while HIV-1 group O (outlier) and HIV type 2 are endemic only in west and central Africa. The failure of HIV-2 and especially HIV-1 group O to spread following the initial zoonotic jumps is not well understood. This study was designed to examine the relative replicative capacities between these human lentiviruses. A pairwise competition experiment was performed with peripheral blood mononuclear cells with eight HIV-2 isolates, 6 group O viruses, and 15 group M viruses of subtype A (2 viruses), B (5 viruses), C (4 viruses), D (2 viruses) and CRF01_AE (2 viruses). HIV-1 group M isolates of any subtype were typically 100-fold-more fit than group O or HIV-2 strains when competed in peripheral blood mononuclear cells from various humans. This order in replicative fitness was also observed when virus pairs were added to human dendritic cells and then cocultured with primary, quiescent T cells, which is the model for HIV-1 transmission. These results suggest that reduced replicative and transmission fitness may be contributing to the low prevalence and limited geographical spread of HIV-2 and group O HIV-1 in the human population.
Molecular Epidemiology of Simian Immunodeficiency Virus SIVsm in U.S. Primate Centers Unravels the Origin of SIVmac and SIVstm
Authors: Apetrei Cristian , Kaur Amitinder , Lerche Nicholas W. , Metzger Michael , Pandrea Ivona , Hardcastle Johnny , Falkenstein Shelley , Bohm Rudolf , Koehler Jeffrey , Traina-Dorge Vicki , Williams Tessa , Staprans Silvija , Plauche Gail , Veazey Ronald S. , McClure Harold , Lackner Andrew A. , Gormus Bobby , Robertson David L. , Marx Preston A.
Published: July 2005

Retrospective molecular epidemiology was performed on samples from four sooty mangabey (SM) colonies in the United States to characterize simian immunodeficiency virus SIVsm diversity in SMs and to trace virus circulation among different primate centers (PCs) over the past 30 years. The following SIVsm sequences were collected from different monkeys: 55 SIVsm isolates from the Tulane PC sampled between 1984 and 2004, 10 SIVsm isolates from the Yerkes PC sampled in 2002, 7 SIVsm isolates from the New Iberia PC sampled between 1979 and 1986, and 8 SIVsm isolates from the California PC sampled between 1975 and 1977. PCR and sequencing were done to characterize the
gag
,
pol
, and
env
gp36 genes. Phylogenetic analyses were correlated with the epidemiological data. Our analysis identified nine different divergent phylogenetic lineages that cocirculated in these four SM colonies in the Unites States in the past 30 years. Lineages 1 to 5 have been identified previously. Two of the newly identified SIVsm lineages found in SMs are ancestral to SIVmac251/SIVmac239/SIVmne and SIVstm. We further identified the origin of these two macaque viruses in SMs from the California National Primate Research Center. The diversity of SIVsm isolates in PCs in the United States mirrors that of human immunodeficiency virus type 1 (HIV-1) group M subtypes and offers a model for the molecular epidemiology of HIV and a new approach to vaccine testing. The cocirculation of divergent SIVsm strains in PCs resulted in founder effects, superinfections, and recombinations. This large array of SIVsm strains showing the same magnitude of diversity as HIV-1 group M subtypes should be extremely useful for modeling the efficacy of vaccination strategies under the real-world conditions of HIV-1 diversity. The genetic variability of SIVsm strains among PCs may influence the diagnosis and monitoring of SIVsm infection and, consequently, may bias the results of pathogenesis studies.
Changes in Human Immunodeficiency Virus Type 1 Fitness and Genetic Diversity during Disease Progression
Authors: Troyer Ryan M. , Collins Kalonji R. , Abraha Awet , Fraundorf Erika , Moore Dawn M. , Krizan Randall W. , Toossi Zahra , Colebunders Robert L. , Jensen Mark A. , Mullins James I. , Vanham Guido , Arts Eric J.
Published: July 2005

This study examined the relationship between ex vivo human immunodeficiency virus type 1 (HIV-1) fitness and viral genetic diversity during the course of HIV-1 disease. Primary HIV-1 isolates from 10 patients at different time points were competed against control HIV-1 strains in peripheral blood mononuclear cell (PBMC) cultures to determine relative fitness values. Patient HIV-1 isolates sequentially gained fitness during disease at a significant rate that directly correlated with viral load and HIV-1
env
C2V3 diversity. A loss in both fitness and viral diversity was observed upon the initiation of antiretroviral therapy. A possible relationship between genotype and phenotype (virus replication efficiency) is supported by the parallel increases in ex vivo fitness and viral diversity during disease, of which the correlation is largely based on specific V3 sequences. Syncytium-inducing, CXCR4-tropic HIV-1 isolates did have higher relative fitness values than non-syncytium-inducing, CCR5-tropic HIV-1 isolates, as determined by dual virus competitions in PBMC, but increases in fitness during disease were not solely powered by a gradual switch in coreceptor usage. These data provide in vivo evidence that increasing HIV-1 replication efficiency may be related to a concomitant increase in HIV-1 diversity, which in turn may be a determining factor in disease progression.
Herpes Simplex Virus Type 1 Latency-Associated Transcript Expression Protects Trigeminal Ganglion Neurons from Apoptosis
Authors: Branco Francisco J. , Fraser Nigel W.
Published: July 2005

Upon infection of murine trigeminal ganglia with herpes simplex virus type 1 (HSV-1), an immune response is initiated resulting in significant infiltration of CD8
+
T cells. Previous investigators have observed a lack of apoptosis in HSV-1 trigeminal ganglia even in the presence of cytotoxic immune cells. To determine the role of the latency-associated transcript (LAT) in inhibiting apoptosis, we examined mice during acute and latent infection with HSV-1 (strain 17 or a LAT-negative deletion mutant strain 17 N/H) by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and fluorescence-activated cell sorting (FACS). FACS analysis revealed CD8
+
T cells in the trigeminal ganglia by day 7, with more being present in 17- than 17 N/H-infected trigeminal ganglia (6.22% versus 3.5%) and a decrease in number through day 30 (2.7% to 1.2%). To detect apoptotic CD8
+
T cells, sections were assayed by TUNEL and stained for CD8
+
T cells. By day 7, ∼10% of CD8
+
T cells in both 17- and 17 N/H-infected trigeminal ganglia had undergone apoptosis. By day 30, 58% and 74% of all T cells had undergone apoptosis in 17- and 17 N/H-infected trigeminal ganglia, respectively. Furthermore, no HSV strain 17-infected trigeminal ganglion neurons were apoptotic, but 0.087% of 17ΔSty and 0.98% of 17 N/H-infected neurons were apoptotic. We conclude that the antiapoptotic effect of LAT appears to require the LAT promoter, with most of the antiapoptotic effect mapping within the StyI (+447) to the HpaI (+1667) region and a minor contribution from the upstream StyI (+76) to StyI (+447) region.
Low Human Immunodeficiency Virus Envelope Diversity Correlates with Low In Vitro Replication Capacity and Predicts Spontaneous Control of Plasma Viremia after Treatment Interruptions
Authors: Joos Beda , Trkola Alexandra , Fischer Marek , Kuster Herbert , Rusert Peter , Leemann Christine , Böni Jürg , Oxenius Annette , Price David A. , Phillips Rodney E. , Wong Joseph K. , Hirschel Bernard , Weber Rainer , Günthard Huldrych F.
Published: July 2005

Genetic diversity of viral isolates in human immunodeficiency virus (HIV)-infected individuals varies substantially. However, it remains unclear whether HIV-related disease progresses more rapidly in patients harboring virus swarms with low or high diversity and, in the same context, whether high or low diversity is required to induce potent humoral and cellular immune responses. To explore whether viral diversity predicts virologic control, we studied HIV-infected patients who received antiretroviral therapy (ART) for years before undergoing structured treatment interruptions (STI). Viral diversity before initiation of ART and the ability of the patients to contain viremia after STI and final cessation of treatment was evaluated. Seven out of 21 patients contained plasma viremia at low levels after the final treatment cessation. Clonal sequences encompassing the envelope C2V3C3 domain derived from plasma prior to treatment, exhibited significantly lower diversity in these patients compared to those derived from patients with poor control of viremia. Viral diversity pre-ART correlated with the viral replication capacity of rebounding virus isolates during STI. Neutralizing antibody activity against autologous virus was significantly higher in patients who controlled viremia and was associated with lower pretreatment diversity. No such association was found with binding antibodies directed to gp120. In summary, lower pretreatment viral diversity was associated with spontaneous control of viremia, reduced viral replication capacity and higher neutralizing antibody titers, suggesting a link between viral diversity, replication capacity, and neutralizing antibody activity.
Heterologous Late-Domain Sequences Have Various Abilities To Promote Budding of Human Immunodeficiency Virus Type 1
Authors: Ott David E. , Coren Lori V. , Gagliardi Tracy D. , Nagashima Kunio
Published: July 2005

Retroviral late (L) domains present within Gag act in conjunction with cellular proteins to efficiently release virions from the surface of the cell. Three different critical core sequences have been identified as required elements for L-domain function: PPPY, PTAP (also PSAP), and YPDL, with different retroviruses utilizing one or two of these core sequences. The human immunodeficiency virus type 1 (HIV-1) L domain is centered around a PTAP sequence in the p6 region of Gag. To assess the ability of heterologous L-domain sequences to be functionally interchanged for those in full-length HIV-1, we produced a series of constructs that replaced PTAP-containing p6
Gag
sequences with those of PPPY- or YPDL-based L domains. While previous studies had found that L domains are interchangeable in other retroviruses, most of the sequences introduced into p6
Gag
failed to substitute for PTAP-mediated L-domain function. One exception was the 11-amino-acid p2b sequence of Rous sarcoma virus (RSV) Gag, which could fully restore HIV-1 budding, while a PPPPY sequence exchange alone did not. This suggests that the RSV L domain consists of more than simply its core L-domain sequence. The HIV-p2b chimera was as infectious as the wild type, produced normal virions, and was sensitive to proteasome inhibitors. These results show that L-domain sequences are not necessarily interchangeable. Thus, HIV-1 Gag might have a more stringent requirement for L-domain function than the other retroviruses previously studied.
Structural and Functional Analysis of the
cis
-Acting Elements Required for Plus-Strand RNA Synthesis of
Bamboo Mosaic Virus
Authors: Lin Jen-Wen , Chiu Hsiao-Ning , Chen I-Hsuan , Chen Tzu-Chi , Hsu Yau-Heiu , Tsai Ching-Hsiu
Published: July 2005

Bamboo mosaic virus
(BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three
cis
-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.
The V1, V2, and V3 Regions of the Human Immunodeficiency Virus Type 1 Envelope Differentially Affect the Viral Phenotype in an Isolate-Dependent Manner
Authors: Saunders Cheryl J. , McCaffrey Ruth A. , Zharkikh Irina , Kraft Zane , Malenbaum Susan E. , Burke Brian , Cheng-Mayer Cecilia , Stamatatos Leonidas
Published: July 2005

It is well documented that removal of the V1V2 region or of the V2 loop alone from the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) increases the susceptibility of these viruses to neutralization by antibodies. The specific role of the V1 loop in defining the neutralization susceptibility of HIV is, however, not well documented. Our current studies indicate that although the V1V2 region is a global modulator of the HIV-1 neutralization susceptibility, the individual roles the V1 and V2 loops have in defining the neutralization susceptibility profile of HIV-1 differ and in some cases are opposite. While deletion of the V2 loop renders the virus more susceptible to neutralization by antibodies that recognize diverse epitopes, in particular certain ones located in the CD4 binding site and the V3 loop, deletion of the V1 loop renders the virus refractory to neutralization, especially by antibodies that recognize CD4-induced epitopes and certain CD4-site binding antibodies. Our current studies also indicate that the relative involvement of the V2 loop of the HIV-1 envelope during virus-cell entry appears to be envelope background dependent. As a result, although deletion of the V2 loop from the clade B, R5-tropic SF162 HIV-1 virus resulted in a virus that was replication competent, the same modification introduced on the background of two other R5-tropic isolates, SF128A (clade B) or SF170 (clade A), abrogated the ability of these envelopes to mediate virus-cell entry.
Defective Replication in Human Immunodeficiency Virus Type 1 When Non-\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document}Primers Are Used for Reverse Transcription
Authors: Wei Min , Cen Shan , Niu Meijuan , Guo Fei , Kleiman Lawrence
Published: July 2005

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document}

, the primer for reverse transcriptase in human immunodeficiency virus type 1 (HIV-1), anneals to the primer binding site (PBS) in HIV-1 RNA. It has been shown that altering the PBS and U5 regions upstream of the PBS in HIV-1 so as to be complementary to sequences in tRNA
Met
or tRNA
His
will allow these tRNA species to be stably used as primers for reverse transcription. We have examined the replication of these mutant viruses in Sup-T1 cells. When Sup-T1 cells are infected by cocultivation with HIV-1-transfected 293T cells, viruses using tRNA
His
or tRNA
Met
are produced at rates that are approximately 1/10 or 1/100, respectively, of rates for wild-type virions that use

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document}

. When Sup-T1 cells are directly infected with equal amounts of these different viruses isolated from the culture supernatant of transfected 293T cells, virions using tRNA
Met
are produced at 1/100 the rate of wild-type viruses, and production of virions using tRNA
His
is not detected. Both wild-type and mutant virions selectively package tRNA
Lys
only, and examination of the ability of total viral RNA to prime reverse transcription in vitro indicates a >80% reduction in the annealing of tRNA
His
or tRNA
Met
to the mutant viral RNAs. PCR analysis of which of the three primer tRNAs is used indicates that only

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{3}^{Lys}\) \end{document}

is detected as primer in wild-type virions and only tRNA
His
is detected as primer in virions containing a PBS complementary to tRNA
His
, while the mutant viruses containing a PBS complementary to tRNA
Met
use both tRNA
Met
and

\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(tRNA_{1,2}^{Lys}\) \end{document}

as primer tRNAs.
Constituents of SH1, a Novel Lipid-Containing Virus Infecting the Halophilic Euryarchaeon
Haloarcula hispanica
Authors: Bamford Dennis H. , Ravantti Janne J. , Rönnholm Gunilla , Laurinavičius Simonas , Kukkaro Petra , Dyall-Smith Mike , Somerharju Pentti , Kalkkinen Nisse , Bamford Jaana K. H.
Published: July 2005

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting
Haloarcula hispanica
, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.
Genetic Variability and Molecular Evolution of the Human Respiratory Syncytial Virus Subgroup B Attachment G Protein
Authors: Zlateva Kalina T. , Lemey Philippe , Moës Elien , Vandamme Anne-Mieke , Van Ranst Marc
Published: July 2005

Human respiratory syncytial virus (HRSV) is the most important cause of acute respiratory disease in infants. Two major subgroups (A and B) have been identified based on antigenic differences in the attachment G protein. Antigenic variation between and within the subgroups may contribute to reinfections with these viruses by evading the host immune responses. To investigate the circulation patterns and mechanisms by which HRSV-B viruses evolve, we analyzed the G protein genetic variability of subgroup B sequences isolated over a 45-year period, including 196 Belgian strains obtained over 22 epidemic seasons (1982 to 2004). Our study revealed that the HRSV-B evolutionary rate (1.95 × 10
−3
nucleotide substitutions/site/year) is similar to that previously estimated for HRSV-A (1.83 × 10
−3
nucleotide substitutions/site/year). However, natural HRSV-B isolates appear to accommodate more drastic changes in their attachment G proteins. The most recent common ancestor of the currently circulating subgroup B strains was estimated to date back to around the year 1949. The divergence between the two major subgroups was calculated to have occurred approximately 350 years ago. Furthermore, we have identified 12 positively selected sites in the G protein ectodomain, suggesting that immune-driven selective pressure operates in certain codon positions. HRSV-A and -B strains have similar phylodynamic patterns: both subgroups are characterized by global spatiotemporal strain dynamics, where the high infectiousness of HRSV permits the rapid geographic spread of novel strain variants.
Infectious Pancreatic Necrosis Virus VP5 Is Dispensable for Virulence and Persistence
Authors: Santi Nina , Song Haichen , Vakharia Vikram N. , Evensen Øystein
Published: July 2005

Infectious pancreatic necrosis virus (IPNV) is the causative agent of infectious pancreatic necrosis (IPN) disease in salmonid fish. Recent studies have revealed variation in virulence between isolates of the Sp serotype, associated with certain residues of the structural protein VP2. The isolates are also highly heterogenic in the coding region of the nonstructural VP5 protein. To study the involvement of this protein in the pathogenesis of disease, we generated three recombinant VP5 mutant viruses using reverse genetics. The “wild-type” recombinant NVI15 (rNVI15) virus is virulent, having a premature stop codon at nucleotide position 427, putatively encoding a truncated 12-kDa VP5 protein, whereas rNVI15-15K virus encodes a 15-kDa protein. Recombinant rNVI15-ΔVP5 virus contains a mutation in the initiation codon of the VP5 gene that ablates the expression of VP5. Atlantic salmon postsmolts were challenged to study the virulence characteristics of the recovered viruses in vivo. The role of VP5 in persistent infection was investigated by challenging Atlantic salmon fry with the recovered viruses, as well as with the low-virulence field strain Sp103 and a naturally occurring VP5-deficient mutant of Sp103. The results show that VP5 is not required for viral replication in vivo, and its absence does not alter the virulence characteristics of the virus or the establishment of persistent IPNV infection.
The Marine Algal Virus PpV01 Has an Icosahedral Capsid with T=219 Quasisymmetry
Authors: Yan Xiaodong , Chipman Paul R. , Castberg Tonje , Bratbak Gunnar , Baker Timothy S.
Published: July 2005

Phaeocystis pouchetii
virus (PpV01) infects and lyses the haptophyte
Phaeocystis pouchetii
(Hariot) Lagerheim and was first isolated from Norwegian coastal waters. We have used electron cryomicroscopy and three-dimensional image reconstruction methods to examine the native morphology of PpV01 at a resolution of 3 nm. The icosahedral capsid of PpV01 has a maximum diameter of 220 nm and is composed of 2,192 capsomers arranged with T=219 quasisymmetry. One specific capsomer in each asymmetric unit contains a fiber-like protrusion. Density attributed to the presence of a lipid membrane appears just below (inside) the capsid. PpV01 is the largest icosahedral virus whose capsid structure has been determined in three dimensions from images of vitrified samples. Striking similarities in the structures of PpV01 and a number of other large double-stranded DNA viruses are consistent with a growing body of evidence that they share a common evolutionary origin.
A Downstream Polyadenylation Element in Human Papillomavirus Type 16 L2 Encodes Multiple GGG Motifs and Interacts with hnRNP H
Authors: Öberg Daniel , Fay Joanna , Lambkin Helen , Schwartz Stefan
Published: July 2005

Production of human papillomavirus type 16 (HPV-16) virus particles is totally dependent on the differentiation-dependent induction of viral L1 and L2 late gene expression. The early polyadenylation signal in HPV-16 plays a major role in the switch from the early to the late, productive stage of the viral life cycle. Here, we show that the L2 coding region of HPV-16 contains RNA elements that are necessary for polyadenylation at the early polyadenylation signal. Consecutive mutations in six GGG motifs located 174 nucleotides downstream of the polyadenylation signal resulted in a gradual decrease in polyadenylation at the early polyadenylation signal. This caused read-through into the late region, followed by production of the late mRNAs encoding L1 and L2. Binding of hnRNP H to the various triple-G mutants correlated with functional activity of the HPV-16 early polyadenylation signal. In addition, the polyadenylation factor CStF-64 was also found to interact specifically with the region in L2 located 174 nucleotides downstream of the early polyadenylation signal. Staining of cervix epithelium with anti-hnRNP H-specific antiserum revealed high expression levels of hnRNP H in the lower layers of cervical epithelium and a loss of hnRNP H production in the superficial layers, supporting a model in which a differentiation-dependent down regulation of hnRNP H causes a decrease in HPV-16 early polyadenylation and an induction of late gene expression.
A Genetically Engineered Attenuated Coxsackievirus B3 Strain Protects Mice against Lethal Infection
Authors: Dan M. , Chantler J. K.
Published: July 2005

Coxsackievirus B3 (CVB3) is a common human pathogen that is endemic throughout the world. There is currently no vaccine available, although the virus is known to be highly lethal to newborns and has been associated with heart disease and pancreatitis in older children and adults. Previously, we showed that the virulence of CVB3 is reduced by a lysine-to-arginine substitution in the capsid protein VP2 (K2168R) or a glutamic acid-to-glycine substitution in VP3 (E3060G). In this report, we show that the double mutant virus CVB3(KR/EG) displays additional attenuation, particularly for the pancreas, in A/J mice. In addition, two other attenuating mutations have been identified in the capsid protein VP1. When either the aspartic acid residue D1155 was replaced with glutamic acid or the proline residue P1126 was replaced with methionine, the resulting mutant also possessed an attenuated phenotype. Moreover, when either of these mutations was incorporated into CVB3(KR/EG), the resulting triple mutant viruses, CVB3(KR/EG/DE) and CVB3(KR/EG/PM), were completely noncardiovirulent and caused only small foci of damage to the pancreas, even at a high dose. Both triple mutants were found to be immunogenic, and a single injection of young A/J mice with either was found to protect them from a subsequent lethal challenge with wild-type CVB3. These findings indicate that the triple mutants could be exploited for the development of a live attenuated vaccine against CVB3.
Cloning and Identification of a MicroRNA Cluster within the Latency-Associated Region of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Samols Mark A. , Hu Jianhong , Skalsky Rebecca L. , Renne Rolf
Published: July 2005

MicroRNAs (miRNAs) are small, noncoding regulatory RNA molecules that bind to 3′ untranslated regions (UTRs) of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by viruses. The human gammaherpesvirus Epstein-Barr virus has been shown to encode miRNAs, which potentially regulate both viral and cellular genes. To determine whether Kaposi's sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV-positive primary effusion lymphoma-derived cells and endothelial cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align with KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latency-associated region of KSHV. These data suggest that viral and host cellular gene expression may be regulated by miRNAs during both latent and lytic KSHV replication.
RNA-Dependent Protein Kinase Is Required for Alpha-1 Interferon Transgene-Induced Resistance to Genital Herpes Simplex Virus Type 2
Authors: Carr Daniel J. J. , Tomanek Lisa , Silverman Robert H. , Campbell Iain L. , Williams Bryan R. G.
Published: July 2005

We investigated the mechanism of resistance to genital herpes simplex virus type 2 (HSV-2) infection in mice transfected with the murine alpha-1 interferon (IFN-α1) transgene. In situ transfection of mice with the IFN-α1 transgene resulted in an elevation in an IFN-responsive gene, RNA-dependent protein kinase (PKR), but not 2′,5′-oligoadenylate synthetases (OAS), in vaginal tissue. Coupled with the finding that mice lacking a functional PKR pathway were no longer resistant to genital HSV-2 infection following transfection with the IFN-α1 transgene in comparison to wild-type mice or mice lacking a functional OAS pathway, these results suggest that PKR is the dominant antiviral pathway activated by the IFN-α1 transgene.
Evidence for NF-κB- and CBP-Independent Repression of p53's Transcriptional Activity by Human T-Cell Leukemia Virus Type 1 Tax in Mouse Embryo and Primary Human Fibroblasts
Authors: Miyazato Akiko , Sheleg Sergey , Iha Hidekatsu , Li Yan , Jeang Kuan-Teh
Published: July 2005

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein can repress the transcriptional activity of the tumor suppressor protein p53. However, it remains controversial whether Tax requires NF-κB factors/activity and/or p300/CBP in order to inactivate p53 function. To address this issue, we have investigated Tax's effect on p53's transcriptional activation in IκB-kinase-deficient mouse embryonic fibroblasts (MEFs); some of which are entirely silent for Tax-induced NF-κB activity. We found that, in IKKα
−/−
, IKKβ
−/−
, and IKKγ
−/−
MEFs, p53 activation of a prototypic responsive plasmid (pG13-luciferase) was repressed by wild-type Tax. Curiously, p53's activity in MEFs was also repressed by a p300/CBP-binding deficient Tax protein. Our results highlight the complex nature of Tax-mediated repression of p53- activity, which requires further investigation.
Reduced Mobilization of Rev-Responsive Element-Deficient Lentiviral Vectors
Authors: Lucke Susann , Grunwald Thomas , Überla Klaus
Published: July 2005

Infection of cells transduced with a lentiviral vector by human immunodeficiency virus (HIV) could lead to packaging of the lentiviral vector RNA into HIV particles and unintended transfer of the vector. To prevent this, the Rev-responsive element (RRE) of an HIV-1 vector was functionally replaced by a heterologous RNA element (MS2). Providing Rev fused to an MS2 binding protein allowed efficient vector production. Mobilization of the vector from infected target cells was below the level of detection and at least 10
3
- to 10
4
-fold lower than for the RRE-containing vector. Thus, RRE-deficient lentiviral vectors provide a novel approach to reduce the risk of vector mobilization.