bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential Environmental Impact of a Hydrogen Economy on the Stratosphere
Authors: Tromp Tracey K. , Shia Run-Lie , Allen Mark , Eiler John M. , Yung Y. L.
Published: June 2003

The widespread use of hydrogen fuel cells could have hitherto unknown environmental impacts due to unintended emissions of molecular hydrogen, including an increase in the abundance of water vapor in the stratosphere (plausibly by as much as ∼1 part per million by volume). This would cause stratospheric cooling, enhancement of the heterogeneous chemistry that destroys ozone, an increase in noctilucent clouds, and changes in tropospheric chemistry and atmosphere-biosphere interactions.
Making Evo-Devo Comparative
Authors: Wake David B.
Published: June 2003

The Development of Animal Form
Ontogeny, Morphology, and Evolution. Alessandro Minelli. Cambridge University Press, Cambridge, 2003. 341 pp. $75, £55. ISBN 0-521-80851-0.

Minelli's overview of animal organization synthesizes traditional morphological approaches and the molecular genetic perspectives that characterize most recent work in evolutionary developmental biology.
Evolution of the Protein Repertoire
Authors: Chothia Cyrus , Gough Julian , Vogel Christine , Teichmann Sarah A.
Published: June 2003

Most proteins have been formed by gene duplication, recombination, and divergence. Proteins of known structure can be matched to about 50% of genome sequences, and these data provide a quantitative description and can suggest hypotheses about the origins of these processes.
Preserving the Tree of Life
Authors: Mace Georgina M. , Gittleman John L. , Purvis Andy
Published: June 2003

Phylogenies provide new ways to measure biodiversity, to assess conservation priorities, and to quantify the evolutionary history in any set of species. Methodological problems and a lack of knowledge about most species have so far hampered their use. In the future, as techniques improve and more data become accessible, we will have an expanded set of conservation options, including ways to prioritize outcomes from evolutionary and ecological processes.
Phylogenomics: Intersection of Evolution and Genomics
Authors: Eisen Jonathan A. , Fraser Claire M.
Published: June 2003

Much has been gained from genomic and evolutionary studies of species. Combining the perspectives of these different approaches suggests that an integrated phylogenomic approach will be beneficial.
Sustained Small Interfering RNA-Mediated HumanImmunodeficiency Virus Type 1 Inhibition in PrimaryMacrophages
Authors: Song Erwei , Lee Sang-Kyung , Dykxhoorn Derek M. , Novina Carl , Zhang Dong , Crawford Keith , Cerny Jan , Sharp Phillip A. , Lieberman Judy , Manjunath N , Shankar Premlata
Published: June 2003

Small
interfering RNAs (siRNAs) can induce potent gene silencing by
degradation of cognate mRNA. However, in dividing cells, the silencing
lasts only 3 to 7 days, presumably because of siRNA dilution with cell
division. Here, we investigated if sustained siRNA-mediated silencing
of human immunodeficiency virus type 1 (HIV-1) is possible in
terminally differentiated macrophages, which constitute an important
reservoir of HIV in vivo. CCR5, the major HIV-1 coreceptor in
macrophages, and the viral structural gene for p24 were targeted either
singly or in combination. When transfected 2 days prior to infection,
both CCR5 and p24 siRNAs effectively reduced HIV-1 infection for the
entire 15-day period of observation, and combined targeting of both
genes abolished infection. To investigate whether exogenously
introduced siRNA is maintained stably in macrophages, we tested the
kinetics of siRNA-mediated viral inhibition by initiating infections at
various times (2 to 15 days) after transfection with CCR5 and p24
siRNAs. HIV suppression mediated by viral p24 siRNA progressively
decreased and was lost by day 7 posttransfection. In contrast, viral
inhibition by cellular CCR5 knockdown was sustained even when
transfection preceded infection by 15 days, suggesting that the
continued presence of target RNA may be needed for persistence of
siRNA. The longer sustenance of CCR5 relative to p24 siRNA in
uninfected macrophages was also confirmed by detection of internalized
siRNA by modified Northern blot analysis. We also tested the potential
of p24 siRNA to stably silence HIV in the setting of an established
infection where the viral target gene is actively transcribed. Under
these circumstances, long-term suppression of HIV replication could be
achieved with p24 siRNA. Thus, siRNAs can induce potent and
long-lasting HIV inhibition in nondividing cells such as
macrophages.
Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes
Authors: Wang Xiuqing , Messerle Martin , Sapinoro Ramil , Santos Kathlyn , Hocknell Peter K. , Jin Xia , Dewhurst Stephen
Published: June 2003

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.
Effects of Mutant Ubiquitin on
ts
1 Retrovirus-Mediated Neuropathology
Authors: Zhang Mei , Thurig Sherry , Tsirigotis Maria , Wong Paul K. Y. , Reuhl Kenneth R. , Gray Douglas A.
Published: June 2003

ts
1 is a temperature-sensitive mutant of Moloney murine leukemia virus that induces a rapid spongiform encephalopathy in mice infected as newborns. The pathological features include the formation of ubiquitinated inclusions resembling Lewy bodies. To determine how perturbation of the ubiquitin-proteasome pathway might affect
ts
1-mediated neurodegeneration, the virus was introduced into transgenic mice in which the assembly of ubiquitin chains was compromised by the expression of dominant-negative mutant ubiquitin. The onset of symptoms was greatly delayed in a transgenic mouse line expressing K48R mutant ubiquitin; no such delay was observed in mice expressing a wild-type ubiquitin transgene or K63R mutant ubiquitin. The extended latency was found to correlate with a delayed increase in viral titers. Pathological findings in K48R transgenic mice at 60 days were found to be similar to those in the other strains at 30 days, suggesting that while delayed, the neurodegenerative process in K48R mice was otherwise similar. These data demonstrate the sensitivity of retroviral replication to the partial disruption of ubiquitin-mediated proteolysis in vivo, a finding that may have therapeutic potential.
Mosaic Genomes of the Six Major Primate Lentivirus Lineages Revealed by Phylogenetic Analyses
Authors: Salemi Marco , De Oliveira Tulio , Courgnaud Valerie , Moulton Vincent , Holland Barbara , Cassol Sharon , Switzer William M. , Vandamme Anne-Mieke
Published: June 2003

To clarify the origin and evolution of the primate lentiviruses (PLVs), which include human immunodeficiency virus types 1 and 2 as well as their simian relatives, simian immunodeficiency viruses (SIVs), isolated from several host species, we investigated the phylogenetic relationships among the six supposedly nonrecombinant PLV lineages for which the full genome sequences are available. Employing bootscanning as an exploratory tool, we located several regions in the PLV genome that seem to have uncertain or conflicting phylogenetic histories. Phylogeny reconstruction based on distance and maximum-likelihood algorithms followed by a number of statistical tests confirms the existence of at least five putative recombinant fragments in the PLV genome with different clustering patterns. Split decomposition analysis also shows that phylogenetic relationships among PLVs may be better represented by network-based graphs, such as the ones produced by SplitsTree. Our findings not only imply that the six so-called pure PLV lineages have in fact mosaic genomes but also make more unlikely the hypothesis of cospeciation of SIVs and their simian hosts.
The Influenza A Virus PB1-F2 Protein Targets the Inner Mitochondrial Membrane via a Predicted Basic Amphipathic Helix That Disrupts Mitochondrial Function
Authors: Gibbs James S. , Malide Daniela , Hornung Felicita , Bennink Jack R. , Yewdell Jonathan W.
Published: June 2003

The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic α-helix and, as such, is similar to the MTS of the p13
II
protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.
A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein
Authors: Venkatesh L. K. , Gettemeier T. , Chinnadurai G.
Published: June 2003

The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.
NF-κB Is Required for Apoptosis Prevention during Herpes Simplex Virus Type 1 Infection
Authors: Goodkin Margot L. , Ting Adrian T. , Blaho John A.
Published: June 2003

Wild-type herpes simplex virus type 1 (HSV-1) infection triggers apoptosis in human cells. The subsequent synthesis of infected cell proteins between 3 and 6 h postinfection (hpi) acts to block this process from killing the cells. The factors produced during this window also prevent cell death induced by environmental staurosporine or sorbitol (M. Aubert, J. O'Toole, and J. A. Blaho, J. Virol. 73:10359-10370, 1999). We now report that (i) during the prevention window, HSV-1(F) also inhibited apoptosis induced by tumor necrosis factor alpha (TNF-α) plus cycloheximide (CHX) treatment. While deciphering the mechanism of this inhibition, we observed that (ii) the transcription factor NF-κB translocated from the cytoplasm into the nuclei of infected cells, and (iii) this migration initiated at 3 hpi. (iv) The complete inhibition of protein synthesis at 3 hpi by the addition of CHX precluded NF-κB translocation, while CHX additions at 6 hpi or later did not elicit this effect. This result confirms that infected cell protein synthesis is required for the nuclear import of NF-κB. (v) The detection of NF-κB in nuclei correlated with the ability of HSV-1(F), HSV-1(KOS1.1), or HSV-1(R7032), a replication-competent recombinant virus containing a deletion in the gene encoding the gE glycoprotein, to prevent apoptosis. (vi) NF-κB did not bind its κB DNA recognition site and remained cytoplasmic in cells actively undergoing apoptosis following infection with HSV-1(vBSΔ27), a virus with the key regulatory protein ICP27 deleted. (vii) Prestimulation of NF-κB by the addition of a phorbol ester prevented HSV-1(vBSΔ27)-induced apoptosis. (viii) Retention of NF-κB in the cytoplasm by the addition of a pharmacological antagonist of its release from IκBα led to an increase in death factor processing during HSV-1(F) infection. (ix) A novel HEp-2 clonal cell line, termed IκBαDN, was generated which expresses a dominant-negative form of IκBα. Treatment of IκBαDN cells with TNF-α in the absence of CHX resulted in apoptotic death due to the inability of NF-κB to become activated in these cells. Finally, (x) infection of IκBαDN cells with HSV-1(F) or HSV-1(KOS1.1) resulted in apoptosis, demonstrating that (xi) the nuclear translocation of NF-κB between 3 and 6 hpi (the prevention window) is necessary to prevent apoptosis in wild-type HSV-1-infected human HEp-2 cells.
Yeast-Based Genetic System for Functional Analysis of Poxvirus mRNA Cap Methyltransferase
Authors: Saha Nayanendu , Shuman Stewart , Schwer Beate
Published: June 2003

Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast
Saccharomyces cerevisiae
as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The
S. cerevisiae
capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of
abd1
Δ cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish
abd1
Δ complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.
Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes
Authors: Marques Sofia , Efstathiou Stacey , Smith K. G. , Haury Matthias , Simas J. Pedro
Published: June 2003

Intranasal infection of mice with murine gammaherpesvirus 68 (MHV-68), a virus genetically related to the human pathogen Kaposi's sarcoma-associated herpesvirus, results in a persistent, latent infection in the spleen and other lymphoid organs. Here, we have determined the frequency of virus infection in splenic dendritic cells, macrophages, and several B-cell subpopulations, and we quantified cell type-dependent virus transcription patterns. The frequencies of virus genome positive cells were maximal at 14 days postinfection in all splenic cell populations analyzed. Marginal zone and germinal center B cells harbored the highest frequency of infection and the former population accounted for approximately half the total number of infected B cells. Analysis of virus transcription during the establishment of latency revealed that virus gene expression in B cells was restricted and dependent on the differentiation stage of the B cell. Notably, transcription of
ORF73
was detected in germinal center B cells, a finding in agreement with the predicted latent genome maintenance function of ORF73 in dividing cells. At late times after infection, virus DNA could only be detected in newly formed and germinal center B cells, which suggests that B cells play a critical role in facilitating life-long latency.
Respiratory Syncytial Virus Nucleoprotein-Specific Cytotoxic T-Cell Epitopes in a South African Population of Diverse HLA Types Are Conserved in Circulating Field Strains
Authors: Venter Marietjie , Rock Michael , Puren Adrian J. , Tiemessen Caroline T. , Crowe James E.
Published: June 2003

This study identifies memory cytotoxic T lymphocyte (CTL) epitopes to respiratory syncytial virus (RSV) in healthy South African adults and demonstrates the conservation of those epitopes in circulating field strains of RSV in South Africa. Thirty-seven healthy adults from a population with diverse HLA backgrounds were screened by gamma interferon (IFN-γ) enzyme-linked immunospot for memory CTL activity in response to overlapping peptides representing the complete nucleoprotein (N) of RSV. Responses of more than 40 spot-forming cells/million cells were detectable in 21 individuals. The significant responses were further characterized, and 14-mer peptides were identified that induced cytolytic activity. Fine mapping of peptides with the highest cytolytic activity identified an HLA-B*08-restricted RSV-specific CTL epitope. The extended 14-mer peptide containing this epitope also induced lysis in the context of A*02-restricted target cells in some individuals. These HLA types are common in the target population; thus, the epitope is useful for studies of CTL responses to RSV in humans. The epitope was detected in healthy adults, reflecting the response generated in the course of previous natural RSV infection. We obtained a large panel of naturally occurring isolates of RSV to determine whether there was evidence of escape from CTL activity in circulating strains. We found that this epitope and a previously identified B*07-restricted N protein epitope were conserved in RSV field strains representing the diversity of circulating genotypes. This work suggests that escape from CTL activity is not common for this acute respiratory infection.
Enhanced Detection of Human Immunodeficiency Virus Type 1-Specific T-Cell Responses to Highly Variable Regions by Using Peptides Based on Autologous Virus Sequences
Authors: Altfeld Marcus , Addo Marylyn M. , Shankarappa Raj , Lee Paul K. , Allen Todd M. , Yu Xu G. , Rathod Almas , Harlow Jason , O'Sullivan Kristin , Johnston Mary N. , Goulder Philip J. R. , Mullins James I. , Rosenberg Eric S. , Brander Christian , Korber Bette , Walker Bruce D.
Published: June 2003

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (
P
= 6 × 10
−7
). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (
P
= 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.
Lentivirus Vectors Incorporating the Immunoglobulin Heavy Chain Enhancer and Matrix Attachment Regions Provide Position-Independent Expression in B Lymphocytes
Authors: Lutzko Carolyn , Senadheera Dinithi , Skelton Dianne , Petersen Denise , Kohn Donald B.
Published: June 2003

In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (Eμ) with and without associated matrix attachment regions (EμMAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34
+
progenitors in vitro transduced with Eμ- and EμMAR-containing lentivectors. Lastly, we evaluated the expression from the EμMAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the Eμ and EμMAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the EμMAR-containing vector and not other cells types or vectors. Proviral genomes with the EμMAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the EμMAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies.
Identification of a Single Amino Acid Change in the Human Respiratory Syncytial Virus L Protein That Affects Transcriptional Termination
Authors: Cartee Tara L. , Megaw A. George , Oomens A. G. P. , Wertz Gail W.
Published: June 2003

Infectious
Human respiratory syncytial virus
(HRSV) with an aberrant RNA synthesis pattern was recovered from a cDNA clone. The virus displayed increased levels of polycistronic readthrough mRNAs resulting from failure of the polymerase to terminate transcription efficiently at the gene ends. An asparagine (N) to aspartic acid (D) change at amino acid 1049 in the large (L) polymerase protein was found to be responsible for the readthrough phenotype. Virus encoding N at position 1049 displayed an RNA synthesis pattern indistinguishable from the A2 strain of HRSV. We compared the transcriptional activities of polymerases that encoded an N or D at position 1049 (L
1049N
or L
1049D
) by using dicistronic subgenomic replicons and found that the alteration of amino acid 1049 specifically affected transcriptional termination but had no effect on genome replication. L
1049N
recognized and terminated transcription at each of the naturally occurring gene junctions with differing efficiencies but at significantly higher efficiency than L
1049D
. The abilities of the polymerases to recognize the
cis
-acting gene end signals required for termination were compared by examining the effect of single nucleotide changes at positions 1 to 8 of the M gene end and were found to be similar. This work identifies a single amino acid position in the polymerase that specifically affects the ability of the polymerase to terminate transcription, but which does not affect genome replication or interaction with the M2-1 protein. This work shows the critical importance of the previously defined
cis
-acting signals for termination, confirms differential termination at the varied gene junctions, and shows that the polymerase responds to the
cis
-acting sequences similarly, whether it has N or D at position 1049.
Second-Strand Genome Conversion of Adeno-Associated Virus Type 2 (AAV-2) and AAV-5 Is Not Rate Limiting following Apical Infection of Polarized Human Airway Epithelia
Authors: Ding Wei , Yan Ziying , Zak Roman , Saavedra Milene , Rodman David M. , Engelhardt John F.
Published: June 2003

Recombinant adeno-associated virus type 5 (rAAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.
Identification of the Murine Coronavirus MP1 Cleavage Site Recognized by Papain-Like Proteinase 2
Authors: Kanjanahaluethai Amornrat , Jukneliene Dalia , Baker Susan C.
Published: June 2003

The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a
trans
-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constructs that extended various distances upstream from the previously defined C-terminal end of MP1. We found that the construct extending from the putative PLP2 cleavage site at glycine 2840-alanine 2841 was most similar in size to the processed MP1 replicase product generated in a
trans
-cleavage assay. To determine which amino acids are critical for PLP2 recognition and processing, we generated 14 constructs with amino acid substitutions upstream and downstream of the putative cleavage site and assessed the effects of the mutations in the PLP2
trans
-cleavage assay. We found that substitutions at phenylalanine 2835, glycine 2839, or glycine 2840 resulted in a reduction in cleavage of MP1. Finally, to unequivocally identify this cleavage site, we isolated radiolabeled MP1 protein and determined the position of [
35
S]methionine residues released by Edman degradation reaction. We found that the amino-terminal residue of MP1 corresponds to alanine 2841. Therefore, murine coronavirus PLP2 cleaves the replicase polyprotein between glycine 2840 and alanine 2841, and the critical determinants for PLP2 recognition and processing occupy the P6, P2, and P1 positions of the cleavage site. This study is the first report of the identification and characterization of a cleavage site recognized by murine coronavirus PLP2 activity.
Analysis of Human Immunodeficiency Virus Type 1 Gene Expression in Latently Infected Resting CD4
+
T Lymphocytes In Vivo
Authors: Hermankova Monika , Siliciano Janet D. , Zhou Yan , Monie Daphne , Chadwick Karen , Margolick Joseph B. , Quinn Thomas C. , Siliciano Robert F.
Published: June 2003

In individuals with human immunodeficiency virus type 1 (HIV-1) infection, a small reservoir of resting memory CD4
+
T lymphocytes carrying latent, integrated provirus persists even in patients treated for prolonged periods with highly active antiretroviral therapy (HAART). This reservoir greatly complicates the prospects for eradicating HIV-1 infection with antiretroviral drugs. Therefore, it is critical to understand how HIV-1 latency is established and maintained. In particular, it is important to determine whether transcriptional or posttranscriptional mechanisms are involved. Therefore, HIV-1 DNA and mRNAs were measured in highly purified populations of resting CD4
+
T lymphocytes from the peripheral blood of patients on long-term HAART. In such patients, the predominant form of persistent HIV-1 is latent integrated provirus. Typically, 100 HIV-1 DNA molecules were detected per 10
6
resting CD4
+
T cells. Only very low levels of unspliced HIV-1 RNA (∼50 copies/10
6
resting CD4
+
T cells) were detected using a reverse transcriptase PCR assay capable of detecting a single molecule of RNA standard. Levels of multiply spliced HIV-1 RNA were below the limit of detection (<50 copies/10
6
cells). Only 1% of the HIV-1 DNA-positive lymphocytes in this compartment could be induced to up-regulate HIV-1 mRNAs after cellular activation, indicating that most of the proviral DNA in resting CD4
+
T cells either carries intrinsic defects precluding transcription or is subjected to transcriptional control mechanisms that preclude high-level production of multiply spliced mRNAs. Nevertheless, by inducing T-cell activation, it is possible to isolate replication-competent virus from resting CD4
+
T lymphocytes of all infected individuals, including those on prolonged HAART. Thus, a subset of integrated proviruses (1%) remains competent for high-level mRNA production after cellular activation, and a subset of these can produce infectious virus. Measurements of steady-state levels of multiply spliced and unspliced HIV-1 RNA prior to cellular activation suggest that infected resting CD4
+
T lymphocytes in blood synthesize very little viral RNA and are unlikely to be capable of producing virus. In these cells, latency appears to reflect regulation at the level of mRNA production rather than at the level of splicing or nuclear export of viral mRNAs.
The Virus-Encoded Chemokine vMIP-II Inhibits Virus-Induced Tc1-Driven Inflammation
Authors: Lindow Morten , Nansen Anneline , Bartholdy Christina , Stryhn Annette , Hansen Nils J. V. , Boesen Thomas P. , Wells Timothy N. C. , Schwartz Thue W. , Thomsen Allan R.
Published: June 2003

The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8+T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8+T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.
The 5′-Proximal Hairpin of Turnip Yellow Mosaic Virus RNA: Its Role in Translation and Encapsidation
Authors: Bink Hugo H. J. , Schirawski Jan , Haenni Anne-Lise , Pleij Cornelis W. A.
Published: June 2003

The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5′-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5′ cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.
Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures
Authors: Uprichard Susan L. , Knipe David M.
Published: June 2003

The herpes simplex virus (HSV) single-stranded DNA-binding protein, ICP8, is required for viral DNA synthesis. Before viral DNA replication, ICP8 colocalizes with other replication proteins at small punctate foci called prereplicative sites. With the onset of viral genome amplification, these proteins become redistributed into large globular replication compartments. Here we present the results of immunocytochemical and biochemical analysis of ICP8 showing that various antibodies recognize distinct forms of ICP8. Using these ICP8-specific antibodies as probes for ICP8 structure, we detected a time-dependent appearance and disappearance of ICP8 epitopes in immunoprecipitation assays. Immunofluorescence staining of ICP8 in cells infected with different HSV mutant viruses as well as cells transfected with a limited number of viral genes demonstrated that these and other antigenic changes occur coincident with ICP8 assembly at intranuclear replication structures. Genetic analysis has revealed a correlation between the ability of various ICP8 mutant proteins to form the 39S epitope and their ability to bind to DNA. These results support the hypothesis that ICP8 undergoes a conformational change upon binding to other HSV proteins and/or to DNA coincident with assembly into viral DNA replication structures.
Intranasal Immunization of Guinea Pigs with an Immunodominant Foot-and-Mouth Disease Virus Peptide Conjugate Induces Mucosal and Humoral Antibodies and Protection against Challenge
Authors: Fischer D. , Rood D. , Barrette R. W. , Zuwallack A. , Kramer E. , Brown F. , Silbart L. K.
Published: June 2003

Guinea pigs immunized intranasally with a keyhole limpet hemocyanin-linked peptide, corresponding to the prominent G-H loop of the VP1 protein of foot-and-mouth disease virus, raised substantial levels of antipeptide and virus-neutralizing antibodies in sera and of peptide-specific secretory immunoglobulin A in nasal secretions. In groups of animals immunized intranasally without adjuvant, 86 percent were fully protected upon challenge with homotypic virus. Surprisingly, animals given the peptide conjugates plus the mucosal adjuvant cholera toxin were afforded only partial protection in that primary lesions were observed in most animals, although spread to other feet was prevented. These results indicate that intranasal inoculation with the peptide offers a potential route of vaccination against foot-and-mouth disease and may be useful for eliciting protection in the upper respiratory tracts of susceptible animals.
Comprehensive Screening Reveals Strong and Broadly Directed Human Immunodeficiency Virus Type 1-Specific CD8 Responses in Perinatally Infected Children
Authors: Feeney M. E. , Roosevelt K. A. , Tang Y. , Pfafferott K. J. , McIntosh K. , Burchett S. K. , Mao C. , Walker B. D. , Goulder P. J. R.
Published: June 2003

Advances in antiviral therapy have dramatically shifted the demographics of pediatric human immunodeficiency virus type 1 (HIV-1) infection in the developed world, and a growing proportion of perinatally HIV-1-infected children are now entering their second or even third decade of life. Although cellular immune responses to HIV are known to be weak in early infancy, the magnitude, breadth, and specificity of responses later in childhood have not been characterized in detail. We performed a comprehensive characterization of HIV-1-specific CD8 responses in 18 perinatally infected children (age range, 6 to 17 years), most of whom were on antiviral therapy, using both previously defined HIV-1 epitopes and overlapping peptides spanning all HIV-1 proteins. Multispecific responses were detected in all subjects and accounted for a median of 0.25 to 0.3% of all peripheral blood mononuclear cells that was similar to the magnitude seen in HIV-infected adults. CD8 responses were broadly directed at an average of 11 epitopes (range, 2 to 27 epitopes) and targeted nearly all HIV-1 proteins, with the highest proportion in Gag. Responses were readily detected even in those children with suppressed viremia on highly active antiretroviral therapy, although the breadth (P= 0.037) and the magnitude (P= 0.021) were significantly lower in these subjects. Each child recognized only a small minority of the HIV-1 optimal epitopes defined for his or her class I HLA alleles. Together, these data indicate that perinatally infected children who survive infancy mount a robust HIV-1-specific CD8 response that is much stronger than previously thought and is comparable in magnitude and breadth to that of adults. Moreover, this response has the potential to be broadened to target more epitopes, making these children attractive candidates for immunotherapeutic interventions.
Replication and IRES-Dependent Translation Are Both Affected by Core Coding Sequences in Subgenomic GB Virus B Replicons
Authors: Pizzuti Maura , De Tomassi Amedeo , Traboni Cinzia
Published: June 2003

The yield of G418-resistant Huh7 cell clones bearing subgenomic dicistronic GB virus B (GBV-B) is significantly affected by the insertion of a portion of the viral core gene between the GBV-B 5′ untranslated region and the exogenous neomycin phosphotransferase selector gene (A. De Tomassi, M. Pizzuti, R. Graziani, A. Sbardellati, S. Altamura, G. Paonessa, and C. Traboni, J. Virol. 76:7736-7746, 2002). In this report, we have dissected this phenomenon, examining the effects of the insertion of core sequences of different lengths on GBV-B IRES-dependent translation and RNA replication by using experimental approaches aimed at analyzing these two aspects independently. The results achieved indicate that an enhancement of translation efficiency does occur and that it correlates with the length of the inserted core sequences. Interestingly, the insertion of these sequences also has a direct similar effect on the efficiency of replication of the GBV-B replicon. These results suggest that in GBV-B replicon RNA and potentially in the complete viral genome, the core coding sequences not only are part of the IRES but also take part in the replication process, independently of the presence of the corresponding whole protein.
N-Linked Glycosylation Is Required for XC Cell-Specific Syncytium Formation by the R Peptide-Containing Envelope Protein of Ecotropic Murine Leukemia Viruses
Authors: Kubo Yoshinao , Ishimoto Akinori , Amanuma Hiroshi
Published: June 2003

The XC cell line undergoes extensive syncytium formation after infection with ecotropic murine leukemia viruses (MLVs) and is frequently used to titrate these viruses. This cell line is unique in its response to the ecotropic MLV envelope protein (Env) in that it undergoes syncytium formation with cells expressing Env protein containing R peptide (R
+
Env), which is known to suppress the fusogenic potential of the Env protein in other susceptible cells. To analyze the ecotropic receptor, CAT1, in XC cells, a mouse CAT1 tagged with the influenza virus hemagglutinin epitope (mCAT1-HA)-expressing retroviral vector was inoculated into XC and NIH 3T3 cells. The molecular size of the mCAT1-HA protein expressed in XC cells was smaller than that in NIH 3T3 cells due to altered N glycosylation in XC cells. Treatment of XC cells with tunicamycin significantly suppressed the formation of XC cell syncytia induced by the R
+
Env protein but not that induced by the R
−
Env protein. This result indicates that N glycosylation is required for XC cell-specific syncytium formation by the R
+
Env protein. The R
+
Env protein induced syncytia in XC cells expressing a mutant mCAT1 lacking both of two N glycosylation sites, and tunicamycin treatment suppressed syncytium formation by R
+
Env in those cells. This suggests that N glycosylation of a molecule(s) other than the receptor is required for the induction of XC cell syncytia by the R
+
Env protein.
Kinetic Analysis of Binding Interaction between the Subgroup A Rous Sarcoma Virus Glycoprotein SU and Its Cognate Receptor Tva: Calcium Is Not Required for Ligand Binding
Authors: Yu Xuemei , Wang Qing-Yin , Guo Ying , Dolmer Klavs , Young John A. T. , Gettins Peter G. W. , Rong Lijun
Published: June 2003

Tva is the receptor for subgroup A Rous sarcoma virus, and it contains a single LDL-A module which is the site of virus interaction. In this study, we expressed the entire extracellular region of Tva (referred to as Ecto-Tva) as a GST fusion protein and characterized its refolding properties. We demonstrated that the correct folding of the Ecto-Tva protein, like that of the Tva LDL-A module, is calcium dependent. We used the IAsys system to measure the kinetics of binding between the surface (SU) subunit of the viral glycoprotein and Tva in real time. We found that the Ecto-Tva protein and the Tva LDL-A module displayed similar affinities for SU, providing direct evidence that the LDL-A module of Tva is the only viral interaction domain of the receptor. Furthermore, misfolded Tva proteins displayed lower binding affinities to SU, largely due to a decrease in their association rates, suggesting that a high association rate between SU and Tva is crucial for efficient virus-host interaction. Furthermore, we found that calcium did not influence the overall binding affinity between Tva and SU. These results indicate that, although calcium is important in facilitating correct folding of the LDL-A module of Tva, it is not essential for ligand binding. Thus, these results may have broad implications for the mechanism of protein folding and ligand recognition of the LDL receptor and other members of the LDL receptor superfamily.