bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Lymphocyte Apoptosis after Exposure to Influenza A Virus
Authors: Nichols Joan E. , Niles Jean A. , Roberts Norbert J.
Published: July 2002

Infection of humans with influenza A virus (IAV) results in a severe transient leukopenia. The goal of these studies was to analyze possible mechanisms behind this IAV-induced leukopenia with emphasis on the potential induction of apoptosis of lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure to IAV showed that a portion of CD3+, CD4+, CD8+, and CD19+lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response but could be a possible mechanism of disease pathogenesis.
Rescue of Very Virulent and Mosaic Infectious Bursal Disease Virus from Cloned cDNA: VP2 Is Not the Sole Determinant of the Very Virulent Phenotype
Authors: Boot Hein J. , ter Huurne A. Agnes H. M. , Hoekman Arjan J. W. , Peeters Ben P. H. , Gielkens Arno L. J.
Published: July 2002

Many recent outbreaks of infectious bursal disease in commercial chicken flocks worldwide are due to the spread of very virulent strains of infectious bursal disease virus (vvIBDV). The molecular determinants for the enhanced virulence of vvIBDV compared to classical IBDV are unknown. The lack of a reverse genetics system to rescue vvIBDV from its cloned cDNA hampers the identification and study of these determinants. In this report we describe, for the first time, the rescue of vvIBDV from its cloned cDNA. Two plasmids containing a T7 promoter and either the full-length A- or B-segment cDNA of vvIBDV (D6948) were cotransfected into QM5 cells expressing T7 polymerase. The presence of vvIBDV could be detected after passage of the transfection supernatant in either primary bursa cells (in vitro) or embryonated eggs (in vivo), but not QM5 cells. Rescued vvIBDV (rD6948) appeared to have the same virulence as the parental isolate, D6948. Segment-reassorted IBDV, in which one of the two genomic segments originated from cDNA of classical attenuated IBDV CEF94 and the other from D6948, could also be rescued by using this system. Segment-reassorted virus containing the A segment of the classical attenuated isolate (CEF94) and the B segment of the very virulent isolate (D6948) is not released until 15 h after an in vitro infection. This indicates a slightly retarded replication, as the first release of CEF94 is already found at 10 h after infection. Next to segment reassortants, we generated and analyzed mosaic IBDVs (mIBDVs). In these mIBDVs we replaced the region of CEF94 encoding one of the viral proteins (pVP2, VP3, or VP4) by the corresponding region of D6948. Analysis of these mIBDV isolates showed that tropism for non-B-lymphoid cells was exclusively determined by the viral capsid protein VP2. However, the very virulent phenotype was not solely determined by this protein, since mosaic virus containing VP2 of vvIBDV induced neither morbidity nor mortality in young chickens.
Effects of Truncation of the Carboxy Terminus of Pseudorabies Virus Glycoprotein B on Infectivity
Authors: Nixdorf Ralf , Klupp Barbara G. , Karger Axel , Mettenleiter Thomas C.
Published: July 2002

Glycoproteins homologous to the type I membrane glycoprotein B (gB) of herpes simplex virus 1 (HSV-1) are the most highly conserved glycoproteins within the family
Herpesviridae
and are present in members of each herpesvirus subfamily. In the alphaherpesvirus pseudorabies virus (PrV), gB is required for entry into target cells and for direct viral cell-to-cell spread. These processes, though related, appear to be distinct, and thus it was interesting to analyze whether they require different functions of gB. To this end, we established cell lines stably expressing different carboxy-terminally truncated versions of PrV gB by deleting either (i) one predicted intracytoplasmic α-helical domain encompassing putative YQRL and dileucine internalization signals, (ii) two predicted intracytoplasmic α-helical domains, (iii) the complete intracytoplasmic domain, or (iv) the intracytoplasmic domain and the transmembrane anchor region. Confocal laser scanning microscopy showed that gB derivatives lacking at least the last 29 amino acids (aa) localize close to the plasma membrane, while the full-length protein accumulates in intracellular aggregations. Trans-complementation studies with a gB-deleted PrV (PrV-gB
−
) demonstrated that the 29-aa truncated form lacking the putative internalization signals and the C-terminal α-helical domain (gB-008) was efficiently incorporated into PrV-gB
−
virions and efficiently complemented infectivity and cell-to-cell spread. Moreover, gB-008 exhibited an enhanced fusogenic activity. In contrast, gB proteins lacking both α-helical domains (gB-007), the complete intracytoplasmic domain, or the intracytoplasmic domain and transmembrane anchor were only inefficiently or not at all incorporated into PrV-gB
−
virions and did not complement infectivity. However, gB-007 was able to mediate cell-to-cell spread of PrV-gB
−
. Similar phenotypes were observed when virus recombinants expressing gB-008 or gB-007, respectively, instead of wild-type gB were isolated and analyzed. Thus, our data show that internalization of gB is not required for gB incorporation into virions nor for its function in either entry or cell-to-cell spread. Moreover, they indicate different requirements for gB in these membrane fusion processes.
The B-Oligomer of Pertussis Toxin Inhibits Human Immunodeficiency Virus Type 1 Replication at Multiple Stages
Authors: Alfano Massimo , Pushkarsky Tatiana , Poli Guido , Bukrinsky Michael
Published: July 2002

We have recently demonstrated that the binding subunit (B-oligomer) of pertussis toxin (PTX-B) deactivates CCR5 and inhibits entry of R5 human immunodeficiency virus type 1 (HIV-1) strains in activated primary T lymphocytes (M. Alfano et al., J. Exp. Med. 190:597–605, 1999). We now present evidence that PTX-B also affects a postentry step of HIV-1 replication. While PTX-B inhibited fusion induced by R5 but not that induced by X4 envelopes, it blocked infection of T cells with recombinant HIV-1 particles pseudotyped with R5, X4, and even murine leukemia virus or vesicular stomatitis virus envelopes. It also suppressed HIV-1 RNA synthesis in cultures of infected peripheral blood mononuclear cells when new infections had been inhibited by zidovudine, and it reduced Tat-dependent expression of the luciferase reporter gene controlled by the HIV-1 long terminal repeat (LTR). Surprisingly, PTX-B did not affect expression from the cytomegalovirus promoter, nor did it reduce the basal (Tat-independent) expression from the LTR promoter. These results indicate that PTX-B inhibits HIV-1 infection at both the entry and the postentry stages of viral replication, with the postentry activity specifically affecting transcription or stability of Tat-stimulated HIV-1 mRNAs.
E2-p7 Region of the Bovine Viral Diarrhea Virus Polyprotein: Processing and Functional Studies
Authors: Harada Takashi , Tautz Norbert , Thiel Heinz-Jürgen
Published: July 2002

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7). It has been shown that cleavage between E2 and p7 is incomplete, resulting in proteins E2-p7, E2, and p7. We found no precursor-product relationship between E2-p7 and E2, which indicates a stable nature of E2-p7. To study the function of the E2-p7 region of the polyprotein, mutations were introduced into an infectious cDNA of bovine viral diarrhea virus (BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletion of p7. To prevent synthesis of E2-p7, a translational stop codon was introduced after the last codon of the E2 gene and an internal ribosome entry site element followed by a signal peptide coding sequence was inserted upstream of the p7 gene. Transfection of RNA transcribed from the bicistronic construct led to the release of infectious virus particles. Thus, synthesis of E2-p7 is not essential for the generation of infectious virions. Cell lines constitutively expressing BVDV p7 and/or E2 were generated for complementation studies. Transfection of BVDV RNAs with point mutations or a deletion in the E2-p7 region into the complementing cell lines led to the generation of infectious virions. According to our studies, p7 as well as E2 can be complemented intrans.
Structural Phosphoprotein M2-1 of the Human Respiratory Syncytial Virus Is an RNA Binding Protein
Authors: Cuesta Isabel , Geng Xuehui , Asenjo Ana , Villanueva Nieves
Published: July 2002

The structural phosphoprotein M2-1 of human respiratory syncytial virus (HRSV) Long strain shows RNA binding capacity in three different assays that detect RNA-protein complexes: cross-linking, gel retardation, and Northern-Western assays. It is able to bind HRSV leader RNA specifically with cooperative kinetics, with an apparent

K
d

of at least 90 nM. It also binds to long RNAs with no sequence specificity. The RNA binding domain has been located between amino acid residues 59 and 85, at the NH
2
terminus of the protein. This region contains the phosphorylatable amino acid residues threonine 56 and serine 58, whose modification decreases the binding capacity of M2-1 protein to long RNAs.
Striking Similarity of Murine Nectin-1α to Human Nectin-1α (HveC) in Sequence and Activity as a Glycoprotein D Receptor for Alphaherpesvirus Entry
Authors: Shukla Deepak , Dal Canto Mauro C. , Rowe Cynthia L. , Spear Patricia G.
Published: July 2002

A cDNA encoding the murine homolog of human nectin-1α (also known as poliovirus receptor-related protein 1 [Prr1] and herpesvirus entry protein C [HveC]) was isolated. The protein encoded by this cDNA proved to be 95% identical in sequence to the human protein and to have similar herpesvirus entry activity. Upon expression of the murine cDNA in hamster cells resistant to alphaherpesvirus entry, the cells became susceptible to the entry of herpes simplex virus types 1 and 2 (HSV-1 and -2), pseudorabies virus, and bovine herpesvirus 1. HSV envelope glycoprotein D (gD), a viral ligand for human nectin-1α, is also a ligand for the murine homolog based on evidence that (i) a soluble hybrid protein composed in part of the murine nectin-1 ectodomain bound specifically to purified soluble forms of HSV-1 and HSV-2 gD as demonstrated by enzyme-linked immunosorbent assay, (ii) a soluble hybrid of HSV-1 gD bound to hamster cells expressing murine nectin-1α but not to control cells, and (iii) cells expressing both murine nectin-1α and one of the alphaherpesvirus gDs were resistant to entry of HSV-1, indicative of interference with entry resulting from interactions of cell-associated gD with the entry receptor. Northern blot analysis revealed that nectin-1 is expressed in most of the mouse tissues examined and at high levels in the brain, skin, and kidneys. Immunocytochemical localization demonstrated the presence of nectin-1 in epithelial cells of the mouse vagina and also in neuronal cells of the central nervous system, suggesting an expression pattern relevant to both infection at a portal of entry and spread of infection to the brain.
Papio cynocephalus Endogenous Retrovirus
among Old World Monkeys: Evidence for Coevolution and Ancient Cross-Species Transmissions
Authors: Mang Rui , Maas Jolanda , van der Kuyl Antoinette C. , Goudsmit Jaap
Published: July 2002

To study the evolutionary history of
Papio cynocephalus endogenous retrovirus
(PcEV), we analyzed the distribution and genetic characteristics of PcEV among 17 different species of primates. The viral
pol-env
and long terminal repeat and untranslated region (LTR-UTR) sequences could be recovered from all Old World species of the papionin tribe, which includes baboons, macaques, geladas, and mangabeys, but not from the New World monkeys and hominoids we tested. The Old World genera
Cercopithecus
and
Miopithecus
hosted either a PcEV variant with an incomplete genome or a virus with substantial mismatches in the LTR-UTR. A complete PcEV was found in the genome of
Colobus guereza
—but not in
Colobus badius
—with a copy number of 44 to 61 per diploid genome, comparable to that seen in papionins, and with a sequence most closely related to a virus of the papionin tribe. Analysis of evolutionary distances among PcEV sequences for synonymous and nonsynonymous sites indicated that purifying selection was operational during PcEV evolution. Phylogenetic analysis suggested that possibly two subtypes of PcEV entered the germ line of a common ancestor of the papionins and subsequently coevolved with their hosts. One strain of PcEV was apparently transmitted from a papionin ancestor to an ancestor of the central African lowland
C. guereza
.
Bovine Herpesvirus 5 Glycoprotein E Is Important for Neuroinvasiveness and Neurovirulence in the Olfactory Pathway of the Rabbit
Authors: Chowdhury S. I. , Lee B. J. , Ozkul A. , Weiss M. L.
Published: July 2002

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEΔ), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295–310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEΔ and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.
Nef-Induced Major Histocompatibility Complex Class I Down-Regulation Is Functionally Dissociated from Its Virion Incorporation, Enhancement of Viral Infectivity, and CD4 Down-Regulation
Authors: Akari Hirofumi , Arold Stefan , Fukumori Tomoharu , Okazaki Toshiyuki , Strebel Klaus , Adachi Akio
Published: July 2002

The N-terminal alpha-helix domain of the human immunodeficiency virus type 1 (HIV-1) Nef protein plays important roles in enhancement of viral infectivity, virion incorporation of Nef, and the down-regulation of major histocompatibility complex class I (MHC-I) expression on cell surfaces. In this study, we demonstrated that Met 20 in the alpha-helix domain was indispensable for the ability of Nef to modulate MHC-I expression but not for other events. We also showed that Met 20 was unnecessary for the down-regulation of CD4. These findings indicate that the region governing MHC-I down-regulation is proximate in the alpha-helix domain but is dissociated functionally from that determining enhancement of viral infectivity, virion incorporation of Nef, and CD4 down-regulation.
E1A Blocks Hyperphosphorylation of p130 and p107 without Affecting the Phosphorylation Status of the Retinoblastoma Protein
Authors: Parreño Matilde , Garriga Judit , Limón Ana , Mayol Xavier , Beck George R. , Moran Elizabeth , Graña Xavier
Published: July 2002

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G
1
/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of “free” hyperphosphorylated p130, which could act as a CDK inhibitor.
Interaction of the Influenza Virus Nucleoprotein with the Cellular CRM1-Mediated Nuclear Export Pathway
Authors: Elton Debra , Simpson-Holley Martha , Archer Kate , Medcalf Liz , Hallam Roger , McCauley John , Digard Paul
Published: July 2002

Influenza virus transcription occurs in the nuclei of infected cells, where the viral genomic RNAs are complexed with a nucleoprotein (NP) to form ribonucleoprotein (RNP) structures. Prior to assembly into progeny virions, these RNPs exit the nucleus and accumulate in the cytoplasm. The mechanisms responsible for RNP export are only partially understood but have been proposed to involve the viral M1 and NS2 polypeptides. We found that the drug leptomycin B (LMB), which specifically inactivates the cellular CRM1 polypeptide, caused nuclear retention of NP in virus-infected cells, indicating a role for the CRM1 nuclear export pathway in RNP egress. However, no alteration was seen in the cellular distribution of M1 or NS2, even in the case of a mutant virus which synthesizes greatly reduced amounts of NS2. Furthermore, NP was distributed throughout the nuclei of infected cells at early times postinfection but, when retained in the nucleus at late times by LMB treatment, was redistributed to the periphery of the nucleoplasm. No such change was seen in the nuclear distribution of M1 or NS2 after drug treatment. Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2. Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution. Consistent with this, NP bound CRM1 in vitro. Overall, these data raise the possibility that RNP export is mediated by a direct interaction between NP and the cellular CRM1 export pathway.
Immunoglobulin G (IgG) and IgA, but also Nonantibody Factors, Account for In Vitro Neutralization of Human Immunodeficiency Virus (HIV) Type 1 Primary Isolates by Serum and Plasma of HIV-Infected Patients
Authors: Burrer Renaud , Salmon-Ceron Dominique , Richert Sophie , Pancino Gianfranco , Spiridon Gabriella , Haessig Sandrine , Roques Virginie , Barre-Sinoussi Francoise , Aubertin Anne-Marie , Moog Christiane
Published: July 2002

The factors present in serum and plasma samples of human immunodeficiency virus (HIV)-infected patients that are responsible for the neutralization of four HIV type 1 (HIV-1) primary isolates in vitro have been analyzed. Purification of immunoglobulins (Ig) by affinity chromatography showed that the activities were mostly attributable to IgG and less frequently to IgA. For two samples, we have shown that the high-level and broad-spectrum inhibitory activity was essentially caused by non-Ig factors interfering with the measurement of antibody-specific neutralizing activity.
Polymerase Slippage at Vesicular Stomatitis Virus Gene Junctions To Generate Poly(A) Is Regulated by the Upstream 3′-AUAC-5′ Tetranucleotide: Implications for the Mechanism of Transcription Termination
Authors: Barr John N. , Wertz Gail W.
Published: July 2002

Termination of mRNA synthesis in vesicular stomatitis virus (VSV), the prototypic rhabdovirus, is controlled by a 13-nucleotide gene end sequence which comprises the conserved tetranucleotide 3′-AUAC-5′, the U
7
tract and the intergenic dinucleotide. mRNAs terminated at this sequence possess 100- to 300-nucleotide-long 3′ poly(A) tails which are thought to result from polymerase slippage (reiterative transcription) by the VSV polymerase on the U
7
tract. Previously we determined that in addition to the AUAC tetranucleotide, the U
7
tract was an essential signal in the termination process. Shortening or interrupting the U
7
tract abolished termination. These altered U tracts also prevented the polymerase from performing reiterative transcription necessary for generation of the mRNA poly(A) tail and thus established seven residues as the minimum length of U tract that allowed reiterative transcription to occur. In this study we investigated whether sequences other than the essential U
7
tract are involved in controlling polymerase slippage. We investigated whether the AUAC tetranucleotide affected the process of reiterative transcription by analyzing the nucleotide sequence of RNAs transcribed from altered subgenomic templates and infectious VSV variants. The tetranucleotide was found to regulate reiterative transcription on the U
7
tract. The extent of polymerase slippage was governed not by specific tetranucleotide sequences but rather by nucleotide composition such that slippage occurred when the tetranucleotide was composed of A or U residues but not when it was composed of G or C residues. This suggested that polymerase slippage was controlled, at least in part, by the strength of base pairing between the template and nascent strands. Further data presented here indicate that the tetranucleotide contains both a signal that directs the VSV polymerase to slip on the downstream U
7
tract and also a signal that directs a slipping polymerase to terminate mRNA synthesis.
Identification of Genotypic Changes in Human Immunodeficiency Virus Protease That Correlate with Reduced Susceptibility to the Protease Inhibitor Lopinavir among Viral Isolates from Protease Inhibitor-Experienced Patients
Authors: Kempf Dale J. , Isaacson Jeffrey D. , King Martin S. , Brun Scott C. , Xu Yi , Real Kathryn , Bernstein Barry M. , Japour Anthony J. , Sun Eugene , Rode Richard A.
Published: July 2002

The association of genotypic changes in human immunodeficiency virus (HIV) protease with reduced in vitro susceptibility to the new protease inhibitor lopinavir (previously ABT-378) was explored using a panel of viral isolates from subjects failing therapy with other protease inhibitors. Two statistical tests showed that specific mutations at 11 amino acid positions in protease (L10F/I/R/V, K20M/R, L24I, M46I/L, F53L, I54L/T/V, L63P, A71I/L/T/V, V82A/F/T, I84V, and L90M) were associated with reduced susceptibility. Mutations at positions 82, 54, 10, 63, 71, and 84 were most closely associated with relatively modest (4- and 10-fold) changes in phenotype, while the K20M/R and F53L mutations, in conjunction with multiple other mutations, were associated with >20- and >40-fold-reduced susceptibility, respectively. The median 50% inhibitory concentrations (IC50) of lopinavir against isolates with 0 to 3, 4 or 5, 6 or 7, and 8 to 10 of the above 11 mutations were 0.8-, 2.7-, 13.5-, and 44.0-fold higher, respectively, than the IC50against wild-type HIV. On average, the IC50of lopinavir increased by 1.74-fold per mutation in isolates containing three or more mutations. Each of the 16 viruses that displayed a >20-fold change in susceptibility contained mutations at residues 10, 54, 63, and 82 and/or 84, along with a median of three mutations at residues 20, 24, 46, 53, 71, and 90. The number of protease mutations from the 11 identified in these analyses (the lopinavir mutation score) may be useful for the interpretation of HIV genotypic resistance testing with respect to lopinavir-ritonavir (Kaletra) regimens and may provide insight into the genetic barrier to resistance to lopinavir-ritonavir in both antiretroviral therapy-naive and protease inhibitor-experienced patients.
Use of Patient-Derived Human Immunodeficiency Virus Type 1 Integrases To Identify a Protein Residue That Affects Target Site Selection
Authors: Harper Amy L. , Skinner Lynn M. , Sudol Malgorzata , Katzman Michael
Published: July 2002

To identify parts of retroviral integrase that interact with cellular DNA, we tested patient-derived human immunodeficiency virus type 1 (HIV-1) integrases for alterations in the choice of nonviral target DNA sites. This strategy took advantage of the genetic diversity of HIV-1, which provided 75 integrase variants that differed by a small number of amino acids. Moreover, our hypothesis that biological pressures on the choice of nonviral sites would be minimal was validated when most of the proteins that catalyzed DNA joining exhibited altered target site preferences. Comparison of the sequences of proteins with the same preferences then guided mutagenesis of a laboratory integrase. The results showed that single amino acid substitutions at one particular residue yielded the same target site patterns as naturally occurring integrases that included these substitutions. Similar results were found with DNA joining reactions conducted with Mn
2+
or with Mg
2+
and were confirmed with a nonspecific alcoholysis assay. Other amino acid changes at this position also affected target site preferences. Thus, this novel approach has identified a residue in the central domain of HIV-1 integrase that interacts with or influences interactions with cellular DNA. The data also support a model in which integrase has distinct sites for viral and cellular DNA.
Phosphorylation of Two Serine Residues Regulates Human T-Cell Leukemia Virus Type 2 Rex Function
Authors: Narayan Murli , Kusuhara Koichi , Green Patrick L.
Published: July 2002

The function of the human T-cell leukemia virus (HTLV) Rex phosphoprotein is to increase the level of the viral structural and enzymatic gene products expressed from the incompletely spliced viral RNAs containing the Rex-responsive element. The phosphorylation of HTLV type 2 Rex (Rex-2), predominantly on serine residues, correlates with an altered conformation, as detected by a gel mobility shift, and is required for specific binding to its viral RNA target sequence. Thus, the phosphorylation state of Rex in the infected cell may be a switch that determines whether the virus exists in a latent or a productive state. A mutational analysis of Rex-2 that focused on serine and threonine residues was performed to identify regions or domains within Rex-2 important for function, with a specific emphasis on identifying Rex-2 phosphorylation mutants. We identified mutations near the carboxy terminus that disrupted a novel region or domain and abrogated Rex-2 function. Mutant M17 (with S151A and S153A mutations) displayed reduced phosphorylation that correlated with reduced function. Replacement of both serine residues 151 and 153 with phosphomimetic aspartic acid restored Rex-2 function and locked Rex-2 in a phosphorylated active conformation. A mutant containing threonine residues at positions 151 and 153 displayed a phenotype indistinguishable from that of wild-type Rex. Furthermore, this same mutant showed increased threonine phosphorylation and decreased serine phosphorylation, providing conclusive evidence that one or both of these residues are phosphorylated in vivo. Our results provide the first direct evidence that the phosphorylation of Rex-2 is important for function. Further understanding of HTLV Rex phosphorylation will provide insight into the regulatory control of HTLV replication and ultimately the pathobiology of HTLV.
Comparison of Predicted Amino Acid Sequences of Measles Virus Strains in the Edmonston Vaccine Lineage
Authors: Parks Christopher L. , Lerch Robert A. , Walpita Pramila , Wang Hai-Ping , Sidhu Mohinder S. , Udem Stephen A.
Published: July 2002

Protein-encoding nucleotide sequences of the N, P, M, F, H, and L genes were determined for a low-passage isolate of the Edmonston wild-type (wt) measles virus and five Edmonston-derived vaccine virus strains, including AIK-C, Moraten, Schwarz, Rubeovax, and Zagreb. Comparative analysis demonstrated a high degree of nucleotide sequence homology; vaccine viruses differed at most by 0.3% from the Edmonston wt strain. Deduced amino acid sequences predicted substitutions in all viral polypetides. Eight amino acid coding changes were common to all vaccine viruses; an additional two were conserved in all vaccine strains except Zagreb. Comparisons made between vaccine strains indicated that commercial vaccine lots of Moraten and Schwarz had identical coding regions and were closely related to Rubeovax, while AIK-C and Zagreb diverged from the Edmonston wt along slightly different paths. These comparisons also revealed amino acid coding substitutions in Moraten and Schwarz that were absent from the closely related reactogenic Rubeovax strain. All of the vaccine viruses contained amino acid coding changes in the core components of the virus-encoded transcription and replication apparatus. This observation, combined with identification of noncoding region nucleotide changes in potential
cis
-acting sequences of the vaccine strains (C. L. Parks, R. A. Lerch, P. Walpita, H.-P. Wang, M. S. Sidhu, and S. A. Udem, J. Virol. 75:921–933, 2001), suggest that modulation of transcription and replication plays an important role in attenuation.
Potential Transmission of Human Polyomaviruses through the Gastrointestinal Tract after Exposure to Virions or Viral DNA
Authors: Bofill-Mas Sı́lvia , Formiga-Cruz Meritxell , Clemente-Casares Pilar , Calafell Francesc , Girones Rosina
Published: July 2002

The mechanism of human-to-human transmission of the polyomaviruses JC virus (JCV) and BK virus (BKV) has not been firmly established with regard to possible human exposure. JCV and BKV have been found in sewage samples from different geographical areas in Europe, Africa, and the United States, with average concentrations of 10
2
to 10
3
JCV particles/ml and 10
1
to 10
2
BKV particles/ml. Selected polyomavirus-positive sewage samples were further characterized. The JCV and BKV present in these samples were identified by sequencing of the intergenic region (the region found between the T antigen and VP coding regions) of JCV and the VP1 region of BKV. The regulatory region of the JCV and BKV strains found in sewage samples presented archetypal or archetype-like genetic structures, as described for urine samples. The stability (the time required for a 90% reduction in the virus concentration) of the viral particles in sewage at 20°C was estimated to be 26.7 days for JCV and 53.6 days for BKV. The presence of JCV in 50% of the shellfish samples analyzed confirmed the stability of these viral particles in the environment. BKV and JCV particles were also found to be stable at pH 5; however, treatment at a pH lower than 3 resulted in the detection of free viral DNA. Since most humans are infected with JCV and BKV, these data indicate that the ingestion of contaminated water or food could represent a possible portal of entrance of these viruses or polyomavirus DNA into the human population.
cisExpression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor
Authors: Lee Benhur , Leslie George , Soilleux Elizabeth , O'Doherty Una , Baik Sarah , Levroney Ernest , Flummerfelt Karen , Swiggard William , Coleman Nicholas , Malim Michael , Doms Robert W.
Published: July 2002

DC-SIGN is a C-type lectin expressed on dendritic cells and restricted macrophage populations in vivo that binds gp120 and acts intransto enable efficient infection of T cells by human immunodeficiency virus type 1 (HIV-1). We report here that DC-SIGN, when expressed inciswith CD4 and coreceptors, allowed more efficient infection by both HIV and simian immunodeficiency virus (SIV) strains, although the extent varied from 2- to 40-fold, depending on the virus strain. Expression of DC-SIGN on target cells did not alleviate the requirement for CD4 or coreceptor for viral entry. Stable expression of DC-SIGN on multiple lymphoid lines enabled more efficient entry and replication of R5X4 and X4 viruses. Thus, 10- and 100-fold less 89.6 (R5/X4) and NL4–3 (X4), respectively, were required to achieve productive replication in DC-SIGN-transduced Jurkat cells when compared to the parental cell line. In addition, DC-SIGN expression on T-cell lines that express very low levels of CCR5 enabled entry and replication of R5 viruses in a CCR5-dependent manner, a property not exhibited by the parental cell lines. Therefore, DC-SIGN expression can boost virus infection incisand can expand viral tropism without affecting coreceptor preference. In addition, coexpression of DC-SIGN enabled some viruses to use alternate coreceptors like STRL33 to infect cells, whereas in its absence, infection was not observed. Immunohistochemical and confocal microscopy data indicated that DC-SIGN was coexpressed and colocalized with CD4 and CCR5 on alveolar macrophages, underscoring the physiological significance of thesecisenhancement effects.
Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice
Authors: Moore Anne C. , Kong Wing-pui , Chakrabarti Bimal K. , Nabel Gary J.
Published: July 2002

The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-γ) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-γ and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-γ and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.
Development of a Vaccine against Murine Cytomegalovirus (MCMV), Consisting of Plasmid DNA and Formalin-Inactivated MCMV, That Provides Long-Term, Complete Protection against Viral Replication
Authors: Morello Christopher S. , Ye Ming , Spector Deborah H.
Published: July 2002

We previously demonstrated that immunization of mice with plasmid DNAs (pDNAs) expressing the murine cytomegalovirus (MCMV) genes IE1-pp89 and
M84
provided synergistic protection against sublethal viral challenge, while immunization with plasmids expressing putative virion proteins provided no or inconsistent protection. In this report, we sought to augment protection by increasing the breadth of the immune response. We identified another MCMV gene (
m04
encoding gp34) that provided strong and consistent protection against viral replication in the spleen. We also found that immunization with a DNA pool containing 10 MCMV genes that individually were nonprotective elicited reproducible protection against low to intermediate doses of challenge virus. Moreover, inclusion of these plasmids into a mixture with gp34, pp89, and
M84
DNAs provided even greater protection than did coimmunization with pp89 and
M84
. The highest level of protection was achieved by immunization of mice with the pool of 13 pDNAs, followed by formalin-inactivated MCMV (FI-MCMV). Immunization with FI-MCMV elicited neutralizing antibodies against salivary gland-derived MCMV, and of greatest importance, mice immunized with both the combined pDNA pool and FI-MCMV had undetectable levels of virus in the spleen and salivary glands after challenge. Intracellular cytokine staining of splenocytes from pDNA- and FI-MCMV-immunized mice showed that pDNA immunization elicited high levels of pp89- and M83-specific CD8
+
T cells, whereas both pDNA and FI-MCMV immunizations generated strong CD8
+
-T-cell responses against virion-associated antigens. Taken together, these results show that immunization with pDNA and inactivated virus provides strong antibody and cell-mediated immunity against CMV infection.
Binding of Transmissible Gastroenteritis Coronavirus to Cell Surface Sialoglycoproteins
Authors: Schwegmann-Weßels Christel , Zimmer Gert , Laude Hubert , Enjuanes Luis , Herrler Georg
Published: July 2002

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.
The Synthesis of Minus-Strand RNA of Bamboo Mosaic Potexvirus Initiates from Multiple Sites within the Poly(A) Tail
Authors: Cheng Jai-Hong , Peng Chi-Weng , Hsu Yau-Heiu , Tsai Ching-Hsiu
Published: July 2002

The 3′ terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5′ portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3′ untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5′ terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5′ rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5′-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from
Nicotiana benthamiana
protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5′ RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3′ UTR of BaMV genomic RNA.
High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish
Authors: Chen Wenbiao , Burgess Shawn , Golling Greg , Amsterdam Adam , Hopkins Nancy
Published: July 2002

Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate
Temporal Effects of Gamma Interferon Deficiency on the Course of Friend Retrovirus Infection in Mice
Authors: Stromnes Ingunn M. , Dittmer Ulf , Schumacher Ton N. M. , Schepers Koen , Messer Ronald J. , Evans Leonard H. , Peterson Karin E. , Race Brent , Hasenkrug Kim J.
Published: July 2002

The current studies demonstrate complex and seemingly contradictory effects by gamma interferon (IFN-γ) on Friend virus (FV) infection. Both temporal and tissue-specific effects were observed. During the first week of infection, IFN-γ-deficiency caused increased levels of FV infection in multiple tissues. Surprisingly, however, by 2 weeks postinfection, IFN-γ-deficient mice had significantly lower levels of infection in both the spleen and bone marrow compared to wild-type mice. The rapid reduction of virus in the IFN-γ-deficient mice correlated with a more rapid virus-neutralizing antibody response than was observed in the wild-type mice. Furthermore, the virus-neutralizing antibody response in wild-type mice could be accelerated by ablation of their IFN-γ response. Although the IFN-γ-deficient mice developed an accelerated virus-neutralizing antibody response, they did not class-switch to immunoglobulin G class immunoglobulins nor could they maintain long-term virus-neutralizing antibody titers. Eventually, all of the IFN-γ-deficient mice failed to keep persistent virus in check and developed fatal FV-induced erythroleukemia.
Recognition of the Measles Virus Nucleocapsid as a Mechanism of IRF-3 Activation
Authors: tenOever Benjamin R. , Servant Marc J. , Grandvaux Nathalie , Lin Rongtuan , Hiscott John
Published: July 2002

The mechanisms of cellular recognition for virus infection remain poorly understood despite the wealth of information regarding the signaling events and transcriptional responses that ensue. Host cells respond to viral infection through the activation of multiple signaling cascades, including the activation of NF-κB, c-Jun/ATF-2 (AP-1), and the interferon regulatory factors (IRFs). Although viral products such as double-stranded RNA (dsRNA) and the processes of viral binding and fusion have been implicated in the activation of NF-κB and AP-1, the mechanism(s) of IRF-1, IRF-3, and IRF-7 activation has yet to be fully elucidated. Using recombinant measles virus (MeV) constructs, we now demonstrate that phosphorylation-dependent IRF-3 activation represents a novel cellular detection system that recognizes the MeV nucleocapsid structure. At low multiplicities of infection, IRF-3 activation is dependent on viral transcription, since UV cross-linking and a deficient MeV containing a truncated polymerase L gene failed to induce IRF-3 phosphorylation. Expression of the MeV nucleocapsid (N) protein, without the requirement for any additional viral proteins or the generation of dsRNA, was sufficient for IRF-3 activation. In addition, the nucleocapsid protein was found to associate with both IRF-3 and the IRF-3 virus-activated kinase, suggesting that it may aid in the colocalization of the kinase and the substrate. Altogether, this study suggests that IRF-3 recognizes nucleocapsid structures during the course of an MeV infection and triggers the induction of interferon production.