bluuebunny/crossref_metadata_2025 huggingface dataset:
vacA
Genotypes in
Helicobacter pylori
Strains Isolated from Children with and without Duodenal Ulcer in Brazil
Authors: De Gusmão Valquiria Ribeiro , Nogueira Mendes Edilberto , De Magalhães Queiroz Dulciene Maria , Aguiar Rocha Gifone , Camargos Rocha Andreia Maria , Ramadan Ashour Abdussalam Ali , Teles Carvalho Anfrisina Sales
Published: December 2019

Data concerning the association between
vacA
genotypes and disease in children in both developed and developing countries are scarce, especially because of the small number of children with a duodenal ulcer studied. The
vacA
genotypes of
Helicobacter pylori
strains obtained from 65 children (24 with a duodenal ulcer and 41 without a duodenal ulcer; 33 girls; mean age, 10.2 years; age range, 1 to 17 years) were investigated as described by J. C. Atherton et al. (J. Clin. Microbiol. 37:2979–2982, 1999). Ten (15.4%) children were infected with more than one
H. pylori
strain. None of these patients were included in our analysis of the relationship between gastric disorders and specific
vacA
genotypes. The s1 allele was detected in all
H. pylori
strains isolated from patients with a duodenal ulcer and from 21 (58.3%) patients without a duodenal ulcer (
P
= 0.003). Strains with the s2 allele were found only in patients without ulcer (
n
= 15; 41.7%). Most s1 strains had the s1b allele (97.5%), a result similar to that reported for adults from the Iberian peninsula, which could reflect the Brazilian population origin. One untypeable s1 strain was isolated. The m1 allele was also more frequently found in strains obtained from duodenal ulcer patients (
P
= 0.028). The m2 allele was found in strains obtained from 20 (36.4%) children, 3 (15.8%) with an ulcer and 17 (47.2%) without an ulcer. Only one m hybrid strain (m1 and m2 hybrid) was detected. It was demonstrated for the first time that the frequencies of colonization with strains with the s1 allele (14.3% in children up to 8 years of age and 85.7% in older patients;
P
= 0.012) and of strains with the m1 allele (11.1% in patients up to the age 8 years and 88.9% in older children;
P
= 0.013) increase with age.
Mutations in the
rpoB
Gene of Multidrug-Resistant
Mycobacterium tuberculosis
Isolates from Brazil
Authors: Valim Andréia Rosane M. , Rossetti Maria Lucia R. , Ribeiro Marta O. , Zaha Arnaldo
Published: December 2019

Mutations in a 69-bp region of the
rpoB
gene associated with rifampin resistance (Rif
r
) in 100 isolates (82 Rif
r
) from three states of Brazil were studied. Twenty-one different kinds of mutations were identified in the Rif
r
isolates, and six new alleles are described.
Characterization of the Mycobacterium bovis Restriction Fragment Length Polymorphism DNA Probe pUCD and Performance Comparison with Standard Methods
Authors: O'Brien Rory , Danilowicz Bret S. , Bailey Louise , Flynn Orla , Costello Eamon , O'Grady Don , Rogers Mark
Published: December 2019

In this study, the newly described Mycobacterium bovisrestriction fragment length polymorphism (RFLP) typing probe pUCD was characterized by sequence analysis and the previously observed polymorphic banding pattern was reproduced with a combination of three oligonucleotide probes in a single, mixed hybridization. In addition, the ability of pUCD to distinguish between 299 M. bovisisolates from the Republic of Ireland was assessed in relation to established methods and a statistical function for objective comparison of RFLP probes was derived. It was found that typing with pUCD alone produced greater discrimination between M. bovis isolates than typing with the commonly used mycobacterial DNA probes IS6110, PGRS, and DR and also by the spoligotyping technique. pUCD and DR in combination produced the highest level of discrimination while maintaining a high level of concordance with known epidemiological data relating to the samples. The reduction of pUCD to the level of oligonucleotides should in future allow pUCD and DR to be included together in a mixed hybridization, thus producing a high level of M. bovis strain type discrimination from a single round of RFLP analysis.
Expression of Foreign Proteins by Poliovirus Polyprotein Fusion: Analysis of Genetic Stability Reveals Rapid Deletions and Formation of Cardioviruslike Open Reading Frames
Authors: Mueller Steffen , Wimmer Eckard
Published: December 2019

Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448–1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (
gfp
) of
Aequorea victoria
or the
gag
gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3C
pro
/3CD
pro
cleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins >10 kDa in size through fusion with the polyprotein needs to be reevaluated.
Changes in the Extracellular Envelope Glycoprotein of Variants That Evolve during the Course of Simian Immunodeficiency Virus SIVMne Infection Affect Neutralizing Antibody Recognition, Syncytium Formation, and Macrophage Tropism but Not Replication, Cytopathicity, or CCR-5 Coreceptor Recognition
Authors: Rudensey Lyle M. , Kimata Jason T. , Long E. Michelle , Chackerian Bryce , Overbaugh Julie
Published: December 2019

Simian immunodeficiency virus SIVMne, like human immunodeficiency virus, evolves from a macrophage-tropic, non-syncytium-inducing virus at early times in infection to a T-cell-tropic, syncytium-inducing, cytopathic virus population over the course of progression to AIDS. Because the viruses isolated late in SIVMne infection of macaques include a complex mixture of variants, the viral determinants of such phenotypic changes have not been defined. To identify genetic changes that are important to virus evolution in the host, we constructed chimeric viruses by introducing variant envelope genes representative of proviruses throughout the course of infection and disease into the SIVMne parental clone (SIVMneCL8) that infected the macaque. The chimeric viruses expressed sequences encoding the surface unit of the envelope glycoprotein (Env-SU) of variants cloned between 35 and 170 weeks postinfection. The chimera with Env-SU from 35 weeks postinfection encoded only four changes in V1 compared to SIVMneCL8, whereas the chimeras encoding Env-SU from variants isolated later in infection encoded progressively more mutations both in V1 and elsewhere. Like SIVMneCL8, the chimeras were infectious for CEMx174 cells and macaque peripheral blood mononuclear cells. However, in contrast to SIVMneCL8, the chimeric viruses did not infect macaque macrophages, although each retained the ability to recognize the CCR-5 coreceptor. Thus, these data provide direct evidence that changes which evolve in Env-SU during the course of SIVMne infection do not alter CCR-5 interactions. Viruses encoding Env-SU from the latest times in infection (121 to 170 weeks postinfection), after disease was apparent, were syncytium inducing. However, these viruses were not highly cytopathic, suggesting that additional viral determinants may be required for the rapidly replicating, cytopathic phenotype of the uncloned mixed variant population. Changes in Env-SU did allow the virus to escape serum neutralizing antibodies that recognized the SIVMneCL8 parent. Moreover, the chimera encoding the Env-SU of a virus from 35 weeks postinfection, which differed from SIVMneCL8 only in V1, was not sensitive to neutralization by infected macaque sera, suggesting that V1 may define a portion of the principal neutralizing determinant for SIVMne. Together, these data suggest that SIV variants with changes in the Env-SU may be selected primarily by virtue of their ability to escape neutralizing antibody recognition.
Antibody-Dependent Cellular Cytotoxicity Directed against Cells Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Epitope Specificities
Authors: Alsmadi Osama , Tilley Shermaine A.
Published: December 2019

The characteristics of antibody-dependent cellular cytotoxicity (ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope specificities were studied; this was accomplished by using target cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of either primary or laboratory-adapted strains. Human MAbs of similar apparent affinities (1 × 109to 2 × 109liters/mol) against either a “cluster II”-overlapping epitope of gp41 or against the CD4 binding site, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. As expected, those MAbs specific for relatively conserved regions of Env generally exhibited ADCC activity against a broader range of HIV-1 strains than those directed against variable epitopes. Significant ADCC activities of selected MAbs against primary isolate Env-expressing cells were demonstrated. In addition, a new ADCC epitope in the V2 domain of gp120 was defined. CD56+cells were demonstrated to be the effector cells in these studies by fluorescence-activated cell sorting followed by ADCC assays. Notably, all anti-Env MAbs tested in this study, including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against targets expressing Env of one or more HIV-1 strains. These results imply that many, if not most, HIV-1-neutralizing human Abs of high affinity (≥3 × 108liters/mol in these studies) and of the immunoglobulin G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of directing ADCC. Since neutralizing Abs have been associated with long-term survival following HIV-1 infection, this suggests that ADCC activity may be beneficial in vivo.
Spontaneous Mutations in the
env
Gene of the Human Immunodeficiency Virus Type 1 NDK Isolate Are Associated with a CD4-Independent Entry Phenotype
Authors: Dumonceaux Julie , Nisole Sébastien , Chanel Chantal , Quivet Laurence , Amara Ali , Baleux Frano̧ise , Briand Pascale , Hazan Uriel
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.
Glycoproteins M and N of Pseudorabies Virus Form a Disulfide-Linked Complex
Authors: Jöns Alice , Dijkstra Johannes M. , Mettenleiter Thomas C.
Published: December 2019

Genes homologous to the herpes simplex virus UL49.5 open reading frame are conserved throughout the
Herpesviridae
. In the alphaherpesvirus pseudorabies virus (PrV), the UL49.5 product is an O-glycosylated structural protein of the viral envelope, glycoprotein N (gN) (A. Jöns, H. Granzow, R. Kuchling, and T. C. Mettenleiter, J. Virol. 70:1237–1241, 1996). For functional characterization of gN, a gN-negative PrV mutant, PrV-gNβ, and the corresponding rescuant, PrV-gNβR, were constructed, gN-negative PrV was able to productively replicate on noncomplementing cells, and one-step growth in cell culture was only slightly reduced compared to that of wild-type PrV. However, penetration was significantly delayed. In indirect immunofluorescence assays with rabbit serum directed against baculovirus-expressed gN, specific staining of wild-type PrV-infected cells occurred only after permeabilization of cells, whereas live cells failed to react with the antiserum. This indicates the lack of surface accessibility of gN in the plasma membrane of a PrV-infected cell. Western blot analyses and radioimmunoprecipitation experiments under reducing and nonreducing conditions led to the discovery of a heteromeric complex composed of gM and gN. The complex was stable in the absence of 2-mercaptoethanol but dissociated after the addition of the reducing agent, indicating that the partners are linked by disulfide bonds. Finally, gN was absent from gM-negative PrV virions, whereas gM was readily detected in virions in the absence of gN. Thus, gM appears to be required for virion localization of gN.
Genetic Determinants of Rous Sarcoma Virus Particle Size
Authors: Krishna Neel K. , Campbell Stephen , Vogt Volker M. , Wills John W.
Published: December 2019

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.
The Mouse Homolog of the Bovine Leukemia Virus Receptor Is Closely Related to the δ Subunit of Adaptor-Related Protein Complex AP-3, Not Associated with the Cell Surface
Authors: Suzuki Takako , Ikeda Hidetoshi
Published: December 2019

A mouse cDNA (mBLVR1) which was highly homologous to the bovine cDNA of the bovine leukemia virus receptor (
BLVR
) gene was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR protein was thought to be a type I transmembrane protein, the deduced protein coded by mBLVR1 did not appear to be a typical transmembrane protein. The ORF of mBLVR1 ended at a site 280 amino acids upstream of the termination codon of the bovine BLVR ORF, so the deduced mouse BLVR protein lacked the corresponding transmembrane and cytoplasmic regions of the predicted bovine BLVR protein. No significant hydrophobic region was found in the mouse protein. Recently, a human cDNA which was highly homologous (69.6% homology) to the mouse
BLVR
gene was reported. The cDNA encodes the δ subunit of the human adaptor-related protein complex AP-3, which aligned almost collinearly with the mouse BLVR protein. AP-3 and all other related adaptor protein complexes have been shown to be associated with intracellular vesicles but not with the cell surface. Thus, the mouse
BLVR
homolog appeared to be the mouse AP-3 δ subunit itself or closely related to it, but the bovine
BLVR
gene seemed slightly different from the adaptor subunit gene family.
The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge
Authors: Peng Tao , Ponce-de-Leon Manuel , Jiang Hongbin , Dubin Gary , Lubinski John M. , Eisenberg Roselyn J. , Cohen Gary H.
Published: December 2019

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.
Inhibition of Human Cytomegalovirus DNA Maturation by a Benzimidazole Ribonucleoside Is Mediated through the UL89 Gene Product
Authors: Underwood Mark R. , Harvey Robert J. , Stanat Sylvia C. , Hemphill Mary Lou , Miller Teresa , Drach John C. , Townsend Leroy B. , Biron Karen K.
Published: December 2019

2-Bromo-5,6-dichloro-1-β-
d
-ribofuranosyl benzimidazole (BDCRB) is a member of a new class of benzimidazole ribonucleosides which inhibit human cytomegalovirus (HCMV) late in the replication cycle without inhibiting viral DNA synthesis. We show here that polygenomic concatemeric HCMV DNA does not mature to unit genome length in the presence of BDCRB. To discover the locus of action, virus resistant to BDCRB was selected by serial passage in the presence of the compound. Genetic mapping experiments with BDCRB-resistant virus demonstrated that the resistance phenotype mapped to one amino acid (Asp
344
Glu; low resistance) or two amino acids (Asp
344
Glu and Ala
355
Thr; high resistance) within the product of exon 2 of the HCMV U
L
89 open reading frame. The HCMV U
L
89 open reading frame and its homologs are among the most conserved open reading frames in the herpesviruses, and their products have sequence similarities to a known ATP-dependent endonuclease from the double-stranded DNA bacteriophage T4. These findings strongly suggest that BDCRB prevents viral DNA maturation by interacting with a U
L
89 gene product and that the U
L
89 open reading frame may encode an endonucleolytic subunit of the putative HCMV terminase. Further, since mammalian cell DNA replication does not involve a DNA maturation step, compounds which inhibit viral DNA maturation should be selective and safe.
CXCR-4 Is Expressed by Primary Macrophages and Supports CCR5-Independent Infection by Dual-Tropic but Not T-Tropic Isolates of Human Immunodeficiency Virus Type 1
Authors: Yi Yanjie , Rana Shalini , Turner Julie D. , Gaddis Nathan , Collman Ronald G.
Published: December 2019

Primary macrophages are infected by macrophage (M)-tropic but not T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, and CCR5 and CXCR-4 are the principal cofactors utilized for CD4-mediated entry by M-tropic and T-tropic isolates, respectively. Macrophages from individuals homozygous for an inactivating mutation of CCR5 are resistant to prototype M-tropic strains that depend on CCR5 but are permissive for a dual-tropic isolate, 89.6, that can use both CCR5 and CXCR-4, as well as CCR2b, CCR3, and CCR8. Here we show that 89.6 entry into CCR5-deficient macrophages is blocked by an anti-CXCR-4 antibody and by the CXCR-4-specific chemokine SDF but not by the ligands to CCR2b or CCR3. Reverse transcription-PCR demonstrated expression of CXCR-4 but not CCR3 or CCR8 in macrophages, while CCR2b was variable. Macrophage surface expression of CXCR-4 was confirmed by immunofluorescence staining and flow cytometry. Thus, CXCR-4 is expressed by primary macrophages and functions as a cofactor for entry by dual-tropic but not T-tropic HIV-1 isolates, and macrophage resistance to T-tropic strains does not result from a lack of the T-tropic entry cofactor CXCR-4. Since CXCR-4 on macrophages can be used by some but not other isolates, these results indicate that HIV-1 strains differ in how they utilize chemokine receptors as cofactors for entry and that the ability of a chemokine receptor to mediate HIV-1 entry differs, depending on the cell type in which it is expressed.
Gene Arrangement within the Unique Long Genome Region of Infectious Laryngotracheitis Virus Is Distinct from That of Other Alphaherpesviruses
Authors: Ziemann Katharina , Mettenleiter Thomas C. , Fuchs Walter
Published: December 2019

The genome of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) comprises ca. 155 kbp of which ca. one-third have been sequenced so far. To gain additional sequence information we analyzed two stretches of 15.5 and 1.9 kbp of the ILTV unique long (U
L
) genome region. The larger fragment contains homologs of the herpes simplex virus (HSV) UL23 (thymidine kinase) and UL22 (glycoprotein H) genes followed by five open reading frames (ORF) encoding putative proteins of 334 to 410 amino acids which exhibit no homology to any known herpesvirus protein. RNA analyses showed that these unique ILTV genes are indeed expressed. An origin of replication separates this cluster of unique genes from a conserved gene cluster consisting of the UL45, UL46, UL48, UL49, UL49.5, and UL50 homologs. The absence of UL47 from this position coincides with the localization of a UL47-homologous ORF within the unique short (U
S
) region of the ILTV genome (M. Wild, S. Cook, and M. Cochran, Virus Genes 12:107–116, 1996). Within the second analyzed region the ILTV UL21 homolog was found adjacent to the UL44 gene. We thus identified five novel herpesvirus genes in ILTV and present evidence for a large internal inversion in the ILTV U
L
region, in contrast to the collinear genomes of other alphaherpesviruses. Interestingly, a similar inversion is also present in the porcine alphaherpesvirus pseudorabies virus.
Effect of Immune Activation on the Dynamics of Human Immunodeficiency Virus Replication and on the Distribution of Viral Quasispecies
Authors: Ostrowski Mario A. , Krakauer David C. , Li Yuexia , Justement Shawn J. , Learn Gerald , Ehler Linda A. , Stanley Sharilyn K. , Nowak Martin , Fauci Anthony S.
Published: December 2019

Virus replication in a human immunodeficiency virus (HIV)-infected individual, as determined by the steady-state level of plasma viremia, reflects a complex balance of viral and host factors. We have previously demonstrated that immunization of HIV-infected individuals with the common recall antigen, tetanus toxoid, disrupts this steady state, resulting in transient bursts of plasma viremia after immunization. The present study defines the viral genetic basis for the transient bursts in viremia after immune activation. Tetanus immunization was associated with dramatic and generally reversible shifts in the composition of plasma viral quasispecies. The viral bursts in most cases reflected a nonspecific increase in viral replication secondary to an expanded pool of susceptible CD4+T cells. An exception to this was in a patient who harbored viruses of differing tropisms (syncytium inducing and non-syncytium inducing [NSI]). In this situation, immunization appeared to select for the replication of NSI viruses. In one of three patients, the data suggested that immune activation resulted in the appearance in plasma of virus induced from latently infected cells. These findings illustrate certain mechanisms whereby antigenic stimulation may influence the dynamics of HIV replication, including the relative expression of different viral variants.
The NH
2
Terminus of the Herpes Simplex Virus Type 1 Regulatory Protein ICP0 Contains a Promoter-Specific Transcription Activation Domain
Authors: Lium Erik K. , Panagiotidis Christos A. , Wen Xiaoshan , Silverstein Saul J.
Published: December 2019

The transcriptional program of herpes simplex virus is regulated by the concerted action of three immediate-early (α) proteins, ICP4, ICP27, and ICP0. The experiments described in this study examine the role of the acidic amino terminus (amino acids 1 to 103) of ICP0 in gene activation. When tethered to a DNA binding domain, this sequence activates transcription in the yeast
Saccharomyces cerevisiae
. Deletion of these amino acids affects the ability of ICP0 to activate α-gene promoter reporters in transient expression assays, while it has little or no effect on a β- and a γ-gene reporter in the same assay. Viruses that express the deleted form of ICP0 (ICP0-NX) have a small-plaque phenotype on both Vero cells and the complementing cell line L7. Transient expression and immunofluorescence analyses demonstrate that ICP0-NX is a dominant negative form of ICP0. Immunoprecipitation of ICP0 from cells coinfected with viruses expressing ICP0-NX and ICP0 revealed that ICP0 oligomerizes in infected cells. These data, in conjunction with the finding that ICP0-N/X is dominant negative, provide both biochemical and genetic evidence that ICP0 functions as a multimer in infected cells.
E1B 55-Kilodalton-Associated Protein: a Cellular Protein with RNA-Binding Activity Implicated in Nucleocytoplasmic Transport of Adenovirus and Cellular mRNAs
Authors: Gabler Stefan , Schütt Holger , Groitl Peter , Wolf Hans , Shenk Thomas , Dobner Thomas
Published: December 2019

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.
Functional Analysis of the Human Cytomegalovirus US28 Gene by Insertion Mutagenesis with the Green Fluorescent Protein Gene
Authors: Vieira Jeffrey , Schall Thomas J. , Corey Lawrence , Geballe Adam P.
Published: December 2019

The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR). Previous studies demonstrated that recombinant US28 protein can bind the β class of chemokines (K. Neote, D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall, Cell 72:415–425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J. L. Gao and P. M. Murphy, J. Biol. Chem. 269:28539–28542, 1994). In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of theEscherichia coli gptgene and the gene for the green fluorescent protein. The US28 gene is not required for growth in human fibroblasts (HF). HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES. In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the β-chemokine binding and induced calcium signaling in HCMV-infected cells. The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells. Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.
Efficient Class II Major Histocompatibility Complex Presentation of Endogenously Synthesized Hepatitis C Virus Core Protein by Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Lines to CD4+T Cells
Authors: Chen Ming , Shirai Mutsunori , Liu Zijuan , Arichi Tatsumi , Takahashi Hidemi , Nishioka Mikio
Published: December 2019

The induction of an efficient CD4+T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4+T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 104transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.
In Vivo Translation of the Triple Gene Block of Potato Virus X Requires Two Subgenomic mRNAs
Authors: Verchot Jeanmarie , Angell Susan M. , Baulcombe David C.
Published: December 2019

The 25-kilodalton (25K), 12K, and 8K movement proteins of potato virus X are derived from overlapping open reading frames (ORFs). Using an in vivo complementation assay, we have shown that the 25K protein is expressed from a functionally monocistronic mRNA, whereas the 12K and 8K proteins are from a bicistronic mRNA. Translation of the 8K ORF is by leaky ribosome scanning through the 12K ORF.
Herpes Simplex Virus 1 Regulatory Protein ICP22 Interacts with a New Cell Cycle-Regulated Factor and Accumulates in a Cell Cycle-Dependent Fashion in Infected Cells
Authors: Bruni Renato , Roizman Bernard
Published: December 2019

The herpes simplex virus 1 infected cell protein 22 (ICP22), the product of the α22 gene, is a nucleotidylylated and phosphorylated nuclear protein with properties of a transcriptional factor required for the expression of a subset of viral genes. Here, we report the following. (i) ICP22 interacts with a previously unknown cellular factor designated p78 in the yeast two-hybrid system. The p78 cDNA encodes a polypeptide with a distribution of leucines reminiscent of a leucine zipper. (ii) In uninfected and infected cells, antibody to p78 reacts with two major bands with an apparentM
r of 78,000 and two minor bands with apparentM
rs of 62,000 and 55,000. (ii) p78 also interacts with ICP22 in vitro. (iii) In uninfected cells, p78 was dispersed largely in the nucleoplasm in HeLa cells and in the nucleoplasm and cytoplasm in HEp-2 cells. After infection, p78 formed large dense bodies which did not colocalize with the viral regulatory protein ICP0. (iv) Accumulation of p78 was cell cycle dependent, being highest very early in S phase. (v) The accumulation of ICP22 in synchronized cells was highest in early S phase, in contrast to the accumulation of another protein, ICP27, which was relatively independent of the cell cycle. (vi) In the course of the cell cycle, ICP22 was transiently modified in an aberrant fashion, and this modification coincided with expression of p78. The results suggest that ICP22 interacts with and may be stabilized by cell cycle-dependent proteins.
The Herpes Simplex Virus US11 Protein Effectively Compensates for the γ134.5 Gene if Present before Activation of Protein Kinase R by Precluding Its Phosphorylation and That of the α Subunit of Eukaryotic Translation Initiation Factor 2
Authors: Cassady Kevin A. , Gross Martin , Roizman Bernard
Published: December 2019

In herpes simplex virus-infected cells, viral γ134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1α to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). The amino acid sequence of the γ134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the α47 promoter next to US11, a γ2(late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the γ134.5 gene. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2α by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2α, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The γ134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a γ2gene because, like many viral proteins, it has multiple functions.
Isolation of an Arabidopsis thalianaMutant in Which the Multiplication of both Cucumber Mosaic Virus and Turnip Crinkle Virus Is Affected
Authors: Yoshii Motoyasu , Yoshioka Norimichi , Ishikawa Masayuki , Naito Satoshi
Published: December 2019

During the systemic infection of plants by viruses, host factors play an important role in supporting virus multiplication. To identify and characterize the host factors involved in this process, we isolated an Arabidopsis thaliana mutant named RB663, in which accumulation of the coat protein (CP) of cucumber mosaic virus (CMV) in upper uninoculated leaves was delayed. Genetic analyses suggested that the phenotype of delayed accumulation of CMV CP in RB663 plants was controlled by a monogenic, recessive mutation designatedcum2-1, which is located on chromosome III and is distinct from the previously characterized cum1 mutation. Multiplication of CMV was delayed in inoculated leaves of RB663 plants, whereas the multiplication in RB663 protoplasts was similar to that in wild-type protoplasts. This suggests that the cum2-1mutation affects the cell-to-cell movement of CMV rather than CMV replication within a single cell. In RB663 plants, the multiplication of turnip crinkle virus (TCV) was also delayed but that of tobacco mosaic virus was not affected. As observed with CMV, the multiplication of TCV was normal in protoplasts and delayed in inoculated leaves of RB663 plants compared to that in wild-type plants. Furthermore, the phenotype of delayed TCV multiplication cosegregated with thecum2-1 mutation as far as we examined. Therefore, thecum2-1 mutation is likely to affect the cell-to-cell movement of both CMV and TCV, implying a common aspect to the mechanisms of cell-to-cell movement in these two distinct viruses.
Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha
Authors: Schneemann Anette , Marshall Dawn
Published: December 2019

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.
A Functional Role for Neutralizing Antibodies in Borna Disease: Influence on Virus Tropism outside the Central Nervous System
Authors: Stitz L. , Nöske K. , Planz O. , Furrer E. , Lipkin W. I. , Bilzer T.
Published: December 2019

Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.
Role of Variable Regions A and B in Receptor Binding Domain of Amphotropic Murine Leukemia Virus Envelope Protein
Authors: Han Jin-Young , Zhao Yi , Anderson W. French , Cannon Paula M.
Published: December 2019

For the amphotropic murine leukemia virus (MuLV), a 208-amino-acid amino-terminal fragment of the surface unit (SU) of the envelope glycoprotein is sufficient to bind to its receptor, Pit2. Within this binding domain, two hypervariable regions, VRA and VRB, have been proposed to be important for receptor recognition. In order to specifically locate residues that are important for the interaction with Pit2, we generated a number of site-specific mutations in both VRA and VRB and analyzed the resulting envelope proteins when expressed on retroviral vectors. Concurrently, we substituted portions of the amphotropic SU with homologous regions from the polytropic MuLV envelope protein. The amphotropic SU was unaffected by most of the point mutations we introduced. In addition, the deletion of eight residues in a region of VRA that was previously suggested to be essential for Pit2 utilization only decreased titer on NIH 3T3 cells by 1 order of magnitude. Although the replacement of the amino-terminal two-thirds of VRA with the polytropic sequence abolished receptor binding, smaller nonoverlapping substitutions did not affect the function of the protein. We were not able to identify a single critical receptor contact point within VRA, and we suggest that the amphotropic receptor binding domain probably makes multiple contacts with the receptor and that the loss of some of these contacts can be tolerated.
Pseudorabies Virus-Induced Leukocyte Trafficking into the Rat Central Nervous System
Authors: Rassnick Stefanie , Enquist Lynn W. , Sved Alan F. , Card J. Patrick
Published: December 2019

When the swine alphaherpesvirus pseudorabies virus (PRV) infects the rat retina, it replicates in retinal ganglion cells and invades the central nervous system (CNS) via anterograde transynaptic spread through axons in the optic nerve. Virus can also spread to the CNS via retrograde transport through the oculomotor nucleus that innervates extraocular muscles of the eye. Since retrograde infection of the CNS precedes anterograde transynaptic infection, the temporal sequence of infection of the CNS depends on the route of invasion. Thus, motor neurons are infected first (retrograde infection), followed by CNS neurons innervated by the optic nerve (anterograde transynaptic infection). This temporal separation in the appearance of virus in separate groups of neurons enabled us to compare the immune responses to different stages of CNS infection in the same animal. The data revealed focal trafficking of peripheral immune cells into areas of the CNS infected by retrograde or anterograde transport after PRV Becker was injected into the vitreous body of the eye. Cells expressing the leukocyte common antigen, CD45+, entered the area of infection from local capillaries prior to any overt expression of neuropathology, and quantitative analysis demonstrated that the number of cells increased in proportion to the number of infected neurons within a given region. Recruitment of cells of monocyte/macrophage lineage began prior to the appearance of CD8+cytotoxic lymphocytes, which were, in turn, followed by CD4+lymphocytes. These data demonstrate that PRV replication in CNS neurons stimulates the focal infiltration of specific classes of CD45+cells in a time-dependent, temporally organized fashion that is correlated directly with the number of infected neurons and the time that a given region has been infected.
Transcriptional Organization of the Avian Adenovirus CELO
Authors: Payet Vincent , Arnauld Claire , Picault Jean-Paul , Jestin André , Langlois Patrick
Published: December 2019

A detailed map of the transcriptional organization of the CELO virus genome was produced. Recent computer analysis of CELO virus has indicated the presence of 38 putative open reading frames (ORFs). This study, based on analysis of the transcriptional products of CELO in vitro, confirmed the presence of RNAs for 26 of these 38 ORFs. All of the results were obtained by cDNA isolation or specific reverse transcriptase PCR. Observation of ORF transcription kinetics postinfection revealed the existence of early and late expression, with the earliest starting at 2 h postinfection. The 5′ untranslated regions of some RNAs were also studied, and this revealed the existence of a bipartite leader sequence, comparable in structure to the tripartite leader of mastadenovirus. The leader most probably involved in transcriptional activity was observed in most of the structural protein genes of the CELO virus genome. This suggests some homology in transcriptional organization between the avian adenovirus CELO and known mastadenoviruses such as human adenovirus.