bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tsg101 Control of Human Immunodeficiency Virus Type 1 Gag Trafficking and Release
Authors: Goff A. , Ehrlich L. S. , Cohen S. N. , Carter C. A. 
Published: August 2003

ABSTRACT

The structural precursor polyprotein of human immunodeficiency virus type 1, Pr55

gag

, contains a proline-rich motif (PTAP) called the “late domain” in its C-terminal p6 region that directs release of mature virus-like particles (VLPs) from the plasma membranes of
gag
-transfected COS-1 cells. The motif binds Tsg101 (vacuolar protein-sorting protein 23, or Vps23), which functions in endocytic trafficking. Here, we show that accumulation of the wild-type (wt) Gag precursor in a fraction of COS-1 cytoplasm enriched in multivesicular bodies and small particulate components of the plasma membrane (P100) is p6 dependent. Cleavage intermediates and mature CA mainly partitioned with more rapidly sedimenting larger material enriched in components of lysosomes and early endosomes (P27), and this also was p6 dependent. Expression of truncated or full-length Tsg101 proteins interfered with VLP assembly and Gag accumulation in the P100 fraction. This correlated with reduced accumulation of Gag tagged with green fluorescent protein (Gag-GFP) at the plasma membrane and colocalization with the tagged Tsg101 in perinuclear early endosomes, as visualized by confocal microscopy. Fractionation analysis and confocal examination both indicated that the N-terminal region of Tsg101, which contains binding sites for PTAP and ubiquitin (Ub), was required for Gag trafficking to the plasma membrane. Expression of FLAG-tagged Tsg101 with a deletion in the Ub-binding pocket inhibited VLP release almost completely and to a significantly greater extent than expression of the wt tagged Tsg101 protein or Tsg101-FLAG containing a deletion in the PTAP-binding region. The results demonstrate that Gag associates with endosomal trafficking compartments and indicate that efficient release of virus particles from the plasma membrane requires both the PTAP- and Ub-binding functions of Tsg101 to recruit the cellular machinery required for budding.
Adenovirus Type 11 Uses CD46 as a Cellular Receptor
Authors: Segerman Anna , Atkinson John P. , Marttila Marko , Dennerquist Veronica , Wadell Göran , Arnberg Niklas 
Published: August 2003

ABSTRACTThe 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.
Association of the Herpes Simplex Virus Type 1 Us11 Gene Product with the Cellular Kinesin Light-Chain-Related Protein PAT1 Results in the Redistribution of Both Polypeptides
Authors: Benboudjema Louisa , Mulvey Matthew , Gao Yuehua , Pimplikar Sanjay W. , Mohr Ian 
Published: August 2003

ABSTRACT
The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components.
Replication of Hepatitis C Virus Subgenomes in Nonhepatic Epithelial and Mouse Hepatoma Cells
Authors: Zhu Qing , Guo Ju-Tao , Seeger Christoph 
Published: August 2003

ABSTRACT
The hepatitis C virus (HCV) pandemic affects the health of more than 170 million people and is the major indication for orthotopic liver transplantations. Although the human liver is the primary site for HCV replication, it is not known whether extrahepatic tissues are also infected by the virus and whether nonprimate cells are permissive for RNA replication. Because HCV exists as a quasispecies, it is conceivable that a viral population may include variants that can replicate in different cell types and in other species. We have tested this hypothesis and found that subgenomic HCV RNAs can replicate in mouse hepatoma and nonhepatic human epithelial cells. Replicons isolated from these cell lines carry new mutations that could be involved in the control of tropism of the virus. Our results demonstrated that translation and RNA-directed RNA replication of HCV do not depend on hepatocyte or primate-specific factors. Moreover, our results could open the path for the development of animal models for HCV infection.
Production of Human Papillomavirus Type 16 Virus-Like Particles in Transgenic Plants
Authors: Biemelt Sophia , Sonnewald Uwe , Galmbacher Petra , Willmitzer Lothar , Müller Martin 
Published: August 2003

ABSTRACTCervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Ω derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals.
Mutations in the N Termini of Herpes Simplex Virus Type 1 and 2 gDs Alter Functional Interactions with the Entry/Fusion Receptors HVEM, Nectin-2, and 3-
O
-Sulfated Heparan Sulfate but Not with Nectin-1
Authors: Yoon Miri , Zago Anna , Shukla Deepak , Spear Patricia G. 
Published: August 2003

ABSTRACT

Multiple cell surface molecules (herpesvirus entry mediator [HVEM], nectin-1, nectin-2, and 3-
O
-sulfated heparan sulfate) can serve as entry receptors for herpes simplex virus type 1 (HSV-1) or HSV-2 and also as receptors for virus-induced cell fusion. Viral glycoprotein D (gD) is the ligand for these receptors. A previous study showed that HVEM makes contact with HSV-1 gD at regions within amino acids 7 to 15 and 24 to 32 at the N terminus of gD. In the present study, amino acid substitutions and deletions were introduced into the N termini of HSV-1 and HSV-2 gDs to determine the effects on interactions with all of the known human and mouse entry/fusion receptors, including mouse HVEM, for which data on HSV entry or cell fusion were not previously reported. A cell fusion assay was used to assess functional activity of the gD mutants with each entry/fusion receptor. Soluble gD:Fc hybrids carrying each mutation were tested for the ability to bind to cells expressing the entry/fusion receptors. We found that deletions overlapping either or both of the HVEM contact regions, in either HSV-1 or HSV-2 gD, severely reduced cell fusion and binding activity with all of the human and mouse receptors except nectin-1. Amino acid substitutions described previously for HSV-1 (L25P, Q27P, and Q27R) were individually introduced into HSV-2 gD and, for both serotypes, were found to be without effect on cell fusion and the binding activity for nectin-1. Each of these three substitutions in HSV-1 gD enhanced fusion with cells expressing human nectin-2 (ordinarily low for wild-type HSV-1 gD), but the same substitutions in HSV-2 gD were without effect on the already high level of cell fusion observed with the wild-type protein. The Q27P or Q27R substitution in either HSV-1 and HSV-2 gD, but not the L25P substitution, significantly reduced cell fusion and binding activity for both human and mouse HVEM. Each of the three substitutions in HSV-1 gD, as well as the deletions mentioned above, reduced fusion with cells bearing 3-
O
-sulfated heparan sulfate. Thus, the N terminus of HSV-1 or HSV-2 gD is not necessary for functional interactions with nectin-1 but is necessary for all of the other receptors tested here. The sequence of the N terminus determines whether nectin-2 or 3-
O
-sulfated heparan sulfate, as well as HVEM, can serve as entry/fusion receptors.
Effects of Modification of the Transcription Initiation Site Context on
Citrus Tristeza Virus
Subgenomic RNA Synthesis
Authors: Ayllón María A. , Gowda Siddarame , Satyanarayana Tatineni , Karasev Alexander V. , Adkins Scott , Mawassi Munir , Guerri José , Moreno Pedro , Dawson William O. 
Published: August 2003

ABSTRACT

Citrus tristeza virus
(CTV), a member of the
Closteroviridae
, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3′-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5′ termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5′ or 3′ of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.
Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus
Authors: Rajendran K. S. , Nagy Peter D. 
Published: August 2003

ABSTRACT

Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the
Escherichia coli
-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat
trans
-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.
Identification of a Mutation in Editing of Defective Newcastle Disease Virus Recombinants That Modulates P-Gene mRNA Editing and Restores Virus Replication and Pathogenicity in Chicken Embryos
Authors: Mebatsion Teshome , de Vaan Leonie T. C. , de Haas Niels , Römer-Oberdörfer Angela , Braber Marian 
Published: August 2003

ABSTRACT

Editing of P-gene mRNA of Newcastle disease virus (NDV) enables the formation of two additional proteins (V and W) by inserting one or two nontemplated G residues at a conserved editing site (5′-AAAAAGGG). The V protein of NDV plays an important role in virus replication and is also a virulence factor presumably due to its ability to counteract the antiviral effects of interferon. A recombinant virus possessing a nucleotide substitution within the A-stretch (5′-AAgAAGGG) produced 20-fold-less V protein and, in consequence, was impaired in replication capacity and completely attenuated in pathogenicity for chicken embryos. However, in a total of seven serial passages, restoration of replication and pathogenic capacity in 9- to 11-day-old chicken embryos was noticed. Determining the sequence around the editing site of the virus at passage 7 revealed a C-to-U mutation at the second nucleotide immediately upstream of the 5′-A
5
stretch (5′-GuUAAgAAGGG). The V mRNA increased from an undetectable level at passage 5 to ca. 1 and 5% at passages 6 and 7, respectively. In addition, similar defects in another mutant possessing a different substitution mutation (5′-AAAcAGGG) were restored in an identical manner within a total of seven serial passages. Introduction of the above C-to-U mutation into the parent virus (5′-GuUAAAAAGGG) altered the frequency of P, V, and W mRNAs from 68, 28, and 4% to 15, 44, and 41%, respectively, demonstrating that the U at this position is a key determinant in modulating P-gene mRNA editing. The results indicate that this second-site mutation is required to compensate for the drop in edited mRNAs and consequently to restore the replication capacity, as well as the pathogenic potential, of editing-defective NDV recombinants.
Local Character of Readthrough Activation in Adenovirus Type 5 Early Region 1 Transcription Control
Authors: Shen Li , Spector David J. 
Published: August 2003

ABSTRACT

Wild-type early activity of the adenovirus 5 E1b gene promoter requires readthrough transcription originating from the adjacent upstream E1a gene. This unusual mode of viral transcription activation was identified by genetic manipulation of the mouse β
maj
-globin gene transcription termination sequence (
GGT
) inserted into the E1a gene. To facilitate further study of the mechanism of readthrough activation, the activities of
GGT
and a composite termination sequence
CT
were tested in recombinant adenoviruses containing luciferase reporters driven by the E1b promoter. There was a strict correlation between readthrough and substantial downstream gene expression, indicating that interference with downstream transcription was not a unique property of
GGT
. Blockage of readthrough transcription of E1a had no apparent effect on early expression of the major late promoter, the next active promoter downstream of E1b. A test for epistatic interaction between termination sequence insertions and E1a enhancer mutations suggested that readthrough activation and E1a enhancer activation of the E1b promoter are mechanistically distinct. In addition, substitution of the human cytomegalovirus major immediate-early promoter for the E1b promoter suppressed the requirement for readthrough. These results suggest that readthrough activation is a “local” effect of a direct interaction between the invading transcription elongation complex and the E1b promoter. DNase I hypersensitivity footprinting provided evidence that this interaction altered an extensive E1b promoter DNA-protein complex that was assembled in the absence of readthrough transcription.
Recombinant Sindbis/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic
Authors: Paessler Slobodan , Fayzulin Rafik Z. , Anishchenko Michael , Greene Ivorlyne P. , Weaver Scott C. , Frolov Ilya 
Published: August 2003

ABSTRACTVenezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and thecis-acting RNA elements derived from Sindbis virus (SINV), one of the least human-pathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.
Human Cytomegalovirus US2 Causes Similar Effects on Both Major Histocompatibility Complex Class I and II Proteins in Epithelial and Glial Cells
Authors: Hegde Nagendra R. , Johnson David C. 
Published: August 2003

ABSTRACT
The human cytomegalovirus (HCMV) glycoprotein US2 specifically binds to major histocompatibility complex (MHC) class I heavy chain (HC) and class II proteins DRα and DMα, triggering their degradation by proteasomes. Effects of US2 on class II proteins were originally characterized in HCMV- or adenovirus vector-infected U373 astroglioma cells. Here, we have extended characterization of US2-mediated degradation of class II DRα to two other cell lines, including biologically relevant epithelial cells. Comparison of the effects of US2 in cells expressing both class I and II proteins demonstrated only a slight preference for class I HC. Moreover, US2 caused degradation of DRα and DMα when these proteins were expressed by transfection without DRβ, invariant chain (Ii), or DMβ. Therefore, US2 binds to α chains of DR and DM and triggers endoplasmic reticulum degradation without formation of class II DR αβ/Ii or DM αβ complexes. Similar levels of degradation of class II α were observed in cells expressing vastly different amounts of class II, suggesting that cellular factors, other than class II, were limiting. We concluded that US2 has broad effects in a variety of cells that express both class I and II proteins and is relevant to HCMV infection in vivo.