bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Cytomegalovirus Infection Leads to Accumulation of Geminin and Inhibition of the Licensing of Cellular DNA Replication
Authors: Biswas Nilima , Sanchez Veronica , Spector Deborah H.
Published: January 2003

Previous studies have shown that infection of G
0
-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis. In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC). We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins. The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells. This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor. Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells. Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required. These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle.
The 64-Kilodalton Capsid Protein Homolog of
Beet Yellows Virus
Is Required for Assembly of Virion Tails
Authors: Napuli Alberto J. , Alzhanova Dina V. , Doneanu Catalin E. , Barofsky Douglas F. , Koonin Eugene V. , Dolja Valerian V.
Published: January 2003

The filamentous virion of the closterovirus
Beet yellows virus
(BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography-tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement.
Characterization of a Cytolytic Strain of Equine Infectious Anemia Virus
Authors: Maury Wendy , Wright Patrick J. , Bradley Sarahann
Published: January 2003

A novel strain of equine infectious anemia virus (EIAV) called vMA-1c that rapidly and specifically killed infected equine fibroblasts (ED cells) but not other infectible cell lines was established. This strain was generated from an avirulent, noncytopathic strain of EIAV, MA-1. Studies with this new cytolytic strain of virus have permitted us to define viral parameters associated with EIAV-induced cell killing and begin to explore the mechanism. vMA-1c infection resulted in induction of rapid cell death, enhanced fusogenic activity, and increased rates of spread in equine fibroblasts compared to other strains of EIAV. The highly cytolytic nature of vMA-1c suggested that this strain might be superinfecting equine fibroblasts. Receptor interference studies demonstrated that prior infection of equine fibroblasts with EIAV did not alter the ability of vMA-1c to infect and kill these cells. In similar studies in a canine fibroblast cell line, receptor interference did occur. vMA-1c infection of equine fibroblasts was also associated with large quantities of unintegrated viral DNA, a well-established hallmark of retroviral superinfection. Cloning of the vMA-1c genome identified nucleotide changes that would result in at least one amino acid change in all viral proteins. A chimeric infectious molecular clone containing the vMA-1c
tat
,
S2
, and
env
open reading frames recapitulated most of the characteristics of vMA-1c, including superinfection, fibroblast killing, and fusogenic activity. In summary, in vitro selection for a strain of EIAV that rapidly killed cells resulted in the generation of a virus that was able to superinfect these cells, presumably by the use of a novel mechanism of cell entry. This phenotype mapped to the 3′ half of the genome.
Mucosal Priming of Simian Immunodeficiency Virus-Specific Cytotoxic T-Lymphocyte Responses in Rhesus Macaques by theSalmonellaType III Secretion Antigen Delivery System
Authors: Evans David T. , Chen Li-Mei , Gillis Jacqueline , Lin Kuei-Chin , Harty Brian , Mazzara Gail P. , Donis Ruben O. , Mansfield Keith G. , Lifson Jeffrey D. , Desrosiers Ronald C. , Galán Jorge E. , Johnson R. Paul
Published: January 2003

Nearly all human immunodeficiency virus (HIV) infections are acquired mucosally, and the gut-associated lymphoid tissues are important sites for early virus replication. Thus, vaccine strategies designed to prime virus-specific cytotoxic T lymphocyte (CTL) responses that home to mucosal compartments may be particularly effective at preventing or containing HIV infection. TheSalmonellatype III secretion system has been shown to be an effective approach for stimulating mucosal CTL responses in mice. We therefore testedΔphoP-phoQattenuated strains ofSalmonella entericaserovar Typhimurium andS. entericaserovar Typhi expressing fragments of the simian immunodeficiency virus (SIV) Gag protein fused to the type III-secreted SopE protein for the ability to prime virus-specific CTL responses in rhesus macaques.Mamu-A*01+macaques were inoculated with three oral doses of recombinantSalmonella, followed by a peripheral boost with modified vaccinia virus Ankara expressing SIV Gag (MVA Gag). Transient low-level CTL responses to the Mamu-A*01 Gag181-189epitope were detected following each dose ofSalmonella. After boosting with MVA Gag, strong Gag-specific CTL responses were consistently detected, and tetramer staining revealed the expansion of Gag181-189-specific CD8+T-cell responses in peripheral blood. A significant percentage of the Gag181-189-specific T-cell population in each animal also expressed the intestinal homing receptor α4β7. Additionally, Gag181-189-specific CD8+T cells were detected in lymphocytes isolated from the colon. Yet, despite these responses,Salmonella-primed/MVA-boosted animals did not exhibit improved control of virus replication following a rectal challenge with SIVmac239. Nevertheless, this study demonstrates the potential of mucosal priming by theSalmonellatype III secretion system to direct SIV-specific cellular immune responses to the gastrointestinal mucosa in a primate model.
Murine Gammaherpesvirus 68 Lacking Thymidine Kinase Shows Severe Attenuation of Lytic Cycle Replication In Vivo but Still Establishes Latency
Authors: Coleman Heather M. , Lima Brigitte de , Morton Victoria , Stevenson Philip G.
Published: January 2003

The lytic cycle functions of gammaherpesviruses have received relatively little attention to date, at least in part due to the lack of a convenient experimental model. The murine gammaherpesvirus 68 (MHV-68) now provides such a model and allows the roles of individual lytic cycle gammaherpesvirus proteins to be evaluated in vivo. We have used MHV-68 to determine the contribution of a gammaherpesvirus thymidine kinase (TK) to viral lytic replication and latency establishment. MHV-68 mutants with a disrupted TK gene grew normally in vitro but showed a severe attenuation of replication in the lungs after intranasal inoculation, with lytic titers at least 1,000-fold lower than those of wild-type and revertant viruses. Nevertheless, the establishment of latency by the TK-deficient mutants, while delayed, was not prevented by their lytic replication deficit. The viral TK clearly plays a crucial role in the capacity of MHV-68 to replicate efficiently in its natural host but does not seem to be essential to establish a persistent infection. The potential of TK-deficient mutants as gammaherpesvirus vaccines is discussed.
TT Virus in the Nasal Secretions of Children with Acute Respiratory Diseases: Relations to Viremia and Disease Severity
Authors: Maggi Fabrizio , Pifferi Massimo , Fornai Claudia , Andreoli Elisabetta , Tempestini Elena , Vatteroni Marialinda , Presciuttini Silvano , Marchi Santino , Pietrobelli Angelo , Boner Attilio , Pistello Mauro , Bendinelli Mauro
Published: January 2003

The natural history and pathogenic potential of the recently identified TT virus (TTV) are currently a matter of intensive investigation. In an attempt to shed some light on these issues, nasal and blood specimens of 1- to 24-month-old children hospitalized with a clinical diagnosis of acute respiratory disease (ARD) were examined for the presence, load, and genetic characteristics of TTV. The results have indicated that at least in young children, the respiratory tract not only represents a route by which abundant TTV can be shed into the environment but also may be a site of primary infection and continual replication. Although we found no compelling evidence that TTV was the direct cause of ARD in some of the children studied, the average loads of TTV were considerably higher in patients with bronchopneumonia (BP) than in those with milder ARD, raising interesting questions about the pathophysiological significance of TTV at this site. Furthermore, group 4 TTV was detected almost exclusively in children with BP.
Baculovirus Alkaline Nuclease Possesses a 5′→3′ Exonuclease Activity and Associates with the DNA-Binding Protein LEF-3
Authors: Mikhailov Victor S. , Okano Kazuhiro , Rohrmann George F.
Published: January 2003

Alkaline nuclease (AN) of the
Autographa californica
multiple-capsid nucleopolyhedrovirus (Ac
M
NPV) (open reading frame 133) was expressed in recombinant baculovirus as a His
6
-tagged fusion and purified by sequential chromatography on Ni-NTA-agarose, DEAE-Toyopearl, and heparin-Sepharose. At all stages of purification, Ac
M
NPV AN was found to copurify with a 44-kDa polypeptide which was identified as the baculovirus single-stranded DNA (ssDNA)-binding (SSB) protein, LEF-3. Sedimentation analysis in glycerol gradients of highly purified samples suggested that AN and LEF-3 are associated in a complex (designated *AN/L3), predominantly as heterodimers, although oligomeric forms containing both proteins were evident. In reactions with single- or double-stranded 62-mer oligonucleotides that were labeled with
32
P at the 5′ or 3′ ends, *AN/L3 carried out exonucleolytic hydrolysis of both substrates exclusively in a 5′→3′ direction. Saturation of ssDNA with an excess of LEF-3 prior to the addition of *AN/L3 resulted in a marked decrease in the rate of ssDNA hydrolysis. This suggests that excess LEF-3 may protect ssDNA from digestion by a AN-LEF-3 complex, thus regulating its activity in infected cells. The association of baculovirus AN with the viral SSB LEF-3 and the 5′→3′ exonuclease activity of this complex suggests that AN and LEF-3 may participate in homologous recombination of the baculovirus genome in a manner similar to that of exonuclease (Redα) and DNA-binding protein (Redβ) of the Red-mediated homologous recombination system of bacteriophage λ.
The Early Expression of Glycoprotein B from Herpes Simplex Virus Can Be Detected by Antigen-Specific CD8+T Cells
Authors: Mueller Scott N. , Jones Claerwen M. , Chen Weisan , Kawaoka Yoshihiro , Castrucci Maria R. , Heath William R. , Carbone Francis R.
Published: January 2003

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, α, β, and γ, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB498-505). While gB is considered a γ or “late” gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of naïve transgenic CD8+T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8+T cells.
The Human Herpesvirus 6 U100 Gene Product Is the Third Component of the gH-gL Glycoprotein Complex on the Viral Envelope
Authors: Mori Yasuko , Akkapaiboon Pilailuk , Yang Xuwei , Yamanishi Koichi
Published: January 2003

The human herpesvirus 6 (HHV-6) variant A U100 gene encodes the third component of the glycoprotein H (gH)-glycoprotein L (gL)-containing complex. Glycosidase digestion analysis showed that the U100 gene products are glycoproteins consisting of an 80-kDa protein with complex N-linked oligosaccharides and a 74-kDa protein with immature, high-mannose N-linked oligosaccharides. Based on these characteristics, we designated the U100 gene products glycoprotein Q (gQ). Only the 80-kDa form of gQ was coimmunoprecipitated with an anti-gH antibody, suggesting that the 80-kDa protein associates with the gH-gL complex in HHV-6-infected cells. Furthermore, the complex was detected in purified virions, suggesting that it may play an important role in viral entry.
Identification and Molecular Characterization of the RNA Polymerase-Binding Motif of Infectious Bursal Disease Virus Inner Capsid Protein VP3
Authors: Maraver Antonio , Clemente Roberto , Rodríguez Jose Francisco , Lombardo Eleuterio
Published: January 2003

Infectious bursal disease virus
(IBDV), a member of the
Birnaviridae
family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufficient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 specifically inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells specifically reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication.
Lack of Tumor Necrosis Factor Alpha Induces Impaired Proliferation of Hepatitis B Virus-Specific Cytotoxic T Lymphocytes
Authors: Kasahara Senji , Ando Kazuki , Saito Kuniaki , Sekikawa Kenji , Ito Hiroyasu , Ishikawa Tetsuya , Ohnishi Hiroo , Seishima Mitsuru , Kakumu Shinichi , Moriwaki Hisataka
Published: January 2003

Recent studies have shown that tumor necrosis factor alpha (TNF-α) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-α knockout (TNF-α−/−) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-α−/−mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-α−/−mice after two sessions of immunization with the HBV plasmid; however, TNF-α was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-α−/−mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-α enhanced the proliferation of a TNF-α−/−clone but suppressed that of a TNF-α+/+clone in vitro, TNF-α also has a direct effect on the proliferation of CTLs. In conclusion, TNF-α is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.
Helicase Motif Ia Is Involved in Single-Strand DNA-Binding and Helicase Activities of the Herpes Simplex Virus Type 1 Origin-Binding Protein, UL9
Authors: Marintcheva Boriana , Weller Sandra K.
Published: January 2003

UL9 is a multifunctional protein essential for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. It is thought that UL9 binds the origin of replication and unwinds it in the presence of ATP and the HSV-1 single-stranded DNA (ssDNA)-binding protein. We have previously characterized the biochemical properties of mutants in all helicase motifs except for motif Ia (B. Marintcheva and S. Weller, J. Biol. Chem. 276:6605-6615, 2001). Structural information for other superfamily I and II helicases indicates that motif Ia is involved in ssDNA binding. By analogy, we hypothesized that UL9 motif Ia is important for the ssDNA-binding function of the protein. On the basis of sequence conservation between several UL9 homologs within the
Herpesviridae
family and distant homology with helicases whose structures have been solved, we designed specific mutations in motif Ia and analyzed them genetically and biochemically. Mutant proteins with residues predicted to be involved in ssDNA binding (R112A and R113A/F115A) exhibited wild-type levels of intrinsic ATPase activity and moderate to severe defects in ssDNA-stimulated ATPase activity and ssDNA binding. The S110T mutation targets a residue not predicted to contact ssDNA directly. The mutant protein with this mutation exhibited wild-type levels of intrinsic ATPase activity and near wild-type levels of ssDNA-stimulated ATPase activity and ssDNA binding. All mutant proteins lack helicase activity but were able to dimerize and bind the HSV-1 origin of replication as well as wild-type UL9. Our results indicate that residues from motif Ia contribute to the ssDNA-binding and helicase activities of UL9 and are essential for viral growth. This work represents the successful application of an approach based on a combination of bioinformatics and structural information from related proteins to deduce valuable information about a protein of interest.
Foot-and-Mouth Disease Virus Receptors: Comparison of Bovine α
V
Integrin Utilization by Type A and O Viruses
Authors: Duque Hernando , Baxt Barry
Published: January 2003

Three members of the α
V
integrin family of cellular receptors, α
V
β
1
, α
V
β
3
, and α
V
β
6
, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the βG-βH (G-H) loop of VP1. Other α
V
integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the α
V
integrins from a susceptible species as viral receptors, we molecularly cloned the bovine β
1
, β
5
, and β
6
integrin subunits. Using these subunits, along with previously cloned bovine α
V
and β
3
subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by α
V
β
1
, α
V
β
3
, α
V
β
5
, and α
V
β
6
among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used α
V
β
3
and α
V
β
6
with relatively high efficiency, while only one virus utilized α
V
β
1
with moderate efficiency. In contrast, both type O viruses utilized α
V
β
6
and α
V
β
1
with higher efficiency than α
V
β
3
. Only low levels of viral replication were detected in α
V
β
5
-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the β subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host.
Effective Vaccination against Long-Term Gammaherpesvirus Latency
Authors: Tibbetts Scott A. , McClellan J. Scott , Gangappa Shivaprakash , Speck Samuel H. , Virgin Herbert W.
Published: January 2003

The fundamental question of whether a primed immune system is capable of preventing latent gammaherpesvirus infection remains unanswered. Recent studies showing that vaccination can reduce acute replication and short-term latency but cannot alter long-term latency further call into question the possibility of achieving sterilizing immunity against gammaherpesviruses. Using the murine gammaherpesvirus 68 (γHV68) system, we demonstrate that it is possible to effectively vaccinate against long-term latency. By immunizing mice with a γHV68 mutant virus that is deficient in its ability to reactivate from latency, we reduced latent infection of wild-type challenge virus to a level below the limit of detection. Establishment of latency was inhibited by vaccination regardless of whether mice were challenged intraperitoneally or intranasally. Passive transfer of antibody from vaccinated mice could partially reconstitute the effect, demonstrating that antibody is an important component of vaccination. These results demonstrate the potential of a memory immune response against gammaherpesviruses to alter long-term latency and suggest that limiting long-term latent infection in a clinically relevant situation is an attainable goal.
Identification of a Receptor-Binding Domain of the Spike Glycoprotein of Human Coronavirus HCoV-229E
Authors: Bonavia Aurelio , Zelus Bruce D. , Wentworth David E. , Talbot Pierre J. , Holmes Kathryn V.
Published: January 2003

Human coronavirus HCoV-229E uses human aminopeptidase N (hAPN) as its receptor (C. L. Yeager et al., Nature357:420-422, 1992). To identify the receptor-binding domain of the viral spike glycoprotein (S), we expressed soluble truncated histidine-tagged S glycoproteins by using baculovirus expression vectors. Truncated S proteins purified by nickel affinity chromatography were shown to be glycosylated and to react with polyclonal anti-HCoV-229E antibodies and monoclonal antibodies to the viral S protein. A truncated protein (S547) that contains the N-terminal 547 amino acids bound to 3T3 mouse cells that express hAPN but not to mouse 3T3 cells transfected with empty vector. Binding of S547to hAPN was blocked by an anti-hAPN monoclonal antibody that inhibits binding of virus to hAPN and blocks virus infection of human cells and was also blocked by polyclonal anti-HCoV-229E antibody. S proteins that contain the N-terminal 268 or 417 amino acids did not bind to hAPN-3T3 cells. Antibody to the region from amino acid 417 to the C terminus of S blocked binding of S547to hAPN-3T3 cells. Thus, the data suggest that the domain of the spike protein between amino acids 417 and 547 is required for the binding of HCoV-229E to its hAPN receptor.
G Protein-Dependent CCR5 Signaling Is Not Required for Efficient Infection of Primary T Lymphocytes and Macrophages by R5 Human Immunodeficiency Virus Type 1 Isolates
Authors: Amara Ali , Vidy Aurore , Boulla Genevieve , Mollier Karine , Garcia-Perez Javier , Alcamí Jose , Blanpain Cedric , Parmentier Marc , Virelizier Jean-Louis , Charneau Pierre , Arenzana-Seisdedos Fernando
Published: January 2003

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4+-transformed cells or pharmacological inhibition of Gαi proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Δ32/Δ32). Expression of wild-type (wt) CCR5 inccr5 Δ32/Δ32cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4+cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4+T lymphocytes or macrophages.
The Coxsackie B Virus and Adenovirus Receptor Resides in a Distinct Membrane Microdomain
Authors: Ashbourne Excoffon Katherine J. D. , Moninger Thomas , Zabner Joseph
Published: January 2003

The coxsackie B virus and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily. In addition to activity as a viral receptor, it may play a role in cellular adhesion. We asked what determines the cell membrane microdomain of CAR. We found that CAR is localized to a novel lipid-rich microdomain similar to that of the low-density lipoprotein receptor (LDLR) but distinct from that of a CAR variant that exhibited traditional lipid raft localization via fusion to a glycosylphosphatidylinositol (GPI) tail. The cytoplasmic tail determines its membrane localization, since deletion of this domain resulted in mislocalization. Results indicate that CAR, CAR-LDLR, and LDLR reside in a novel lipid raft that is distinct from caveolin-1-containing caveolae and GPI-linked proteins. Residence in a lipid-rich domain provides a mechanism that allows CAR to interact with other cell adhesion proteins and yet function as an adenovirus receptor.
An Alternate Pathway for Recruiting Template RNA to the Brome Mosaic Virus RNA Replication Complex
Authors: Chen Jianbo , Noueiry Amine , Ahlquist Paul
Published: January 2003

The multidomain RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, plays key roles in assembly and function of the viral RNA replication complex. 1a, which encodes RNA capping and helicase-like domains, localizes to endoplasmic reticulum membranes, recruits BMV 2a polymerase and viral RNA templates, and forms membrane-bound, capsid-like spherules in which RNA replication occurs.
cis
-acting signals necessary and sufficient for RNA recruitment by 1a have been mapped in BMV genomic RNA2 and RNA3. Both signals comprise an extended stem-loop whose apex matches the conserved sequence and structure of the TΨC stem-loop in tRNAs (box B). Mutations show that this box B motif is crucial to 1a responsiveness of wild-type RNA2 and RNA3. We report here that, unexpectedly, some chimeric mRNAs expressing the 2a polymerase open reading frame from RNA2 were recruited by 1a to the replication complex and served as templates for negative-strand RNA synthesis, despite lacking the normally essential, box B-containing 5′ signal. Further studies showed that this template recruitment required high-efficiency translation of the RNA templates. Moreover, multiple small frameshifting insertion or deletion mutations throughout the N-terminal region of the open reading frame inhibited this template recruitment, while an in-frame insertion did not. Providing 2a in
trans
did not restore template recruitment of RNAs with frameshift mutations. Only those deletions in the N-terminal region of 2a that abolished 2a interaction with 1a abolished template recruitment of the RNA. These and other results indicate that this alternate pathway for 1a-dependent RNA recruitment involves 1a interaction with the translating mRNA via the 1a-interactive N-terminal region of the nascent 2a polypeptide. Interaction with nascent 2a also may be involved in 1a recruitment of 2a polymerase to membranes.
B Cells and Antibody Play Critical Roles in the Immediate Defense of Disseminated Infection by West Nile Encephalitis Virus
Authors: Diamond Michael S. , Shrestha Bimmi , Marri Anantha , Mahan Darby , Engle Michael
Published: January 2003

West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (μMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, a ∼500-fold difference in serum viral load was detected in μMT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected μMT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.
Molecular Characteristics of Human Immunodeficiency Virus Type 1 Subtype C Viruses from KwaZulu-Natal, South Africa: Implications for Vaccine and Antiretroviral Control Strategies
Authors: Gordon M. , De Oliveira T. , Bishop K. , Coovadia H. M. , Madurai L. , Engelbrecht S. , Janse van Rensburg E. , Mosam A. , Smith A. , Cassol S.
Published: January 2003

The KwaZulu-Natal region of South Africa is experiencing an explosive outbreak of human immunodeficiency virus type 1 (HIV-1) subtype C infections. Understanding the genetic diversity of C viruses and the biological consequences of this diversity is important for the design of effective control strategies. We analyzed the protease gene, the first 935 nucleotides of reverse transcriptase, and the C2V5 envelope region of a representative set of 72 treatment-naïve patients from KwaZulu-Natal and correlated the results with amino acid signature and resistance patterns. Phylogenetic analysis revealed multiple clusters or “lineages” of HIV-1 subtype C that segregated with other C viruses from southern Africa. The same pattern was observed for both black and Indian subgroups and for retrospective specimens collected prior to 1990, indicating that multiple sublineages of HIV-1 C have been present in KwaZulu-Natal since the early stages of the epidemic. With the exception of three nonnucleoside reverse transcriptase inhibitor mutations, no primary resistance mutations were identified. Numerous accessory polymorphisms were present in the protease, but none were located at drug-binding or active sites of the enzyme. One frequent polymorphism, I93L, was located near the protease/reverse transcriptase cleavage site. In the envelope, disruption of the glycosylation motif at the beginning of V3 was associated with the presence of an extra protein kinase C phosphorylation site at codon 11. Many polymorphisms were embedded within cytotoxic T lymphocyte or overlapping cytotoxic T-lymphocyte/T-helper epitopes, as defined for subtype B. This work forms a baseline for future studies aimed at understanding the impact of genetic diversity on vaccine efficacy and on natural susceptibility to antiretroviral drugs.
A Functional Epitope Determinant on Domain III of the Japanese Encephalitis Virus Envelope Protein Interacted with Neutralizing-Antibody Combining Sites
Authors: Lin Cheng-Wen , Wu Suh-Chin
Published: January 2003

The envelope (E) protein of Japanese encephalitis virus (JEV) is associated with viral binding to cellular receptors, membrane fusion, and the induction of protective neutralizing-antibody responses in hosts. Most previous studies have not provided detailed molecular information about the spatial configuration of the functional epitopes on domain III of the E protein. Here site-directed mutagenesis was performed to demonstrate that the functional epitope determinants at Ser331 and Asp332 on domain III of the JEV E protein interacted with neutralizing monoclonal antibody (MAb) E3.3. Bacterial expression of the recombinant Fab E3.3 confirmed the molecular interactions of Arg94 in complementary determining region H3 with Ser331 and Asp332 on domain III. This study elucidates the detailed molecular structures of the neutralizing epitope determinants on JEV domain III, which can provide useful information for designing new vaccines.
Cell Penetration and Trafficking of Polyomavirus
Authors: Gilbert Joanna M. , Goldberg Ilya G. , Benjamin Thomas L.
Published: January 2003

The murine polyomavirus (Py) enters mouse fibroblasts and kidney epithelial cells via an endocytic pathway that is caveola-independent (as well as clathrin-independent). In contrast, uptake of simian virus 40 into the same cells is dependent on caveola. Following the initial uptake of Py, both microtubules and microfilaments play roles in trafficking of the virus to the nucleus. Colcemid, which disrupts microtubules, inhibits the ability of Py to reach the nucleus and replicate. Paclitaxel, which stabilizes microtubules and prevents microtubule turnover, has no effect, indicating that intact but not dynamic microtubules are required for Py infectivity. Compounds that disrupt actin filaments enhance Py uptake while stabilization of actin filaments impedes Py infection. Virus particles are seen in association with actin in cells treated with microfilament-disrupting or filament-stabilizing agents at levels comparable to those in untreated cells, suggesting that a dynamic state of the microfilament system is important for Py infectivity.
Molluscum Contagiosum Virus Interleukin-18 (IL-18) Binding Protein Is Secreted as a Full-Length Form That Binds Cell Surface Glycosaminoglycans through the C-Terminal Tail and a Furin-Cleaved Form with Only the IL-18 Binding Domain
Authors: Xiang Yan , Moss Bernard
Published: January 2003

Some poxviruses and their mammalian hosts encode homologous proteins that bind interleukin-18 (IL-18) with high affinity and inhibit IL-18-mediated immune responses. MC54L, the IL-18 binding protein of the human poxvirus that causes molluscum contagiosum, is unique in having a C-terminal tail of nearly 100 amino acids that is dispensable for IL-18 binding. When recombinant MC54L was expressed and purified via a C-terminal six-histidine tag, a shorter fragment was detected in addition to the full-length protein. This C-terminal fragment resulted from the cleavage of MC54L by cellular furin, as it was greatly diminished when furin was specifically inhibited or when a furin-deficient cell line was used for expression. Furthermore, the N- and C-terminal fragments of MC54L were generated by cleavage of the recombinant protein with furin in vitro. The furin cleavage site was mapped within a 32-amino-acid segment that is C terminal to the IL-18 binding domain. Full-length MC54L, but not the N-terminal IL-18 binding fragment, bound to cells and to purified heparin and other glycosaminoglycans that are commonly found on the cell surface and in the extracellular matrix. MC54L bound to heparin with a nanomolar

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and could simultaneously bind to IL-18. Their different glycosaminoglycan and cell binding properties may allow the long and short forms of MC54L to inactivate IL-18 near the site of infection and at more distal locations, respectively.
Developing Novel Oncolytic Adenoviruses through Bioselection
Authors: Yan Wen , Kitzes Galila , Dormishian Farid , Hawkins Lynda , Sampson-Johannes Adam , Watanabe Josh , Holt Jenny , Lee Vivian , Dubensky Thomas , Fattaey Ali , Hermiston Terry , Balmain Allan , Shen Yuqiao
Published: January 2003

Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed.
E1A Sensitizes Cells to Tumor Necrosis Factor Alpha by Downregulating c-FLIPS
Authors: Perez Denise , White Eileen
Published: January 2003

Tumor necrosis factor alpha (TNF-α) activates both apoptosis and NF-κB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested.c-FLIPis a TNF-α-induced gene that inhibits caspase-8 activation during TNF-α signaling. Adenovirus infection and E1A expression sensitize cells to TNF-α by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-α-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIPSexpression and prevented its induction by TNF-α. c-FLIPSand viral FLIP expression rescued E1A-mediated sensitization to TNF-α by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-α-dependent induction of c-FLIPSmRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIPSprotein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-κB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor cells.
Early Induction and Maintenance of Env-Specific T-Helper Cells following Human Immunodeficiency Virus Type 1 Infection
Authors: Malhotra Uma , Holte Sarah , Zhu Tuofu , Delpit Elizabeth , Huntsberry Claire , Sette Alessandro , Shankarappa Raj , Maenza Janine , Corey Lawrence , McElrath M. Juliana
Published: January 2003

Mounting evidence points to a role for CD4+T-helper (Th) cell activities in controlling human immunodeficiency virus type 1 (HIV-1) infection. To determine the induction and evolution of Th responses following acute infection, we prospectively analyzed Env- and Gag-specific Th responses longitudinally for 92 patients with acute (n= 28) or early (n= 64) HIV-1 infection (median, 55 days postinfection [DPI]). The probability of detecting HIV-1-specific lymphoproliferative responses was remarkably low, and when present, the responses were more likely to be Gag specific than Env specific (16 versus 5%). Env-specific responses were significantly more common in patients presenting at <30 DPI than in those presenting at 30 to 365 DPI (21 versus 0.5%,P= 0.001). By contrast, Gag-specific responses occurred with similar frequencies among subjects presenting at <30 DPI and 30 to 365 DPI (13 versus 17%,P= 0.6). After treatment, and regardless of the duration of infection before therapy, Gag-specific Th responses predominated. Furthermore, some acutely infected subjects lost detectable Env-specific Th proliferative responses, which failed to reemerge upon treatment. Detailed analysis for one such subject revealed Env-specific lymphoproliferation at 11 DPI but no detectable Env-specific lymphoproliferation or ex vivo gamma interferon (IFN-γ) secretion at multiple subsequent time points. Env-specific CD4+T-cell clones from 11 DPI recognized six epitopes in both conserved and variable regions within gp120 and gp41, exhibited major histocompatibility complex-restricted cytotoxicity, and secreted high levels of antiviral cytokines. T-cell receptor clonal transcript analyses and autologous virus sequencing revealed that Th cells induced during acute infection were maintained and there were no Th escape mutations. Subsequent analysis for this subject and six of seven others revealed detectable IFN-γ-secreting cells, but only following in vitro gp160 stimulation. In summary, we conclude that Env-specific Th responses are elicited very early in acute infection and may precede Gag-specific responses. The inability to detect Env-specific Th responses over time and despite antiretroviral therapy may reflect low frequencies and impaired proliferative capacity, and viral escape is not necessary for this to occur.
Herpes Simplex Virus gE/gI Expressed in Epithelial Cells Interferes with Cell-to-Cell Spread
Authors: Collins Wendy J. , Johnson David C.
Published: January 2003

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.
PrP Polymorphisms Tightly Control Sheep Prion Replication in Cultured Cells
Authors: Sabuncu Elifsu , Petit Stéphanie , Le Dur Annick , Lan Lai Thanh , Vilotte Jean-Luc , Laude Hubert , Vilette Didier
Published: January 2003

Prion diseases are fatal neurodegenerative disorders of animals and humans that are characterized by the conversion of the host-encoded prion protein (PrP) to an abnormal isoform. In several species, including humans, polymorphisms in the gene encoding the PrP protein tightly control susceptibility of individuals toward this disease. In the present study we show that Rov cells expressing an ovine PrP allele (VRQPrP) associated with high susceptibility of sheep to scrapie were very sensitive to sheep prion transmission and replicated the agent to high titers. In contrast, we did not find any evidence of infection when Rov cells expressed similar levels of a PrP variant (ARRPrP) linked to resistance. Our data provide the first direct evidence that natural PrP polymorphisms may affect prion susceptibility by controlling prion replication at the cell level. The study of how PrP polymorphisms influence the genetic control of prion propagation in cultured Rov cells may help elucidate basic mechanisms of prion replication.
Amino Acid Substitutions in VP2 Residues Contacting Sialic Acid in Low-Neurovirulence BeAn Virus Dramatically Reduce Viral Binding and Spread of Infection
Authors: Kumar A. S. Manoj , Kallio Patricia , Luo Ming , Lipton Howard L.
Published: January 2003

Theiler's murine encephalomyelitis viruses (TMEV) consist of two groups, the high- and low-neurovirulence groups, based on lethality in intracerebrally inoculated mice. Low-neurovirulence TMEV result in a persistent central nervous system infection in mice, leading to an inflammatory demyelinating pathology and disease. Low- but not high-neurovirulence strains use sialic acid as an attachment factor. The recent resolution of the crystal structure of the low-neurovirulence DA virus in complex with the sialic acid mimic sialyllactose demonstrated that four capsid residues make contact with sialic acid through noncovalent hydrogen bonds. To systematically test the importance of these sialic acid-binding residues in viral entry and infection, we mutated three VP2 puff B amino acids proposed to make contact with sialic acid and analyzed the consequences of each amino acid substitution on viral entry and spread. The fourth residue is in the VP3-VP1 cleavage dipeptide and could not be mutated. Our data suggest that residues Q2161 and G2174 are directly involved in BeAn virus attachment to sialic acid and that substitutions of these two residues result in the loss of or reduced viral binding and hemagglutination and in the inability to spread among BHK-21 cells. In addition, a gain of function-revertant virus was recovered with the Q2161A mutation after prolonged passage in cells.