bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Recognition by Foot-and-Mouth Disease Virus That Lacks the RGD Integrin-Binding Motif: Flexibility in Aphthovirus Receptor Usage
Authors: Baranowski Eric , Ruiz-Jarabo Carmen M. , Sevilla Noemi , Andreu David , Beck Ewald , Domingo Esteban
Published: July 2002

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143→Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin α
v
β
3
. In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.
Bovine Herpesvirus 1 U
L
3.5 Interacts with Bovine Herpesvirus 1 α-Transinducing Factor
Authors: Lam Ngan , Letchworth Geoffrey J.
Published: July 2002

The bovine herpesvirus 1 (BHV-1) U
L
3.5 gene encodes a 126-amino-acid tegument protein. Homologs of U
L
3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking U
L
3.5 is deficient in viral egress but can be complemented by BHV-1 U
L
3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886–8892, 1997). The function of BHV-1 U
L
3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 U
L
3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 α-transinducing factor (αBTIF) as a BHV-1 U
L
3.5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, U
L
3.5 and αBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of U
L
3.5, but not 40 amino acids from the C terminus, abolished the U
L
3.5-αBTIF interaction both in vitro and in vivo. The interaction between U
L
3.5 and αBTIF may be important for BHV-1 maturation and regulation of αBTIF transactivation activity.
Rotavirus Spike Protein VP4 Is Present at the Plasma Membrane and Is Associated with Microtubules in Infected Cells
Authors: Nejmeddine M. , Trugnan G. , Sapin C. , Kohli E. , Svensson L. , Lopez S. , Cohen J.
Published: July 2002

VP4 is an unglycosylated protein of the outer layer of the capsid of rotavirus. It forms spikes that project from the outer layer of mature virions, which is mainly constituted by glycoprotein VP7. VP4 has been implicated in several important functions, such as cell attachment, penetration, hemagglutination, neutralization, virulence, and host range. Previous studies indicated that VP4 is located in the space between the periphery of the viroplasm and the outside of the endoplasmic reticulum in rotavirus-infected cells. Confocal microscopy of infected MA104 monolayers, immunostained with specific monoclonal antibodies, revealed that a significant fraction of VP4 was present at the plasma membrane early after infection. Another fraction of VP4 is cytoplasmic and colocalizes with β-tubulin. Flow cytometry analysis confirmed that at the early stage of viral infection, VP4 was present on the plasma membrane and that its N-terminal region, the VP8* subunit, was accessible to antibodies. Biotin labeling of the infected cell surface monolayer with a cell-impermeable reagent allowed the identification of the noncleaved form of VP4 that was associated with the glycoprotein VP7. The localization of VP4 was not modified in cells transfected with a plasmid allowing the expression of a fusion protein consisting of VP4 and the green fluorescent protein. The present data suggest that VP4 reaches the plasma membrane through the microtubule network and that other viral proteins are dispensable for its targeting and transport.
Pathogenesis of Herpes Simplex Virus-Induced Ocular Immunoinflammatory Lesions in B-Cell-Deficient Mice
Authors: Deshpande Shilpa P. , Zheng Mei , Daheshia Massoud , Rouse Barry T.
Published: July 2002

The role of B cells and humoral immunity in herpes simplex virus (HSV) ocular infections was studied in immunoglobulin μ chain gene-targeted B-cell-deficient mice (μK/O). At doses of virus well tolerated by immunocompetent mice, heightened susceptibility of μK/O mice to herpetic encephalitis as well as to herpetic stromal keratitis (HSK) was observed. An explanation was sought for the increased severity of HSK in the μK/O mice. First, the lack of antibody responses in μK/O mice resulted in longer viral persistence and dissemination to the corneal stroma, the site of inflammation. Prolonged virus expression in the corneal stroma was suggested to cause bystander activation of Th1-type CD4+T cells, further contributing to the severity of HSK lesion expression in μK/O mice. Second, μK/O mice generated minimal Th2 cytokine responses compared to wild-type mice. Such responses might serve to downregulate the severity of Th1-mediated HSK lesions.
A Block to Human Immunodeficiency Virus Type 1 Assembly in Murine Cells
Authors: Mariani Roberto , Rutter Gabriel , Harris Matthew E. , Hope Thomas J. , Kräusslich Hans-Georg , Landau Nathaniel R.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) does not replicate in murine cells. We investigated the basis of this block by infecting a murine NIH 3T3 reporter cell line that stably expressed human CD4, CCR5, and cyclin T1 and contained a transactivatable HIV-1 long terminal repeat (LTR)-green fluorescent protein (GFP) cassette. Although the virus entered efficiently, formed provirus, and was expressed at a level close to that in a highly permissive human cell line, the murine cells did not support M-tropic HIV-1 replication. To determine why the virus failed to replicate, the efficiency of each postentry step in the virus replication cycle was analyzed using vesicular stomatitis virus G pseudotypes. The murine cells supported reverse transcription and integration at levels comparable to those in the human osteosarcoma-derived cell line GHOST.R5, and human cyclin T1 restored provirus expression, consistent with earlier findings of others. The infected murine cells contained nearly as much virion protein as did the human cells but released less than 1/500 the amount of p24

gag

into the culture medium. A small amount of p24

gag

was released and was in the form of fully infectious virus. Electron microscopy suggested that aberrantly assembled virion protein had accumulated in cytoplasmic vesicular structures. Virions assembling at the cell membrane were observed but were rare. The entry of M-tropic JR.FL-pseudotyped reporter virus was moderately reduced in the murine cells, suggesting a minor reduction in coreceptor function. A small reduction in the abundance of full-length viral mRNA transcripts was also noted; however, the major block was at virion assembly. This could have been due to a failure of Gag to target to the cell membrane. This block must be overcome before a murine model for HIV-1 replication can be developed.
Structural Analysis of a Fiber-Pseudotyped Adenovirus with Ocular Tropism Suggests Differential Modes of Cell Receptor Interactions
Authors: Chiu Charles Y. , Wu Eugene , Brown Swati L. , Von Seggern Dan J. , Nemerow Glen R. , Stewart Phoebe L.
Published: July 2002

Adenovirus (Ad) entry into cells is initiated by the binding of the fiber knob to a cell surface receptor. The coxsackie- and adenovirus receptor (CAR) functions as the attachment receptor for many, but not all, Ad serotypes. Ad type 37 (Ad37), a subgroup D virus that causes keratoconjunctivitis in humans, does not infect cells via CAR despite demonstrated binding of the Ad37 knob to CAR. We have pseudotyped a fiber deletion Ad5 vector with the Ad37 fiber (Ad37f), and this vector retains the ocular tropism of Ad37. Here we present a cryo-electron microscopy reconstruction of Ad37f that shows the entire Ad37 fiber, including the shaft and knob domains. We have previously proposed that Ad37 may not utilize CAR for cell entry because of the geometric constraints imposed by a rigid fiber (E. Wu, J. Fernandez, S. K. Fleck, D. Von Seggern, S. Huang, and G. R. Nemerow, Virology 279:78–89, 2001). Consistent with this hypothesis, our structural results show that the Ad37 fiber is straight and rigid. Modeling of the interaction between Ad37f and host cell receptors indicates that fiber flexibility or rigidity, as well as length, can affect receptor usage and cellular tropism.
Immunogenicity and Protective Efficacy of Recombinant Human T-Cell Leukemia/Lymphoma Virus Type 1 NYVAC and Naked DNA Vaccine Candidates in Squirrel Monkeys (Saimiri sciureus)
Authors: Kazanji Mirdad , Tartaglia James , Franchini Genoveffa , de Thoisy Benoit , Talarmin Antoine , Contamin Hugues , Gessain Antoine , de Thé Guy
Published: July 2002

We assessed the immunogenicities and efficacies of two highly attenuated vaccinia virus-derived NYVAC vaccine candidates encoding the human T-cell leukemia/lymphoma virus type 1 (HTLV-1)envgene or both theenvandgaggenes in prime-boost pilot regimens in combination with naked DNA expressing the HTLV-1 envelope. Three inoculations of NYVAC HTLV-1envat 0, 1, and 3 months followed by a single inoculation of DNAenvat 9 months protected against intravenous challenge with HTLV-1-infected cells in one of three immunized squirrel monkeys. Furthermore, humoral and cell-mediated immune responses against HTLV-1 Env could be detected in this protected animal. However, priming the animal with a single dose ofenvDNA, followed by immunization with the NYVAC HTLV-1gagandenvvaccine at 6, 7, and 8 months, protected all three animals against challenge with HTLV-1-infected cells. With this protocol, antibodies against HTLV-1 Env and cell-mediated responses against Env and Gag could also be detected in the protected animals. Although the relative superiority of a DNA prime-NYVAC boost regimen over addition of the Gag component as an immunogen cannot be assessed directly, our findings nevertheless show that an HTLV-1 vaccine approach is feasible and deserves further study.
Both
Lymantria dispar
Nucleopolyhedrovirus
Enhancin
Genes Contribute to Viral Potency
Authors: Popham Holly J. R. , Bischoff David S. , Slavicek James M.
Published: July 2002

Enhancins are a group of proteins first identified in granuloviruses (GV) that have the ability to enhance nuclear polyhedrosis virus potency. We had previously identified an
enhancin
gene (E1) in the
Lymantria dispar
multinucleocapsid nucleopolyhedrovirus (Ld
M
NPV) (D. S. Bischoff and J. M. Slavicek, J. Virol. 71:8133–8140, 1997). Inactivation of the
E1
gene product within the viral genome lowered viral potency by an average of 2.9-fold. A second
enhancin
gene (E2) was identified when the entire genome of Ld
M
NPV was sequenced (Kuzio et al., Virology 253:17–34, 1999). The E2 protein exhibits approximately 30% amino acid identity to the Ld
M
NPV E1 protein as well as the enhancins from
Trichoplusia ni
GV,
Pseudaletia unipuncta
GV,
Helicoverpa armigera
GV, and
Xestia c-nigrum
GV. Northern analysis of viral RNA indicated that the
E2
gene transcripts are expressed at late times postinfection from a consensus baculovirus late promoter. The effect of the enhancin proteins on viral potency was investigated through bioassay using two recombinant viruses, one with a deletion in the
E2
gene (E2del) and a second with deletion mutations in both
enhancin
genes (E1delE2del). The
enhancin
gene viral constructs were verified by Southern analysis and shown not to produce
enhancin
gene transcripts by Northern analysis. The E2del virus exhibited an average decrease in viral potency of 1.8-fold compared to wild-type virus. In the same bioassays, the recombinant virus E1cat, which does not produce an
E1
gene transcript, exhibited an average decrease in viral potency of 2.3-fold compared to control virus. The E1delE2del virus exhibited an average decrease in viral potency of 12-fold compared to wild-type virus. Collectively, these results suggest that both Ld
M
NPV
enhancin
genes contribute to viral potency, that each enhancin protein can partially compensate for the lack of the other protein, and that both enhancin genes are necessary for wild-type viral potency.
Adenovirus Protein V Induces Redistribution of Nucleolin and B23 from Nucleolus to Cytoplasm
Authors: Matthews David A.
Published: July 2002

Adenovirus infection inhibits synthesis and processing of rRNA and redistributes nucleolar antigens. Adenovirus protein V associates with nucleoli in infected cells. This study delineates regions of protein V independently capable of nucleolar targeting. Also, evidence is presented that protein V has the unique property of relocating nucleolin and B23 to the cytoplasm when transiently expressed on its own in uninfected cells. Point mutation analysis indicates a role for the C terminus of protein V in the redirection of nucleolin and B23 to the cytoplasm. This is the first time an adenovirus protein has been shown to have a direct effect on nucleolar antigens in isolation from viral infection. Moreover, adenovirus protein V is the first protein demonstrated to be capable of redirecting nucleolin and B23 to the cytoplasm.
Central Role of Hemocytes in
Autographa californica M
Nucleopolyhedrovirus Pathogenesis in
Heliothis virescens
and
Helicoverpa zea
Authors: Trudeau Dominique , Washburn Jan O. , Volkman Loy E.
Published: July 2002

Autographa californica M
nucleopolyhedrovirus (Ac
M
NPV) can infect and kill a wide range of larval lepidopteran hosts, but the dosage required to achieve mortal infection varies greatly. Using a reporter gene construct, we identified key differences between Ac
M
NPV pathogenesis in
Heliothis virescens
and
Helicoverpa zea
, a fully permissive and a semipermissive host, respectively. Even though there was more than a 1,000-fold difference in the susceptibilities of these two species to mortal infection, there was no significant difference in their susceptibilities to primary infections in the midgut or secondary infections in the tracheal epidermis. Foci of infection within the tracheal epidermis of
H. zea
, however, were melanized and encapsulated by 48 h after oral inoculation, a host response not observed in
H. virescens
. Further,
H. zea
hemocytes, unlike those of
H. virescens
, were highly resistant to Ac
M
NPV infection; reporter gene expression was observed only rarely even though virus was taken up readily, and nucleocapsids were transported to the nucleus. Collectively, these results demonstrated that hemocytes—by removing virus from the hemolymph instead of amplifying it and by participating in the encapsulation of infection foci—together with the host's melanization response, formed the basis of
H. zea
's resistance to fatal infection by Ac
M
NPV.
Functional Roles of Equine Infectious Anemia Virus Gag p9 in Viral Budding and Infection
Authors: Chen Chaoping , Li Feng , Montelaro Ronald C.
Published: July 2002

Previous studies utilizing Gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (EIAV) Gag p9 protein provides a late assembly function mediated by a critical Y
23
P
24
D
25
L
26
motif (L-domain) to release viral particles from the plasma membrane. To elucidate further the role of EIAV p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of a reference infectious molecular proviral clone, EIAV
uk
. Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K
30
K
31
motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAV
uk
in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication.
Alternative Mechanisms of Respiratory Syncytial Virus Clearance in Perforin Knockout Mice Lead to Enhanced Disease
Authors: Aung Sandra , Rutigliano John A. , Graham Barney S.
Published: July 2002

Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzyme-mediated lysis of infected cells is thought to be the major molecular mechanism used by CD8+cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.
African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis
Authors: Vallée Isabelle , Tait Stephen W. G. , Powell Penelope P.
Published: July 2002

African swine fever (ASF) is an asymptomatic infection of warthogs and bushpigs, which has become an emergent disease of domestic pigs, characterized by hemorrhage, lymphopenia, and disseminated intravascular coagulation. It is caused by a large icosohedral double-stranded DNA virus, African swine fever virus (ASFV), with infection of macrophages well characterized in vitro and in vivo. This study shows that virulent isolates of ASFV also infect primary cultures of porcine aortic endothelial cells and bushpig endothelial cells (BPECs) in vitro. Kinetics of early and late gene expression, viral factory formation, replication, and secretion were similar in endothelial cells and macrophages. However, ASFV-infected endothelial cells died by apoptosis, detected morphologically by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and nuclear condensation and biochemically by poly(ADP-ribose) polymerase (PARP) cleavage at 4 h postinfection (hpi). Immediate-early proinflammatory responses were inhibited, characterized by a lack of E-selectin surface expression and interleukin 6 (IL-6) and IL-8 mRNA synthesis. Moreover, ASFV actively downregulated interferon-induced major histocompatibility complex class I surface expression, a strategy by which viruses evade the immune system. Significantly, Western blot analysis showed that the 65-kDa subunit of the transcription factor NF-κB, a central regulator of the early response to viral infection, decreased by 8 hpi and disappeared by 18 hpi. Both disappearance of NF-κB p65 and cleavage of PARP were reversed by the caspase inhibitor z-VAD-fmk. Interestingly, surface expression and mRNA transcription of tissue factor, an important initiator of the coagulation cascade, increased 4 h after ASFV infection. These data suggest a central role for vascular endothelial cells in the hemorrhagic pathogenesis of the disease. Since BPECs infected with ASFV also undergo apoptosis, resistance of the natural host must involve complex pathological factors other than viral tropism.
Molecular Determinants of Peptide Binding to Two Common Rhesus Macaque Major Histocompatibility Complex Class II Molecules
Authors: Dzuris John L. , Sidney John , Horton Helen , Correa Rose , Carter Donald , Chesnut Robert W. , Watkins David I. , Sette Alessandro
Published: July 2002

Major histocompatibility complex class II molecules encoded by two common rhesus macaque alleles Mamu-DRB1*0406 and Mamu-DRB*w201 have been purified, and quantitative binding assays have been established. The structural requirements for peptide binding to each molecule were characterized by testing panels of single-substitution analogs of the two previously defined epitopes HIV Env242 (Mamu-DRB1*0406 restricted) and HIV Env482 (Mamu-DRB*w201 restricted). Anchor positions of both macaque DR molecules were spaced following a position 1 (P1), P4, P6, P7, and P9 pattern. The specific binding motif associated with each molecule was distinct, but largely overlapping, and was based on crucial roles of aromatic and/or hydrophobic residues at P1, P6, and P9. Based on these results, a tentative Mamu class II DR supermotif was defined. This pattern is remarkably similar to a previously defined human HLA-DR supermotif. Similarities in binding motifs between human HLA and macaque Mamu-DR molecules were further illustrated by testing a panel of more than 60 different single-substitution analogs of the HLA-DR-restricted HA 307–319 epitope for binding to Mamu-DRB*w201 and HLA-DRB1*0101. The Mamu-DRB1*0406 and -DRB*w201 binding capacity of a set of 311 overlapping peptides spanning the entire simian immunodeficiency virus (SIV) genome was also evaluated. Ten peptides capable of binding both molecules were identified, together with 19 DRB1*0406 and 43 DRB*w201 selective binders. The Mamu-DR supermotif was found to be present in about 75% of the good binders and in 50% of peptides binding with intermediate affinity but only in approximately 25% of the peptides which did not bind either Mamu class II molecule. Finally, using flow cytometric detection of antigen-induced intracellular gamma interferon, we identify a new CD4+T-lymphocyte epitope encoded within the Rev protein of SIV.
Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60
Authors: Parissi Vincent , Calmels Christina , De Soultrait Vaea Richard , Caumont Anne , Fournier Michel , Chaignepain Stéphane , Litvak Simon
Published: July 2002

Integration of human immunodeficiency virus type 1 (HIV-1) proviral DNA in the nuclear genome is catalyzed by the retroviral integrase (IN). In addition to IN, viral and cellular proteins associated in the high-molecular-weight preintegration complex have been suggested to be involved in this process. In an attempt to define host factors interacting with IN, we used an in vitro system to identify cellular proteins in interaction with HIV-1 IN. The yeast
Saccharomyces cerevisiae
was chosen since (i) its complete sequence has been established and the primary structure of all the putative proteins from this eucaryote has been deduced, (ii) there is a significant degree of homology between human and yeast proteins, and (iii) we have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype. Strong evidences suggest that this lethality is linked to IN activity in infected human cells where integration requires the cleavage of genomic DNA. Using IN-affinity chromatography we identified four yeast proteins interacting with HIV-1 IN, including the yeast chaperonin yHSP60, which is the counterpart of human hHSP60. Yeast lethality induced by HIV-1 IN was abolished when a mutated HSP60 was coexpressed, therefore suggesting that both proteins interact in vivo. Besides interacting with HIV-1 IN, the hHSP60 was able to stimulate the in vitro processing and joining activities of IN and protected this enzyme from thermal denaturation. In addition, the functional human HSP60-HSP10 complex in the presence of ATP was able to recognize the HIV-1 IN as a substrate.
Amino Acids Responsible for the Absolute Sialidase Activity of the Influenza A Virus Neuraminidase: Relationship to Growth in the Duck Intestine
Authors: Kobasa Darwyn , Wells Krisna , Kawaoka Yoshihiro
Published: July 2002

The 1957 human pandemic strain of influenza A virus contained an avian virus hemagglutinin (HA) and neuraminidase (NA), both of which acquired specificity for the human receptor,
N
-acetylneuraminic acid linked to galactose of cellular glycoconjugates via an α2-6 bond (NeuAcα2-6Gal). Although the NA retained considerable specificity for NeuAcα2-3Gal, its original substrate in ducks, it lost the ability to support viral growth in the duck intestine, suggesting a growth-restrictive change other than a shift in substrate specificity. To test this possibility, we generated a panel of reassortant viruses that expressed the NA genes of human H2N2 viruses isolated from 1957 to 1968 with all other genes from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of A/Singapore/1/57 supported efficient viral growth in the intestines of orally inoculated ducks. The growth-supporting capacity of the NA correlated with a high level of enzymatic activity, comparable to that found to be associated with avian virus NAs. The specific activities of the A/Ann Arbor/6/60 and A/England/12/62 NAs, which showed greatly restricted abilities to support viral growth in ducks, were only 8 and 5%, respectively, of the NA specific activity for A/Singapore/1/57. Using chimeric constructs based on A/Singapore/1/57 and A/England/12/62 NAs, we localized the determinants of high specific NA activity to a region containing six amino acid substitutions in A/England/12/62: Ser331→Arg, Asp339→Asn, Asn367→Ser, Ser370→Leu, Asn400→Ser, and Pro431→Glu. Five of these six residues (excluding Asn400) were required and sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in addition to a change in substrate specificity, a reduction in high specific activity may be required for the adaptation of avian virus NAs to growth in humans. This change is likely needed to maintain an optimal balance between NA activity and the lower affinity shown by human virus HAs for their cellular receptor.
Adenovirus Serotype 7 Retention in a Late Endosomal Compartment prior to Cytosol Escape Is Modulated by Fiber Protein
Authors: Miyazawa Naoki , Crystal Ronald G. , Leopold Philip L.
Published: July 2002

The intracellular trafficking of adenovirus (Ad) subgroup B (e.g., Ad7) differs from that of subgroup C (e.g., Ad5) in that Ad5 rapidly escapes from endocytic compartments following infection whereas Ad7 accumulates in organelles. To assess the hypothesis that Ad7 is targeted to the lysosomal pathway, Ad7 and Ad5 were conjugated with fluorophores and their trafficking in A549 epithelial cells was analyzed by fluorescence microscopy. Within 1 h after infection, Ad7, but not Ad5, accumulated in the cytoplasm of A549 cells. The pH in the environment of Ad5 was nearly neutral (pH 7), while Ad7 occupied acidic compartments (pH 5) over the first 2 h with a gradual shift toward neutrality by 8 h. Ad7 partially colocalized with α
2
-macroglobulin and late endosomal and lysosomal marker proteins, including Rab7, mannose-6-phosphate receptor, and LAMP-1. The pH optimum for membrane lysis by Ad7, as well as a chimeric Ad5 capsid that expressed the Ad7 fiber (Ad5fiber7), was pH 5.5, while that for lysis by Ad5 was pH 6.0. Thus, the native trafficking pathway for Ad7 involves residence in late endosomes and lysosomes, with information encoded in the Ad7 fiber acting as a pH-dependent trigger for membrane lysis and escape to the cytosol.
GLI-2 Modulates Retroviral Gene Expression
Authors: Smith Michael J. , Gitlin Scott D. , Browning Catherine M. , Lane Brian R. , Clark Nina M. , Shah Nilesh , Rainier Shirley , Markovitz David M.
Published: July 2002

GLI proteins are involved in the development of mice, humans, zebrafish,
Caenorhabditis elegans, Xenopus
, and
Drosophila
. While these zinc finger-containing proteins bind to TG-rich promoter elements and are known to regulate gene expression in
C. elegans
and
Drosophila
, mechanistic understanding of how regulation is mediated through naturally occurring transcriptional promoters is lacking. One isoform of human GLI-2 appears to be identical to a factor previously called Tax helper protein (THP), thus named due to its ability to interact with a TG-rich element in the human T-lymphotropic virus type 1 (HTLV-1) enhancer thought to mediate transcriptional stimulation by the Tax protein of HTLV-1. We now demonstrate that, working through its TG-rich binding site and adjacent elements, GLI-2/THP actually suppresses gene expression driven by the HTLV-1 promoter. GLI-2/THP has no effect on the HTLV-2 promoter, activates expression from the promoters of human immunodeficiency virus types 1 and (HIV-1 and -2), and stimulates HIV-1 replication. Both effective suppression and activation of gene expression and viral replication require the first of the five zinc fingers, which is not necessary for DNA binding, to be intact. Thus, not only can GLI-2/THP either activate or suppress gene expression, depending on the promoter, but the same domain (first zinc finger) mediates both effects. These findings suggest a role for GLI-2 in retroviral gene regulation and shed further light on the mechanisms by which GLI proteins regulate naturally occurring promoters.
Human Immunodeficiency Virus Type 1 Nef-Induced CD4 Cell Surface Downregulation Is Inhibited by Ikarugamycin
Authors: Luo Tianci , Fredericksen Brenda L. , Hasumi Keiji , Endo Akira , Garcia J. Victor
Published: July 2002

One well-characterized in vitro function of Nef is its ability to remove CD4, the human immunodeficiency virus (HIV) receptor, from the cell surface. Nef accomplishes this by accelerating the internalization and degradation of CD4. Current models propose that Nef promotes CD4 internalization via an increased association of CD4 with clathrin-coated pits (CCP). Here, we investigated the effect of a naturally occurring antiprotozoan antibiotic, ikarugamycin (IKA), on CD4 cell surface expression in human monocytic cells stably expressing HIV type 1 SF2 Nef. IKA was able to efficiently restore CD4 cell surface expression in Nef-expressing cells without affecting either CD4 synthesis or Nef expression. In addition, we demonstrate that IKA is also capable of efficiently blocking CD4 down-modulation in response to phorbol myristate acetate. Our data suggest that IKA may be an efficient and useful inhibitor of CCP-dependent endocytosis.
Bacteriophage K1-5 Encodes Two Different Tail Fiber Proteins, Allowing It To Infect and Replicate on both K1 and K5 Strains of
Escherichia coli
Authors: Scholl Dean , Rogers Scott , Adhya Sankar , Merril Carl R.
Published: July 2002

A virulent double-stranded DNA bacteriophage, ΦK1-5, has been isolated and found to be capable of infecting
Escherichia coli
strains that possess either the K1 or the K5 polysaccharide capsule. Electron micrographs show that the virion consists of a small icosohedral head with short tail spikes, similar to members of the
Podoviridae
family. DNA sequence analysis of the region encoding the tail fiber protein showed two open reading frames encoding previously characterized hydrolytic phage tail fiber proteins. The first is the K5 lyase protein gene of ΦK5, which allows this phage to specifically infect K5
E. coli
strains. A second open reading frame encodes a protein almost identical in amino acid sequence to the
N
-acetylneuraminidase (endosialidase) protein of ΦK1E, which allows this phage to specifically infect K1 strains of
E. coli
. We provide experimental evidence that mature phage particles contain both tail fiber proteins, and mutational analysis indicates that each protein can be independently inactivated. A comparison of the tail gene regions of ΦK5, ΦK1E, and ΦK1-5 shows that the genes are arranged in a modular or cassette configuration and suggests that this family of phages can broaden host range by horizontal gene transfer.
Deleterious Effects of Hepatitis Delta Virus Replication on Host Cell Proliferation
Authors: Wang David , Pearlberg Joseph , Liu Yu-Tsueng , Ganem Don
Published: July 2002

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.
Jaagsiekte Sheep Retrovirus Proviral Clone JSRV
JS7
, Derived from the JS7 Lung Tumor Cell Line, Induces Ovine Pulmonary Carcinoma and Is Integrated into the Surfactant Protein A Gene
Authors: DeMartini James C. , Bishop Jeanette V. , Allen Thomas E. , Jassim F. A. , Sharp J. Michael , de las Heras Marcelo , Voelker Dennis R. , Carlson Jonathan O.
Published: July 2002

Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRV
JS7
) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRV
JS7
is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.
Open Reading Frame UL26 of Human Cytomegalovirus Encodes a Novel Tegument Protein That Contains a Strong Transcriptional Activation Domain
Authors: Stamminger Thomas , Gstaiger Matthias , Weinzierl Konstanze , Lorz Kerstin , Winkler Michael , Schaffner Walter
Published: July 2002

A selection strategy, the activator trap, was used in order to identify genes of human cytomegalovirus (HCMV) that encode strong transcriptional activation domains in mammalian cells. This approach is based on the isolation of activation domains from a GAL4 fusion library by means of selective plasmid replication, which is mediated in transfected cells by a GAL4-inducible T antigen gene. With this screening strategy, we were able to isolate two types of plasmids encoding transactivating fusion proteins from a library of random HCMV DNA inserts. One plasmid contained the exon 3 of the HCMV IE-1/2 gene region, which has previously been identified as a strong transcriptional activation domain. In the second type of plasmid, the open reading frame (ORF) UL26 of HCMV was fused to the GAL4 DNA-binding domain. By quantitative RNA mapping using S1 nuclease analysis, we were able to classify UL26 as a strong enhancer-type activation domain with no apparent homology to characterized transcriptional activators. Western blot analysis with a specific polyclonal antibody raised against a prokaryotic UL26 fusion protein revealed that two protein isoforms of 21 and 27 kDa are derived from the UL26 ORF in both infected and transfected cells. Both protein isoforms, which arise via alternative usage of two in-frame translational start codons, showed a nuclear localization and could be detected as early as 6 h after infection of primary human fibroblasts. By performing Western blot analysis with purified virions combined with fractionation experiments, we provide evidence that pUL26 is a novel tegument protein of HCMV that is imported during viral infection. Furthermore, we observed transactivation of the HCMV major immediate-early enhancer-promoter by pUL26, whereas several early and late promoters were not affected. Our data suggest that pUL26 is a novel tegument protein of HCMV with a strong transcriptional activation domain that could play an important role during initiation of the viral replicative cycle.
Cowpea Mosaic Virus 32- and 60-Kilodalton Replication Proteins Target and Change the Morphology of Endoplasmic Reticulum Membranes
Authors: Carette Jan E. , van Lent Jan , MacFarlane Stuart A. , Wellink Joan , van Kammen Ab
Published: July 2002

Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have expressed individual CPMV proteins encoded by RNA1 in cowpea protoplasts by transient expression and in
Nicotiana benthamiana
plants by using the tobacco rattle virus (TRV) expression vector. The 32-kDa protein (32K) and 60K, when expressed individually, accumulate in only low amounts but are found associated with membranes mainly derived from the endoplasmic reticulum (ER). 24K and 110K are freely soluble and accumulate to high levels. With the TRV vector, expression of 32K and 60K results in rearrangement of ER membranes. Besides, expression of 32K and 60K results in necrosis of the inoculated
N. benthamiana
leaves, suggesting that 32K and 60K are cytotoxic proteins. On the other hand, during CPMV infection 32K and 60K accumulate to high levels without causing necrosis.
Molecular Characterization of VP6 Genes of Human Rotavirus Isolates: Correlation of Genogroups with Subgroups and Evidence of Independent Segregation
Authors: Iturriza Gómara Miren , Wong Cecilia , Blome Sandra , Desselberger Ulrich , Gray Jim
Published: July 2002

A reverse transcription-PCR (RT-PCR) was established to amplify a 379-bp cDNA fragment (nucleotides 747 to 1126, coding for amino acids 241 to 367) of the VP6 gene of group A rotaviruses associated with subgroup (SG) specificity. Thirty-eight human rotavirus strains characterized with SG-specific monoclonal antibodies were subjected to VP6-specific RT-PCR, and PCR amplicons were used for sequencing. Nucleic acid sequencing and phylogenetic analysis of the VP6 amplicons revealed two clusters, or genogroups. Two genetic lineages were distinguished within genogroup I, consisting of strains serologically characterized as SG I, and three genetic lineages were distinguished within genogroup II, composed of strains serologically characterized as SG II, SG I + II, and SG non-I, non-II. Subgrouping of rotaviruses by means of serological methods may result in strains not being assigned the correct SG or in a failure of strains to subgroup. Molecular characterization of the SG-defining region of VP6 provided evidence for independent segregation of the rotavirus genes encoding VP4, VP6, and VP7.
Administration of Recombinant Rhesus Interleukin-12 during Acute Simian Immunodeficiency Virus (SIV) Infection Leads to Decreased Viral Loads Associated with Prolonged Survival in SIVmac251-Infected Rhesus Macaques
Authors: Ansari A. A. , Mayne A. E. , Sundstrom J. B. , Bostik P. , Grimm B. , Altman J. D. , Villinger F.
Published: July 2002

The ability of recombinant rhesus interleukin-12 (rMamu-IL-12) administration during acute simian immunodeficiency virus SIVmac251 infection to influence the quality of the antiviral immune responses was assessed in rhesus macaques. Group I (n= 4) was the virus-only control group. Group II and III received a conditioning regimen of rMamu-IL-12 (10 and 20 μg/kg, respectively, subcutaneously [s.c.]) on days −2 and 0. Thereafter, group II received 2 μg of IL-12 per kg and group III received 10 μg/kg s.c. twice a week for 8 weeks. On day 0 all animals were infected with SIVmac251 intravenously. While all four group I animals and three of four group II animals died by 8 and 10 months post infection (p.i.), all four group III animals remained alive for >20 months p.i. The higher IL-12 dose led to lower plasma viral loads and markedly lower peripheral blood mononuclear cell and lymph node proviral DNA loads. During the acute viremia phase, the high-IL-12-dose monkeys showed an increase in CD3−CD8α/α+and CD3+CD8 α/α+cells and, unlike the control and low-IL-12-dose animals, did not demonstrate an increase in CD4+CD45RA+CD62L+naive cells. The high-IL-12-dose animals also demonstrated that both CD8α/α+and CD8α/β+cells produced antiviral factors early p.i., whereas only CD8α/β+cells retained this function late p.i. Long-term survival correlated with sustained high levels of SIVgag/poland SIVenvcytotoxic T lymphocytes and retention of high memory responses against nominal antigens. This is the first study to demonstrate the capacity of IL-12 to significantly protect macaques from SIV-induced disease, and it provides a useful model to more precisely identify correlates of virus-specific disease-protective responses.
Identification of Temperature-Sensitive Mutations in the Phosphoprotein of Respiratory Syncytial Virus That Are Likely Involved in Its Interaction with the Nucleoprotein
Authors: Lu Bin , Brazas Robert , Ma Chien-Hui , Kristoff Tina , Cheng Xing , Jin Hong
Published: July 2002

The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37°C in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39°C. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as wild-type A2 virus in both Vero and HEp-2 cells at 33°C, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37°C. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37°C, the E176G mutation in rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.
Mapping of Genes Involved in Murine Herpes Simplex Virus Keratitis: Identification of Genes and Their Modifiers
Authors: Norose Kazumi , Yano Akihiko , Zhang Xiang-Ming , Blankenhorn Elizabeth , Heber-Katz Ellen
Published: July 2002

Herpes simplex keratitis (HSK) is an inflammatory response to viral infection and self antigens in the cornea and is a major cause of blindness. Using two strains of mice which are susceptible (129/SVEV) and resistant (C57BL/6) to herpes simplex virus (HSV) strain KOS, (129/SVEV × C57BL/6)F2mice were generated and examined for their disease susceptibility in terms of clinical symptoms, ocular disease, and antibody production following corneal scarification with HSV (KOS). A genome-wide screen was carried out using microsatellite markers to determine the genetic loci involved in this response. Loci on chromosomes 4, 5, 12, 13, and 14 were shown to be involved in general susceptibility to clinical disease, whereas loci on chromosomes 10 and 17 were shown to be unique to ocular disease.