bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
Autographa californica
Multiple Nucleopolyhedrovirus
ie0
-
ie1
Gene Complex Is Essential for Wild-Type Virus Replication, but either IE0 or IE1 Can Support Virus Growth
Authors: Stewart Taryn M. , Huijskens Ilse , Willis Leslie G. , Theilmann David A. 
Published: March 2005

ABSTRACT

The immediate-early
ie0
-
ie1
gene complex expresses the only baculovirus spliced gene that produces an alternate protein product.
Autographa californica
multiple nucleopolyhedrovirus (AcMNPV) IE1 is a potent transcriptional transactivator that is essential for viral replication in transient assays. IE1 contains 582 amino acids that are arranged into different domains, including an acidic activation domain at the N terminus, a DNA binding domain, and an oligomerization domain at the C terminus. IE0 is a 52-amino-acid N-terminally elongated form of IE1. We investigated the functions of IE0 and IE1 in virus-infected cells by constructing the first
ie1
open reading frame knockout virus. An infectious AcMNPV bacmid was used to generate the
ie1
knockout, and the resulting virus, AcBacIE1KO, effectively deletes both
ie0
and
ie1
. AcBacIE1KO does not infect
Spodoptera frugiperda
cells, showing that the
ie0
-
ie1
gene complex is essential for viral infection. Rescue viruses of AcBacIE1KO were constructed that express only IE1, IE1 and IE0, or only IE0. Our results show that both IE0 and IE1 can function independently, but not equivalently, to support replication, producing infectious virus. Viruses expressing predominately, or only, IE0 produced significantly fewer cells with polyhedra than either the IE1 counterpart or wild-type virus. In addition, DNA replication was prolonged and budded virus and late gene expression were delayed. Viruses expressing only IE1 also produced fewer polyhedra, but replication was slightly faster and achieved higher levels than that of the wild-type virus. Both IE0 and IE1 are therefore required and must be expressed in the correct quantitative ratios to achieve a wild-type infection.
Requirements at the 3′ End of the
Sindbis Virus
Genome for Efficient Synthesis of Minus-Strand RNA
Authors: Hardy Richard W. , Rice Charles M. 
Published: March 2005

ABSTRACT

The 3′-untranslated region of the
Sindbis virus
genome is 0.3 kb in length with a 19-nucleotide conserved sequence element (3′ CSE) immediately preceding the 3′-poly(A) tail. The 3′ CSE and poly(A) tail have been assumed to constitute the core promoter for minus-strand RNA synthesis during genome replication; however, their involvement in this process has not been formally demonstrated. Utilizing both in vitro and in vivo analyses, we have examined the role of these elements in the initiation of minus-strand RNA synthesis. The major findings of this study with regard to efficient minus-strand RNA synthesis are the following: (i) the wild-type 3′ CSE and the poly(A) tail are required, (ii) the poly(A) tail must be a minimum of 11 to 12 residues in length and immediately follow the 3′ CSE, (iii) deletion or substitution of the 3′ 13 nucleotides of the 3′ CSE severely inhibits minus-strand RNA synthesis, (iv) templates possessing non-wild-type 3′ sequences previously demonstrated to support virus replication do not program efficient RNA synthesis, and (v) insertion of uridylate residues between the poly(A) tail and a non-wild-type 3′ sequence can restore promoter function to a limited extent. This study shows that the optimal structure of the 3′ component of the minus-strand promoter is the wild-type 3′ CSE followed a poly(A) tail of at least 11 residues. Our findings also show that insertion of nontemplated bases can restore function to an inactive promoter.
Inhibition of Epstein-Barr Virus OriP Function by Tankyrase, a Telomere-Associated Poly-ADP Ribose Polymerase That Binds and Modifies EBNA1
Authors: Deng Zhong , Atanasiu Constandache , Zhao Kehao , Marmorstein Ronen , Sbodio Juan I. , Chi Nai-Wen , Lieberman Paul M. 
Published: March 2005

ABSTRACT
Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.
Activation of CD21 and CD23 Gene Expression by Kaposi's Sarcoma-Associated Herpesvirus RTA
Authors: Chang Heesoon , Gwack Yousang , Kingston Dior , Souvlis John , Liang Xiaozhen , Means Robert E. , Cesarman Ethel , Hutt-Fletcher Lindsey , Jung Jae U. 
Published: March 2005

ABSTRACTEpstein-Barr virus (EBV) EBNA2 and Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) are recruited to their responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jκ. In particular, RTA and EBNA2 interactions with RBP-Jκ are essential for the lytic replication of KSHV and expression of B-cell activation markers CD21 and CD23a, respectively. Here, we demonstrate that like EBV EBNA2, KSHV RTA strongly induces CD21 and CD23a expression through RBP-Jκ binding sites in the first intron of CD21 and in the CD23a core promoter, respectively. However, unlike EBV EBNA2, which alters immunoglobulin μ (Igμ) and c-mycgene expression, RTA did not affect Igμ and c-mycexpression, indicating that KSHV RTA targets the Notch signal transduction pathway in a manner similar to but distinct from that of EBV EBNA2. Furthermore, RTA-induced expression of CD21 glycoprotein, which is an EBV receptor, efficiently facilitated EBV infection. In addition, RTA-induced CD23 glycoprotein underwent proteolysis and gave rise to soluble CD23 (sCD23) molecules in B lymphocytes and KSHV-infected primary effusion lymphocytes. sCD23 then stimulated primary human lymphocytes. These results demonstrate that cellular CD21 and CD23a are common targets for B lymphotropic gammaherpesviruses and that KSHV RTA regulates RBP-Jκ-mediated cellular gene expression, which ultimately provides a favorable milieu for viral reproduction in the infected host.
Precise Identification of Endogenous Proviruses of NFS/N Mice Participating in Recombination with Moloney Ecotropic Murine Leukemia Virus (MuLV) To Generate Polytropic MuLVs
Authors: Alamgir A. S. M. , Owens Nick , Lavignon Marc , Malik Frank , Evans Leonard H. 
Published: March 2005

ABSTRACT

Polytropic murine leukemia viruses (MuLVs) are generated by recombination of ecotropic MuLVs with
env
genes of a family of endogenous proviruses in mice, resulting in viruses with an expanded host range and greater virulence. Inbred mouse strains contain numerous endogenous proviruses that are potential donors of the
env
gene sequences of polytropic MuLVs; however, the precise identification of those proviruses that participate in recombination has been elusive. Three different structural groups of proviruses in NFS/N mice have been described and different ecotropic MuLVs preferentially recombine with different groups of proviruses. In contrast to other ecotropic MuLVs such as Friend MuLV or Akv that recombine predominantly with a single group of proviruses, Moloney MuLV (M-MuLV) recombines with at least two distinct groups. In this study, we determined that only three endogenous proviruses, two of one group and one of another group, are major participants in recombination with M-MuLV. Furthermore, the distinction between the polytropic MuLVs generated by M-MuLV and other ecotropic MuLVs is the result of recombination with a single endogenous provirus. This provirus exhibits a frameshift mutation in the 3′ region of the surface glycoprotein-encoding sequences that is excluded in recombinants with M-MuLV. The sites of recombination between the
env
genes of M-MuLV and endogenous proviruses were confined to a short region exhibiting maximum homology between the ecotropic and polytropic
env
sequences and maximum stability of predicted RNA secondary structure. These observations suggest a possible mechanism for the specificity of recombination observed for different ecotropic MuLVs.
Novel Rabies Virus-Neutralizing Epitope Recognized by Human Monoclonal Antibody: Fine Mapping and Escape Mutant Analysis
Authors: Marissen Wilfred E. , Kramer R. Arjen , Rice Amy , Weldon William C. , Niezgoda Michael , Faber Milosz , Slootstra Jerry W. , Meloen Rob H. , Clijsters-van der Horst Marieke , Visser Therese J. , Jongeneelen Mandy , Thijsse Sandra , Throsby Mark , de Kruif John , Rupprecht Charles E. , Dietzschold Bernhard , Goudsmit Jaap , Bakker Alexander B. H. 
Published: March 2005

ABSTRACTAnti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.
Expression of CD154 by a Simian Immunodeficiency Virus Vector Induces Only Transitory Changes in Rhesus Macaques
Authors: Hodara Vida L. , Velasquillo M. Cristina , Parodi Laura M. , Giavedoni Luis D. 
Published: March 2005

ABSTRACTHuman immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an activenefgene, our results suggest that, in CD4+T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154.
A Substitution in Rous Sarcoma Virus Integrase That Separates Its Two Biologically Relevant Enzymatic Activities
Authors: Konsavage Wesley M. , Burkholder Stephen , Sudol Malgorzata , Harper Amy L. , Katzman Michael 
Published: March 2005

ABSTRACT

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn
2+
. We now report that specificity for the biologically relevant “−2” site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of −2 nicks to −3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn
2+
. Even with Mg
2+
, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn
2+
and joining was undetectable in reactions with Mg
2+
. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.
Virus-Specific and Bystander CD8 T Cells Recruited during Virus-Induced Encephalomyelitis
Authors: Chen Audrey M. , Khanna Nivedita , Stohlman Stephen A. , Bergmann Cornelia C. 
Published: March 2005

ABSTRACTNeurotropic coronavirus-induced encephalitis was used to evaluate recruitment, functional activation, and retention of peripheral bystander memory CD8+T cells. Mice were first infected with recombinant vaccinia virus expressing a non-cross-reactive human immunodeficiency virus (HIV) epitope, designated p18. Following establishment of an endogenous p18-specific memory CD8+T-cell population, mice were challenged with coronavirus to directly compare recruitment, longevity, and activation characteristics of both primary coronavirus-specific and bystander memory populations trafficking into the central nervous system (CNS). HIV-specific memory CD8+T cells were recruited early into the CNS as components of the innate immune response, preceding CD8+T cells specific for the dominant coronavirus epitope, designated pN. Although pN-specific T-cell numbers gradually exceeded bystander p18-specific CD8+T-cell numbers, both populations peaked concurrently within the CNS. Nevertheless, coronavirus-specific CD8+T cells were preferentially retained. By contrast, bystander CD8+T-cell numbers declined to background numbers following control of CNS virus replication. Furthermore, in contrast to highly activated pN-specific CD8+T cells, bystander p18-specific CD8+T cells recruited to the site of inflammation maintained a nonactivated memory phenotype and did not express ex vivo cytolytic activity. Therefore, analysis of host CD8+T-cell responses to unrelated infections demonstrates that bystander memory CD8+T cells can comprise a significant proportion of CNS inflammatory cells during virus-induced encephalitis. However, transient CNS retention and the absence of activation suggest that memory bystander CD8+T cells may not overtly contribute to pathology in the absence of antigen recognition.
Association of Ebola Virus Matrix Protein VP40 with Microtubules
Authors: Ruthel Gordon , Demmin Gretchen L. , Kallstrom George , Javid Melodi P. , Badie Shirin S. , Will Amy B. , Nelle Timothy , Schokman Rowena , Nguyen Tam L. , Carra John H. , Bavari Sina , Aman M. Javad 
Published: March 2005

ABSTRACT
Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.
Role of the Specific Amino Acid Sequence of the Membrane-Spanning Domain of Human Immunodeficiency Virus Type 1 in Membrane Fusion
Authors: Miyauchi Kosuke , Komano Jun , Yokomaku Yoshiyuki , Sugiura Wataru , Yamamoto Naoki , Matsuda Zene 
Published: March 2005

ABSTRACTFusion between cell and virus membranes mediated by gp41 initiates the life cycle of human immunodeficiency virus type 1. In contrast to the many studies that have elucidated the structure-function relationship of the ectodomain, the study of the membrane-spanning domain (MSD) has been rather limited. In particular, the role that the MSD's specific amino acid sequences may have in membrane fusion as well as other gp41 functions is not well understood. The MSD of gp41 contains well-conserved glycine residues that form the GXXXG motif (G, glycine; X, other amino acid residues), a motif often found at the helix-helix interface of membrane spanning α-helices. Here we examined the role that the specific amino acid sequence of the gp41 MSD has in gp41 function, particularly in membrane fusion, by making two types of MSD mutants: (i) glycine substitution mutants in which glycine residues of the MSD were mutated to alanine or leucine residues, and (ii) replacement mutants in which the entire MSD was replaced with one derived from glycophorin A or from vesicular stomatitis virus G. The substitution of glycines did not affect gp41 function. MSD-replacement mutants, however, showed severely impaired fusion activity. The assay using the Env expression vector revealed defects in membrane fusion after CD4 binding steps in the MSD-replacement mutants. In addition, the change in Env processing was noted for MSD-replacement mutants. These results suggest that the MSD of gp41 has a relatively wide but not unlimited tolerance for mutations and plays a critical role in membrane fusion as well as in other steps of Env biogenesis.
Nuclear Localizations of the Herpes Simplex Virus Type 1 Tegument Proteins VP13/14, vhs, and VP16 Precede VP22-Dependent Microtubule Reorganization and VP22 Nuclear Import
Authors: Yedowitz Jamie C. , Kotsakis Anna , Schlegel Elisabeth F. M. , Blaho John A. 
Published: March 2005

ABSTRACT

Herpes simplex virus type 1 (HSV-1) induces microtubule reorganization beginning at approximately 9 h postinfection (hpi), and this correlates with the nuclear localization of the tegument protein VP22. Thus, the active retention of this major virion component by cytoskeletal structures may function to regulate its subcellular localization (A. Kotsakis, L. E. Pomeranz, A. Blouin, and J. A. Blaho, J. Virol.
75:
8697-8711, 2001). The goal of this study was to determine whether the subcellular localization patterns of other HSV-1 tegument proteins are similar to that observed with VP22. To address this, we performed a series of indirect immunofluorescence analyses using synchronously infected cells. We observed that tegument proteins VP13/14, vhs, and VP16 localized to the nucleus as early as 5 hpi and were concentrated in nuclei by 9 hpi, which differed from that seen with VP22. Microtubule reorganization was delayed during infection with HSV-1(RF177), a recombinant virus that does not produce full-length VP22. These infected cells did not begin to lose microtubule-organizing centers until 13 hpi. Repair of the unique long 49 (U
L
49) locus in HSV-1(RF177) yielded HSV-1(RF177R). Microtubule reorganization in HSV-1(RF177R)-infected cells occurred with the same kinetics as HSV-1(F). Acetylated tubulin remained unchanged during infection with either HSV-1(F) or HSV-1(RF177). Thus, while α-tubulin reorganized during infection, acetylated tubulin was stable, and the absence of full-length VP22 did not affect this stability. Our findings indicate that the nuclear localizations of tegument proteins VP13/14, VP16, and vhs do not appear to require HSV-1-induced microtubule reorganization. We conclude that full-length VP22 is needed for optimal microtubule reorganization during infection. This implies that VP22 mainly functions to reorganize microtubules later, rather than earlier, in infection. That acetylated tubulin does not undergo restructuring during VP22-dependent, virus-induced microtubule reorganization suggests that it plays a role in stabilizing the infected cells. Our results emphasize that VP22 likely plays a key role in cellular cytopathology during HSV-1 infection.