bluuebunny/crossref_metadata_2025 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

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

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

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

Epstein-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

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

Anti-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

Human 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

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

Neurotropic 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

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

Fusion 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

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.
Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion
Authors: Senkevich Tatiana G. , Moss Bernard
Published: March 2005

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.
Visualization and Characterization of the Intracellular Movement of Vaccinia Virus Intracellular Mature Virions
Authors: Ward Brian M.
Published: March 2005

Previous work indicated that vaccinia intracellular mature virus (IMV) utilizes microtubules to move from the viral factory to the site of intracellular envelopment and that expression of the viral A27 protein is required for this transport. To investigate further the role of A27 in IMV intracellular transport, a recombinant vaccinia virus was constructed that had the A27L gene deleted and expressed a yellow fluorescent protein (YFP)-A4 chimera in place of the normal A4 protein. The resulting recombinant, vYFP-A4/ΔA27, produced relatively normal quantities of virus in a one-step growth curve but had a small plaque phenotype. Subsequent experiments demonstrated that vYFP-A4/ΔA27 was severely defective in envelope virus production. Despite the absence of A27, live digital video fluorescent microscopy visualized YFP-labeled IMV movement in cells infected with the recombinant. Virion movement approached 3 μm/s and was sensitive to the microtubule depolymerizing drug nocodazole. In addition, IMV could be discerned transiting away from and back towards viral factories. Immunofluorescent staining determined that the distance traveled by A27-deficient virions was sufficient for transport to the site of envelopment. These results indicate that IMVs are capable of bidirectional movement on microtubules, suggesting that they are able to interact with both kinesin and dynein microtubule motors in the absence of A27 and that the distance traveled is sufficient to deliver IMV to the site of wrapping.
Expression of Matrix Metalloproteinases and Their Tissue Inhibitor during Viral Encephalitis
Authors: Zhou Jiehao , Marten Norman W. , Bergmann Cornelia C. , Macklin Wendy B. , Hinton David R. , Stohlman Stephen A.
Published: March 2005

Matrix metalloproteinases (MMPs) participate in remodeling the extracellular matrix and facilitate entry of inflammatory cells into tissues. Infection of the murine central nervous system (CNS) with a neurotropic coronavirus induces encephalitis associated with increased levels of mRNA encoding MMP-3 and MMP-12. Whereas virus-induced MMP-3 expression was restricted to CNS resident astrocytes, MMP-12 mRNA was expressed by both inflammatory cells and CNS resident cells. Immunosuppression increased both MMP-3 and MMP-12 mRNA levels in CNS resident cells, suggesting that the presence of virus rather than inflammation induced protease up-regulation. MMP activity is partially regulated by a small family of genes encoding tissue inhibitors of matrix metalloproteinases (TIMPs); among the TIMPs, only TIMP-1 mRNA expression increased in the CNS following coronavirus infection. During inflammation TIMP-1 mRNA was most prominently expressed by infiltrating cells. By contrast, in the immunosuppressed host TIMP-1 mRNA was expressed by CNS resident cells. Analysis of cytokine and chemokine mRNA induction within the infected CNS of healthy and immunocompromised mice suggested a possible correlation between increased viral replication and increased levels of beta interferon, MMP-3, MMP-12, and TIMP-1 mRNA. CD4+T cells which localize to the perivascular and subarachnoid spaces were identified as the primary source of TIMP-1 protein. By contrast, protein expression was undetectable in astrocytes or CD8+T cells, the primary antiviral effectors that localize to the CNS parenchyma in response to infection. These data suggest that in contrast to the results seen with MMPs, inhibition of protease activity via TIMP-1 expression correlates with the differential tissue distribution of T-cell subsets during acute coronavirus-induced encephalitis.
Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120
Authors: Desmezieres Emmanuel , Gupta Nidhi , Vassell Russell , He Yong , Peden Keith , Sirota Lev , Yang Zhongning , Wingfield Paul , Weiss Carol D.
Published: March 2005

Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.
Inhibition of Murine Leukemia Virus Envelope Protein (Env) Processing by Intracellular Expression of the Env N-Terminal Heptad Repeat Region
Authors: Ou Wu , Silver Jonathan
Published: March 2005

A conserved structural motif in the envelope proteins of several viruses consists of an N-terminal, alpha-helical, trimerization domain and a C-terminal region that refolds during fusion to bind the N-helix trimer. Interaction between the N and C regions is believed to pull viral and target membranes together in a crucial step during membrane fusion. For several viruses with type I fusion proteins, C regions pack as alpha-helices in the grooves between N-helix monomers, and exogenously added N- and C-region peptides block fusion by inhibiting the formation of the six-helix bundle. For other viruses, including influenza virus and murine leukemia virus (MLV), there is no evidence for comparably extended C-region alpha-helices, although a short, non-alpha-helical interaction structure has been reported for influenza virus. We tested candidate N-helix and C-region peptides from MLV for their ability to inhibit cell fusion but found no inhibitory activity. In contrast, intracellular expression of the MLV N-helix inhibited fusion by efficiently blocking proteolytic processing and intracellular transport of the envelope protein. The results highlight another mechanism by which the N-helix peptides can inhibit fusion.
Comprehensive Analysis of Ebola Virus GP1 in Viral Entry
Authors: Manicassamy Balaji , Wang Jizhen , Jiang Haiqing , Rong Lijun
Published: March 2005

Ebola virus infection is initiated by interactions between the viral glycoprotein GP1 and its cognate receptor(s), but little is known about the structure and function of GP1 in viral entry, partly due to the concern about safety when working with the live Ebola virus and the difficulty of manipulating the RNA genome of Ebola virus. In this study, we have used a human immunodeficiency virus-based pseudotyped virus as a surrogate system to dissect the role of Ebola virus GP1 in viral entry. Analysis of more than 100 deletion and amino acid substitution mutants of GP1 with respect to protein expression, processing, viral incorporation, and viral entry has allowed us to map the region of GP1 responsible for viral entry to the N-terminal 150 residues. Furthermore, six amino acids in this region have been identified as critical residues for early events in Ebola virus entry, and among these, three are clustered and are implicated as part of a potential receptor-binding pocket. In addition, substitutions of some 30 residues in GP1 are shown to adversely affect GP1 expression, processing, and viral incorporation, suggesting that these residues are involved in the proper folding and/or overall conformation of GP. Sequence comparison of the GP1 proteins suggests that the majority of the critical residues for GP folding and viral entry identified in Ebola virus GP1 are conserved in Marburg virus. These results provide information for elucidating the structural and functional roles of the filoviral glycoproteins and for developing potential therapeutics to block viral entry.
The Mitotic Chromosome Binding Activity of the Papillomavirus E2 Protein Correlates with Interaction with the Cellular Chromosomal Protein, Brd4
Authors: Baxter Michael K. , McPhillips Maria G. , Ozato Keiko , McBride Alison A.
Published: March 2005

The papillomavirus transcriptional activator, E2, is involved in key functions of the viral life cycle. These include transcriptional regulation, viral DNA replication, and viral genome segregation. The transactivation domain of E2 is required for each of these functions. To identify the regions of the domain that mediate binding to mitotic chromosomes, a panel of mutations has been generated and their effect on various E2 functions has been analyzed. A structural model of the bovine papillomavirus type 1 (BPV1) E2 transactivation domain was generated based on its homology with the solved structure of the human papillomavirus type 16 (HPV16) domain. This model was used to identify distinct surfaces of the domain to be targeted by point mutation to further delineate the functional region of the transactivation domain responsible for mitotic chromosome association. The mutated E2 proteins were assessed for mitotic chromosome binding and, in addition, transcriptional activation and transcriptional repression activities. Mutation of amino acids R37 and I73, which are located on a surface of the domain that in HPV16 E2 is reported to mediate self-interaction, completely eliminated mitotic chromosome binding. Mitotic chromosome binding activity was found to correlate well with the ability to interact with the cellular chromosomal associated factor Brd4, which has recently been proposed to mediate the association between BPV1 E2 and mitotic chromosomes.
Role of the Varicella-Zoster Virus Gene Product Encoded by Open Reading Frame 35 in Viral Replication In Vitro and in Differentiated Human Skin and T Cells In Vivo
Authors: Ito Hideki , Sommer Marvin H. , Zerboni Leigh , Baiker Armin , Sato Bunji , Liang Ruibin , Hay John , Ruyechan William , Arvin Ann M.
Published: March 2005

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaΔ35(#1) and rOkaΔ35(#2), and replaced ORF35 at a nonnative site, generating two rOkaΔ35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaΔ35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaΔ35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaΔ35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaΔ35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaΔ35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaΔ35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.
Role of Low CD4 Levels in the Influence of Human Immunodeficiency Virus Type 1 Envelope V1 and V2 Regions on Entry and Spread in Macrophages
Authors: Walter Brandon L. , Wehrly Kathy , Swanstrom Ronald , Platt Emily , Kabat David , Chesebro Bruce
Published: March 2005

Human immunodeficiency virus type 1 (HIV-1) isolates vary in their ability to infect macrophages. Previous experiments have mapped viral determinants of macrophage infectivity to the V3 hypervariable region of the HIV-1 envelope glycoprotein. In our earlier studies, V1 and V2 sequences of HIV-1 were also shown to alter the ability of virus to spread in macrophage cultures, whereas no effect was seen in lymphocyte cultures. In the present study, determinants that allowed certain HIV-1 clones to infect and spread in macrophages were primarily mapped to the V2 region and were found to act by influencing early events of viral infection. By an assay of viral entry into macrophages, it was shown that viruses with the V2 region from the Ba-L strain of HIV-1 had >10-fold-higher entry efficiency than viruses with the V2 region derived from the NL4-3 strain. V1 region differences between these groups caused a twofold difference in entry. The known low expression of CD4 on macrophages appeared to be important in this process. In entry assays conducted with HeLa cell lines expressing various levels of CD4 and CCR5, low levels of CD4 influenced the efficiency of entry and fusion which were dependent on viral V1 and V2 envelope sequences. In contrast, no effect of V1 or V2 was seen in HeLa cells expressing high levels of CD4. Thus, the limited expression of CD4 on macrophages or other cell types could serve as a selective factor for V1 and V2 envelope sequences, and this selection could in turn influence many aspects of AIDS pathogenesis in vivo.
Constitutive Expression of Alpha Interferon by Skin Dendritic Cells Confers Resistance to Infection by Foot-and-Mouth Disease Virus
Authors: Bautista Elida M. , Ferman Geoffrey S. , Gregg Douglas , Brum Mario C. S. , Grubman Marvin J. , Golde William T.
Published: March 2005

The role of dendritic cells (DC) in the initiation of immune responses against foot-and-mouth disease virus (FMDV) is poorly understood. We analyzed the innate response of freshly isolated swine skin DC to the virus and show a rapid induction of beta interferon (IFN-β) mRNA but not IFN-α mRNA. However, these DC secreted both IFN-α and IFN-β proteins in response to live virus but not killed virus. Furthermore, the surface expression of swine major histocompatibility complex class II (SLA II) or CD80/CD86 molecules and antigen processing functions were not affected by FMDV exposure. Given the demonstrated sensitivity of FMDV to IFN-α/β, there was no productive or nonproductive infection of these cells. Finally, freshly isolated skin DC constitutively expressed intracellular IFN-α protein in the absence of stimulation, with no detectable secretion of the cytokine until virus exposure. In situ analysis of these DC showed that these cells express and store IFN-α in uninfected animals. This is the first demonstration of the constitutive expression of IFN-α in resident, tissue-derived DC and indicates that skin DC can play an important role in the innate immune response of swine to viral infections.
The p92 Polymerase Coding Region Contains an Internal RNA Element Required at an Early Step in Tombusvirus Genome Replication
Authors: Monkewich Sandra , Lin Han-Xin , Fabian Marc R. , Xu Wei , Na Hong , Ray Debashish , Chernysheva Olena A. , Nagy Peter D. , White K. Andrew
Published: March 2005

The replication of positive-strand RNA viral genomes involves various
cis
-acting RNA sequences. Generally, regulatory RNA sequences are present at or near genomic termini; however, internal replication elements (IREs) also exist. Here we report the structural and functional characterization of an IRE present in the readthrough portion of the p92 polymerase gene of
Tomato bushy stunt virus
. Analysis of this element in the context of a noncoding defective interfering RNA revealed a functional core structure composed of two noncontiguous segments of sequence that interact with each other to form an extended helical conformation. IRE activity required maintenance of several base-paired sections as well as two distinct structural features: (i) a short, highly conserved segment that can potentially form two different and mutually exclusive structures and (ii) an internal loop that contains a critical CC mismatch. The IRE was also shown to play an essential role within the context of the viral genome. In vivo analysis with novel RNA-based temperature-sensitive genomic mutants and translationally active subgenomic viral replicons revealed the following about the IRE: (i) it is active in the positive strand, (ii) it is dispensable late in the viral RNA replication process, and (iii) it is functionally inhibited by active translation over its sequence. Together, these results suggest that IRE activity is required in the cytosol at an early step in the viral replication process, such as template recruitment and/or replicase complex assembly.
Specific Binding of Tombusvirus Replication Protein p33 to an Internal Replication Element in the Viral RNA Is Essential for Replication
Authors: Pogany Judit , White K. Andrew , Nagy Peter D.
Published: March 2005

The mechanism of template selection for genome replication in plus-strand RNA viruses is poorly understood. Using the prototypical tombusvirus,
Tomato bushy stunt virus
(TBSV), we show that recombinant p33 replicase protein binds specifically to an internal replication element (IRE) located within the p92 RNA-dependent RNA polymerase coding region of the viral genome. Specific binding of p33 to the IRE in vitro depends on the presence of a C · C mismatch within a conserved RNA helix. Interestingly, the absence of the p33:p33/p92 interaction domain in p33 prevented specific but allowed nonspecific RNA binding, suggesting that a multimeric form of this protein is involved in the IRE-specific interaction. Further support for the selectivity of p33 binding in vitro was provided by the inability of the replicase proteins of the closely related
Turnip crinkle virus
and distantly related
Hepatitis C virus
to specifically recognize the TBSV IRE. Importantly, there was also a strong correlation between p33:IRE complex formation in vitro and viral replication in vivo, where mutations in the IRE that disrupted selective p33 binding in vitro also abolished TBSV RNA replication both in plant and in
Saccharomyces cerevisiae
cells. Based on these findings and the other known properties of p33 and the IRE, it is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes. Since all genera in
Tombusviridae
encode comparable replicase proteins, these results may be relevant to other members of this large virus family.
Escape Mutations Alter Proteasome Processing of Major Histocompatibility Complex Class I-Restricted Epitopes in Persistent Hepatitis C Virus Infection
Authors: Kimura Yoichi , Gushima Toshifumi , Rawale Sharad , Kaumaya Pravin , Walker Christopher M.
Published: March 2005

Mutations in hepatitis C virus (HCV) genomes facilitate escape from virus-specific CD8+T lymphocytes in persistently infected chimpanzees. Our previous studies demonstrated that many of the amino acid substitutions in HCV epitopes prevented T-cell receptor recognition or binding to class I major histocompatibility complex molecules. Here we report that mutations within HCV epitopes also cause their destruction by changing the pattern of proteasome digestion. This mechanism of immune evasion provides further evidence of the potency of CD8+T-cell selection pressure against HCV and should be considered when evaluating the significance of mutations in viral genomes from persistently infected chimpanzees and humans.
Interleukin-15 Increases Effector Memory CD8+T Cells and NK Cells in Simian Immunodeficiency Virus-Infected Macaques
Authors: Mueller Yvonne M. , Petrovas Constantinos , Bojczuk Paul M. , Dimitriou Ioannis D. , Beer Brigitte , Silvera Peter , Villinger Francois , Cairns J. Scott , Gracely Edward J. , Lewis Mark G. , Katsikis Peter D.
Published: March 2005

Interleukin-15 (IL-15) in vitro treatment of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected individuals specifically enhances the function and survival of HIV-specific CD8+T cells, while in vivo IL-15 treatment of mice preferentially expands memory CD8+T cells. In this study, we investigated the in vivo effect of IL-15 treatment in 9 SIVmac251-infected cynomolgus macaques (low dose of IL-15, 10 μg/kg of body weight,n= 3; high dose of IL-15, 100 μg/kg,n= 3; control [saline],n= 3; dose administered twice weekly for 4 weeks). IL-15 treatment induced a nearly threefold increase in peripheral blood CD8+CD3−NK cells. Furthermore, CD8+T-cell numbers increased more than twofold, mainly due to an increase in the CD45RA−CD62L−and CD45RA+CD62L−effector memory CD8+T cells. Expression of Ki-67 in the CD8+T cells indicated expansion of CD8+T cells and not redistribution. IL-15 did not affect CD4+T-cell, B-cell, and CD14+macrophage numbers. No statistically significant differences in changes from baseline in the viral load were observed when control-, low-dose-, and high-dose-treated animals were compared. No clinical adverse effects were observed in any of the animals studied. The selective expansion of effector memory CD8+T cells and NK cells by IL-15 further supports IL-15's possible therapeutic use in viral infections such as HIV infection.