bluuebunny/crossref_metadata_2025 huggingface dataset:
Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus
Authors: Cuconati Andrea , Molla Akhteruzzaman , Wimmer Eckard
Published: December 2019

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system.
Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro
Authors: Lemm Julie A. , Bergqvist Anders , Read Carol M. , Rice Charles M.
Published: December 2019

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123
C>S
, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23
C>S
, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123
C>S
, thus providing a functional assay for purification of the nsP4 RNA polymerase.
Characterization of a Live-Attenuated Retroviral Vaccine Demonstrates Protection via Immune Mechanisms
Authors: Dittmer Ulf , Brooks Diane M. , Hasenkrug Kim J.
Published: December 2019

Live-attenuated retroviruses have been shown to be effective retroviral vaccines, but currently little is known regarding the mechanisms of protection. In the present studies, we used Friend virus as a model to analyze characteristics of a live-attenuated vaccine in protection against virus-induced disease. Highly susceptible mice were immunized with nonpathogenic Friend murine leukemia helper virus (F-MuLV), which replicates poorly in adult mice. Further attenuation of the vaccine virus was achieved by crossing the Fv-1 genetic resistance barrier. The minimum dose of vaccine virus required to protect 100% of the mice against challenge with pathogenic Friend virus complex was determined to be 103focus-forming units of attenuated virus. Live vaccine virus was necessary for induction of immunity, since inactivated F-MuLV did not induce protection. To determine whether immune cells mediated protection, spleen cells from vaccinated donor mice were adoptively transferred into syngeneic recipients. The results indicated that immune mechanisms rather than viral interference mediated protection.
The Human Homolog of HAVcr-1 Codes for a Hepatitis A Virus Cellular Receptor
Authors: Feigelstock Dino , Thompson Peter , Mattoo Pravina , Zhang Yuan , Kaplan Gerardo G.
Published: December 2019

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA was isolated from a cDNA expression library of African green monkey kidney (AGMK) cells by using protective monoclonal antibody (MAb) 190/4, which blocks the binding of hepatitis A virus (HAV) to AGMK cells. The HAVcr-1 cDNA codes for havcr-1, a 451-amino-acid class I integral-membrane mucin-like glycoprotein of unknown natural function. To determine the existence of a human homolog(s) of HAVcr-1 (huHAVcr-1), we used HAVcr-1-specific primers to amplify cDNAs from human liver and kidney mRNA by reverse transcription-PCR. Nucleotide sequence analysis revealed that the amplified liver and kidney huHAVcr-1 cDNAs were identical and that they coded for a 359-amino-acid glycoprotein, termed huhavcr-1, which was approximately 79% identical to havcr-1. The six Cys residues of the extracellular domain of havcr-1 and its first N-glycosylation site were conserved in huhavcr-1. However, the number of hexameric repeats of the mucin-like region was reduced from 27 in havcr-1 to 13 in huhavcr-1. In addition, 12 C-terminal amino acids in the cytoplasmic domain of huhavcr-1 were deleted. Northern blot analysis of poly(A) RNA showed that huhavcr-1 is expressed in every organ analyzed, including the liver, small intestine, colon, and spleen, and that it is expressed at higher levels in the kidney and testis. Although dog cells transfected with the huHAVcr-1 cDNA did not express the protective 190/4 epitope, they bound hepatitis A virus (HAV) and gained limited susceptibility to HAV infection. Treatment with MAb 190/4 did not protect AGMK cell transfectants expressing huhavcr-1 against HAV, suggesting that HAV infected these cells via the huhavcr-1 receptor and not the endogenously expressed havcr-1, which was blocked by MAb 190/4. Our data demonstrate that huhavcr-1 is a binding receptor for HAV and suggest that it is also a functional receptor for HAV.
Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpolwith VPg and with Genetic Variants of 3AB
Authors: Xiang Wenkai , Cuconati Andrea , Hope Debra , Kirkegaard Karla , Wimmer Eckard
Published: December 2019

Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpolthat suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpolinteraction. The viral proteinase 3Cprowas not found to interact with other 3Cpromolecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDproscored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.
Fowlpox Virus Encodes Nonessential Homologs of Cellular Alpha-SNAP, PC-1, and an Orphan Human Homolog of a Secreted Nematode Protein
Authors: Laidlaw Stephen M. , Anwar M. Arif , Thomas Warren , Green Philip , Shaw Kathy , Skinner Michael A.
Published: December 2019

The genome of fowlpox virus (FWPV), type species of theAvipoxviridae, is considerably rearranged compared with that of vaccinia virus (the prototypic poxvirus and type species of theOrthopoxviridae) and is 30% larger. It is likely that the genome of FWPV contains genes in addition to those found in vaccinia virus, probably involved with its replication and survival in the chicken. A 7,470-bp segment of the FWPV genome has five open reading frames (ORFs), two of which encode ankyrin repeat proteins, many examples of which have been found in poxviruses. The remaining ORFs encode homologs of cellular genes not reported in any other virus. ORF-2 encodes a homolog of the yeast Sec17p and mammalian SNAP proteins, crucial to vesicular transport in the exocytic pathway. ORF-3 encodes a homolog of an orphan human protein, R31240_2, encoded on 19p13.2. ORF-3 is also homologous to three proteins (YLS2, YMV6, and C07B5.5) from the free-living nematodeCaenorhabditis elegansand to a 43-kDa antigen from the parasitic nematodeTrichinella spiralis. ORF-5 encodes a homolog of the mammalian plasma cell antigen PC-1, a type II glycoprotein with exophosphodiesterase activity. The ORFs are present in the virulent precursor, HP1, of the sequenced attenuated virus (FP9) and are conserved in other strains of FWPV. They were shown, by deletion mutagenesis, to be nonessential to virus replication in tissue culture. RNA encoding the viral homolog of PC-1 is expressed strongly early and late in infection, but RNAs encoding the homologs of SNAP and R31240_2 are expressed weakly and late.
Priming with
tat
-Deleted Caprine Arthritis Encephalitis Virus (CAEV) Proviral DNA or Live Virus Protects Goats from Challenge with Pathogenic CAEV
Authors: Harmache Abdallah , Vitu Christian , Guiguen François , Russo Pierre , Bertoni Giuseppe , Pepin Michel , Vigne Robert , Suzan Marie
Published: December 2019

We previously reported that infection of goats with caprine arthritis encephalitis virus (CAEV)
tat
− proviral DNA or virus results in persistent infection, since the animals seroconverted and direct virus isolation from cultures of blood-derived macrophages was positive. In this study we wanted to determine whether goats injected with CAEV
tat
− proviral DNA or virus were protected against challenge with the pathogenic homologous virus and to investigate whether CAEV
tat
− was still pathogenic. All animals injected with CAEV
tat
− became infected as indicated by seroconversion and virus isolation. Challenge at 8 or 9 months postinfection demonstrated protection in four of four animals injected with CAEV
tat
− but did not in three of three mock-inoculated challenged goats. Challenge virus was undetectable in the blood macrophages of protected animals during a period of 6 or 10 months postchallenge. In two of four protected animals, however, we were able to detect the challenge wild-type virus by reverse transcriptase PCR on RNA directly extracted from synovial membrane cells surrounding the inoculation site. This result suggests that protection was achieved without complete sterilizing immunity. Animals injected with CAEV
tat
− and mock challenged developed inflammatory lesions in the joints, although these lesions were not as severe as those in CAEV wild-type-injected goats. These results confirm the dispensable role of Tat in CAEV replication in vivo for the establishment of infection and pathogenesis and demonstrate in another lentivirus infection model the efficacy of live attenuated viruses to induce resistance to superinfection.
p53 Protein Is a Suppressor of Papillomavirus DNA Amplificational Replication
Authors: Lepik Dina , Ilves Ivar , Kristjuhan Arnold , Maimets Toivo , Ustav Mart
Published: December 2019

p53 protein was able to block human and bovine papillomavirus DNA amplificational replication while not interfering with Epstein-Barr virus oriP once-per-cell cycle replication. Oligomerization, intact DNA-binding, replication protein A-binding, and proline-rich domains of the p53 protein were essential for efficient inhibition, while the N-terminal transcriptional activation and C-terminal regulatory domains were dispensable for the suppressor activity of the p53 protein. The inhibition of replication was caused neither by the downregulation of expression of the E1 and E2 proteins nor by cell cycle block or apoptosis. Our data suggest that the intrinsic activity of p53 to suppress amplificational replication of the papillomavirus origin may have an important role in the virus life cycle and in virus-cell interactions.
Kinetics of Antiviral Activity by Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes (CTL) and Rapid Selection of CTL Escape Virus In Vitro
Authors: Van Baalen C. A. , Schutten M. , Huisman R. C. , Boers P. H. M. , Gruters R. A. , Osterhaus A. D. M. E.
Published: December 2019

The antiviral activity of a CD8+cytotoxic T-lymphocyte (CTL) clone (TCC108) directed against a newly identified HLA-B14-restricted epitope, human immunodeficiency virus type 1 (HIV-1) Rev(67-75) SAEPVPLQL, was analyzed with respect to its kinetics of target cell lysis and inhibition of HIV-1 production. Addition of TCC108 cells or CD8+reverse transcriptase-specific CTLs to HLA-matched CD4+T cells at different times after infection with HIV-1 IIIB showed that infected cells became susceptible to CTL-mediated lysis before peak virus production but after the onset of progeny virus release. When either of these CTLs were added to part of the infected cells immediately after infection, p55 expression and virus production were significantly suppressed. These data support a model in which CTLs, apart from exerting cytolytic activity which may prevent continued virus release, can interfere with viral protein expression during the eclipse phase via noncytolytic mechanisms. TCC108-mediated inhibition of virus replication in peripheral blood mononuclear cells caused rapid selection of a virus with a mutation (69E→K) in the Rev(67-75) CTL epitope which abolished recognition by TCC108 cells. Taken together, these data suggest that both cytolytic and noncytolytic antiviral mechanisms of CTLs can be specifically targeted to HIV-1-infected cells.
Binding of Murine Leukemia Virus Gag Polyproteins to KIF4, a Microtubule-Based Motor Protein
Authors: Kim Wankee , Tang Yao , Okada Yasushi , Torrey Ted A. , Chattopadhyay Sisir K. , Pfleiderer Michael , Falkner Falko G. , Dorner Friedrich , Choi Wonja , Hirokawa Nobutaka , Morse Herbert C.
Published: December 2019

A cDNA clone encoding a cellular protein that interacts with murine leukemia virus (MuLV) Gag proteins was isolated from a T-cell lymphoma library. The sequence of the clone is identical to the C terminus of a cellular protein, KIF4, a microtubule-associated motor protein that belongs to the kinesin superfamily. KIF4-MuLV Gag associations have been detected in vitro and in vivo in mammalian cells. We suggest that KIF4 could be involved in Gag polyprotein translocation from the cytoplasm to the cell membrane.
Interleukin-12 p40 mRNA Expression in Bovine Leukemia Virus-Infected Animals: Increase in Alymphocytosis but Decrease in Persistent Lymphocytosis
Authors: Pyeon Dohun , Splitter Gary A.
Published: December 2019

Interleukin-12 (IL-12), a key cytokine in immune regulation, has an important role in activating the cell-mediated immune response in infectious diseases. Recently, a dichotomy between IL-12 and IL-10 regarding progression of a variety diseases has emerged. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, by using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus-infected animals in the alymphocytotic stage of disease express an increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by cells from animals with late-stage disease, termed persistent lymphocytosis, was significantly decreased compared to that by normal and alymphocytotic animals. Interestingly, IL-12 p40 mRNA was also detected in tumor-bearing animals. IL-12 p40 expression occurred only in monocytes/macrophages, not B or T lymphocytes. The present study combined with previous findings suggest that IL-12 in bovine leukemia virus-infected animals may regulate production of other cytokines such as gamma interferon and IL-10 and the progression of bovine leukosis in animals that develop more advanced disease such as a persistent lymphocytosis of B cells or B-cell lymphosarcoma.
Identification of Persistent RNA-DNA Hybrid Structures within the Origin of Replication of Human Cytomegalovirus
Authors: Prichard Mark N. , Jairath Sanju , Penfold Mark E. T. , St. Jeor Stephen , Bohlman Marlene C. , Pari Gregory S.
Published: December 2019

Human cytomegalovirus (HCMV) lytic-phase DNA replication initiates at the
cis
-acting origin of replication, oriLyt. oriLyt is a structurally complex region containing repeat elements and transcription factor binding sites. We identified two site-specific alkali-labile regions within oriLyt which flank an alkali-resistant DNA segment. These alkali-sensitive regions were the result of the degradation of two RNA species embedded within oriLyt and covalently linked to viral DNA. The virus-associated RNA, vRNA, was identified by DNase I treatment of HCMV DNA obtained from sucrose gradient purified virus. This heterogeneous population of vRNA was end labeled and used as a hybridization probe to map the exact location of vRNAs within oriLyt. vRNA-1 is localized between restriction endonuclease sites
Xho
I at nucleotide (nt) 93799 and
Sac
I at nt 94631 and is approximately 500 bases long. The second vRNA, vRNA-2, lies within a region which exhibits a heterogeneous restriction pattern located between the
Sph
I (nt 92636) and
Bam
HI (nt 93513) and is approximately 300 bases long. This region was previously shown to be required for oriLyt replication (D. G. Anders, M. A. Kacica, G. S. Pari, and S. M. Punturieri, J. Virol. 66:3373–3384, 1992). RNase H analysis determined that vRNA-2 forms a persistent RNA-DNA hybrid structure in the context of the viral genome and in an oriLyt-containing plasmid used in the transient-replication assay.
Herpes Simplex Virus Glycoprotein D Can Bind to Poliovirus Receptor-Related Protein 1 or Herpesvirus Entry Mediator, Two Structurally Unrelated Mediators of Virus Entry
Authors: Krummenacher Claude , Nicola Anthony V. , Whitbeck J. Charles , Lou Huan , Hou Wangfang , Lambris John D. , Geraghty Robert J. , Spear Patricia G. , Cohen Gary H. , Eisenberg Roselyn J.
Published: December 2019

Several cell membrane proteins have been identified as herpes simplex virus (HSV) entry mediators (Hve). HveA (formerly HVEM) is a member of the tumor necrosis factor receptor family, whereas the poliovirus receptor-related proteins 1 and 2 (PRR1 and PRR2, renamed HveC and HveB) belong to the immunoglobulin superfamily. Here we show that a truncated form of HveC directly binds to HSV glycoprotein D (gD) in solution and at the surface of virions. This interaction is dependent on the native conformation of gD but independent of its N-linked glycosylation. Complex formation between soluble gD and HveC appears to involve one or two gD molecules for one HveC protein. Since HveA also mediates HSV entry by interacting with gD, we compared both structurally unrelated receptors for their binding to gD. Analyses of several gD variants indicated that structure and accessibility of the N-terminal domain of gD, essential for HveA binding, was not necessary for HveC interaction. Mutations in functional regions II, III, and IV of gD had similar effects on binding to either HveC or HveA. Competition assays with neutralizing anti-gD monoclonal antibodies (MAbs) showed that MAbs from group Ib prevented HveC and HveA binding to virions. However, group Ia MAbs blocked HveC but not HveA binding, and conversely, group VII MAbs blocked HveA but not HveC binding. Thus, we propose that HSV entry can be mediated by two structurally unrelated gD receptors through related but not identical binding with gD.
NS1-Binding Protein (NS1-BP): a Novel Human Protein That Interacts with the Influenza A Virus Nonstructural NS1 Protein Is Relocalized in the Nuclei of Infected Cells
Authors: Wolff Thorsten , O’Neill Robert E. , Palese Peter
Published: December 2019

We used the yeast interaction trap system to identify a novel human 70-kDa protein, termed NS1-binding protein (NS1-BP), which interacts with the nonstructural NS1 protein of the influenza A virus. The genetic interaction was confirmed by the specific coprecipitation of the NS1 protein from solution by a glutathione
S
-transferase–NS1-BP fusion protein and glutathione-Sepharose. NS1-BP contains an N-terminal BTB/POZ domain and five kelch-like tandem repeat elements of ∼50 amino acids. In noninfected cells, affinity-purified antibodies localized NS1-BP in nuclear regions enriched with the spliceosome assembly factor SC35, suggesting an association of NS1-BP with the cellular splicing apparatus. In influenza A virus-infected cells, NS1-BP relocalized throughout the nucleoplasm and appeared distinct from the SC35 domains, which suggests that NS1-BP function may be disturbed or altered. The addition of a truncated NS1-BP mutant protein to a HeLa cell nuclear extract efficiently inhibited pre-mRNA splicing but not spliceosome assembly. This result could be explained by a possible dominant-negative effect of the NS1-BP mutant protein and suggests a role of the wild-type NS1-BP in promoting pre-mRNA splicing. These data suggest that the inhibition of splicing by the NS1 protein may be mediated by binding to NS1-BP.
The Neural Cell Adhesion Molecule Is a Receptor for Rabies Virus
Authors: Thoulouze Maria-Isabel , Lafage Mireille , Schachner Melitta , Hartmann Ursula , Cremer Harold , Lafon Monique
Published: December 2019

Previous reports strongly suggest that, in addition to the nicotinic acetylcholine receptor, rabies virus can use other, as-yet-unidentified receptors. We found that laboratory cell lines susceptible to rabies virus infection express the neural cell adhesion molecule (NCAM) (CD56) on their surface, whereas resistant cells do not, supporting the idea that NCAM could be a rabies virus receptor. We observed that (i) incubation with rabies virus decreases the surface expression of NCAM; (ii) treatment of susceptible cells with heparan sulfate, a ligand for NCAM, or with NCAM antibodies significantly reduces the rabies virus infection; and (iii) preincubation of rabies virus inoculum with soluble NCAM protein as a receptor decoy drastically neutralizes the capacity of rabies virus to infect susceptible cells. Moreover, we demonstrated that transfection of resistant L fibroblasts with the NCAM-encoding gene induces rabies virus susceptibility whereas absence of NCAM in the primary cortical cell cultures prepared from NCAM-deficient mice reduces the rabies virus infection and virus production. This provides evidence that NCAM is an in vitro receptor for the rabies virus. Moreover, the in vivo relevance for the use of NCAM as a receptor was demonstrated by the infection of NCAM-deficient mice, in which rabies mortality was delayed and brain invasion by rabies virus was drastically restricted. Our results showed that NCAM, which is expressed mainly in the adult nervous system, plays an important role in rabies infection. However, it cannot be excluded that receptors other than NCAM are utilized. Thus, the description of NCAM as a new rabies virus receptor would be another example of the use by viruses of more than one receptor to gain entry into the host.
Dissociation of the CD4 and CXCR4 Binding Properties of Human Immunodeficiency Virus Type 1 gp120 by Deletion of the First Putative Alpha-Helical Conserved Structure
Authors: Missé Dorothée , Cerutti Martine , Schmidt Isabelle , Jansen Aline , Devauchelle Gérard , Jansen Franz , Veas Francisco
Published: December 2019

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 ΔαHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1α, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4+/CD4−cells but not to CXCR4−/CD4−cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 ΔαHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4+cells. Nevertheless, the soluble gp120 ΔαHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking αHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 ΔαHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.
Oral Administration of Human T-Cell Leukemia Virus Type 1 Induces Immune Unresponsiveness with Persistent Infection in Adult Rats
Authors: Kato Hirotomo , Koya Yoshihiro , Ohashi Takashi , Hanabuchi Shino , Takemura Fumiyo , Fujii Masahiro , Tsujimoto Hajime , Hasegawa Atsuhiko , Kannagi Mari
Published: December 2019

The major route of human T-cell leukemia virus type 1 (HTLV-1) infection is mother-to-child transmission caused by breast-feeding. We investigated the host immune responses to orally established persistent HTLV-1 infection in adult rats. HTLV-1-producing MT-2 cells were inoculated into immunocompetent adult rats either orally, intravenously, or intraperitoneally. HTLV-1 proviruses were detected in the peripheral blood and several organs for at least 12 weeks. Transmission of HTLV-1 to these animals was confirmed by analysis of HTLV-1 flanking regions. Despite persistent HTLV-1 presence, none of the orally inoculated rats produced detectable levels of anti-HTLV-1 antibodies, whereas all intravenously or intraperitoneally inoculated rats showed significant anti-HTLV-1 antibody responses. T-cell proliferative responses against HTLV-1 were also absent in orally inoculated rats. Our findings suggest that gastrointestinal exposure of adult rats to HTLV-1-infected cells induces persistent HTLV-1 infection in the absence of both humoral and cellular immune responses against HTLV-1. This immune unresponsiveness at primary infection may subsequently affect the host defense ability against HTLV-1.
Measles Virus Spread and Pathogenesis in Genetically Modified Mice
Authors: Mrkic Branka , Pavlovic Jovan , Rülicke Thomas , Volpe Pietro , Buchholz Christian J. , Hourcade Dennis , Atkinson John P. , Aguzzi Adriano , Cattaneo Roberto
Published: December 2019

Attenuated Edmonston measles virus (MV-Edm) is not pathogenic in standard mice. We show here that MV-Edm inoculated via the natural respiratory route has a limited propagation in the lungs of mice with a targeted mutation inactivating the alpha/beta interferon receptor. A high dose of MV-Edm administered intracerebrally is lethal for about half of these mice. To study the consequences of the availability of a high-affinity receptor for MV propagation, we generated alpha/beta interferon-defective mice expressing human CD46 with human-like tissue specificity. Intranasal infection of these mice with MV-Edm resulted in enhanced spread to the lungs and more prominent inflammatory response. Virus replication was also detected in peripheral blood mononuclear cells, the spleen, and the liver. Moreover, intracerebral inoculation of adult animals with low MV-Edm doses caused encephalitis with almost inevitably lethal outcome. We conclude that in mice alpha/beta interferon controls MV infection and that a high-affinity receptor facilitates, but is not strictly required for, MV spread and pathogenesis.
Herpes Simplex Virus Type 1 Cleavage and Packaging Proteins UL15 and UL28 Are Associated with B but Not C Capsids during Packaging
Authors: Yu Dong , Weller Sandra K.
Published: December 2019

At least seven viral genes encode proteins (UL6, UL15, UL17, UL25, UL28, UL32, and UL33) that are required for DNA cleavage and packaging of herpes simplex virus type 1 (HSV-1) DNA. Sequence analysis reveals that UL15 shares homology with gp17, the large catalytic subunit of the bacteriophage T4 terminase. Thus, UL15 may play a direct role in the cleavage of viral DNA replication intermediates into monomers. In this study, we asked whether UL15 and other cleavage and packaging proteins could be detected in capsids isolated from infected cells. Consistent with previous studies showing that UL6 and UL25 are minor protein constituents of the capsids, we detected these proteins in both B and C capsids. In contrast, the previously identified full-length version (81 kDa) of UL15 was found predominantly in B capsids and in much smaller amounts in C capsids. In addition, the UL28 protein was found predominantly in B but not C capsids in a distribution similar to that of the 81-kDa version of UL15. These results suggest that UL28 and the 81-kDa form of UL15 are transiently associated with capsid intermediates during the packaging process. Surprisingly, however, a previously unidentified 87-kDa form of UL15 was found in the B and C capsids and in virions. Analysis of cells infected with mutants individually lacking UL6, UL15, UL25, UL28, or UL32 demonstrates that the lack of one cleavage and packaging protein does not affect the expression of the others. Furthermore, this analysis, together with guanidine HCl extraction analysis of purified capsids, indicates that UL6, UL25, and UL28 are able to associate with B capsids in the absence of other DNA cleavage and packaging proteins. On the other hand, the two UL15-related proteins (81 and 87 kDa) do not associate efficiently with B capsids in cells infected with UL6 and UL28 mutants. These results suggest that the ability of the UL15-related proteins to bind to B capsids may be mediated through interactions with UL6 and UL28.
Influence of theCCR2-V64IPolymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4
Authors: Lee Benhur , Doranz Benjamin J. , Rana Shalini , Yi Yanji , Mellado Mario , Frade Jose M. R. , Martinez-A. Carlos , O’Brien Stephen J. , Dean Michael , Collman Ronald G. , Doms Robert W.
Published: December 2019

The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism inCCR2(CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor activities, and their effects on the expression and receptor activities of the major HIV-1 coreceptors. CCR2b and CCR2b-V64I were expressed at similar levels, and neither molecule affected the expression or coreceptor activity of CCR3, CCR5, or CXCR4 in cotransfected cell lines. Peripheral blood mononuclear cells (PBMCs) fromCCR2-V64Iheterozygotes had normal levels of CCR2b and CCR5 but slightly reduced levels of CXCR4. CCR2b and CCR2b-V64I functioned equally well as HIV-1 coreceptors, and CCR2-V64I PBMCs were permissive for HIV-1 infection regardless of viral tropism. The MCP-1-induced calcium mobilization mediated by CCR2b signaling was unaffected by the polymorphism, but MCP-1 signaling mediated by eitherCCR2b- orCCR2-V64I-encoded receptors resulted in heterologous desensitization (i.e., limiting the signal response of other receptors) of both CCR5 and CXCR4. The heterologous desensitization of CCR5 and CXCR4 signaling by bothCCR2allele receptor types provides a mechanistic link that might help explain the in vivo effects ofCCR2gene variants on progression to AIDS as well as the reported antiviral activity of natural CCR2 ligands.
Retention of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein in the Endoplasmic Reticulum Does Not Redirect Virus Assembly from the Plasma Membrane
Authors: Salzwedel Karl , West John T. , Mulligan Mark J. , Hunter Eric
Published: December 2019

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) has been shown to redirect the site of virus assembly in polarized epithelial cells. To test whether localization of the glycoprotein exclusively to the endoplasmic reticulum (ER) could redirect virus assembly to that organelle in nonpolarized cells, an ER -retrieval signal was engineered into an epitope-tagged variant of Env. The epitope tag, attached to the C terminus of Env, did not affect the normal maturation and transport of the glycoprotein or the incorporation of Env into virions. The epitope-tagged Env was also capable of mediating syncytium formation and virus entry with a similar efficiency to that of wild-type Env. When the epitope was modified to contain a consensus K(X)KXX ER retrieval signal, however, the glycoprotein was no longer proteolytically processed into its surface and transmembrane subunits and Env could not be detected at the cell surface by biotinylation. Endoglycosidase H analysis revealed that the modified Env was not transported to the Golgi apparatus. Immunofluorescent staining patterns were also consistent with the exclusion of Env from the Golgi. As expected, cells expressing the modified Env failed to form syncytia with CD4
+
permissive cells. Despite this tight localization of Env to the ER, when the modified Env was expressed in the context of virus, virions continued to be produced efficiently from the plasma membrane of transfected cells. However, these virions contained no detectable glycoprotein and were noninfectious. Electron microscopy revealed virus budding from the plasma membrane of these cells, but no virus was seen assembling at the ER membrane and no assembled virions were found within the cell. These results suggest that the accumulation of Env in an intracellular compartment is not sufficient to redirect the assembly of HIV Gag in nonpolarized cells.
Incorporation of the Green Fluorescent Protein into the Herpes Simplex Virus Type 1 Capsid
Authors: Desai Prashant , Person Stanley
Published: December 2019

The herpes simplex virus type 1 (HSV-1) UL35 open reading frame (ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located on the outer surface of the capsid specifically on the tips of the hexons that constitute the capsid shell. The bioluminescent jellyfish (
Aequorea victoria
) green fluorescent protein (GFP) was fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein. This fusion protein was fluorescent and localized to distinct regions within the nuclei of transfected cells following infection with wild-type virus. The VP26-GFP marker was introduced into the HSV-1 (KOS) genome resulting in recombinant plaques that were fluorescent. A virus, designated K26GFP, was isolated and purified and was shown to grow as well as the wild-type virus in cell culture. An analysis of the intranuclear capsids formed in K26GFP-infected cells revealed that the fusion protein was incorporated into A, B, and C capsids. Furthermore, the fusion protein incorporated into the virion particle was fluorescent as judged by fluorescence-activated cell sorter (FACS) analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at early times in the replication cycle. At later times during infection a generalized cytoplasmic and nuclear fluorescence, including fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.
Leptomycin B Inhibits Equine Infectious Anemia Virus Rev and Feline Immunodeficiency Virus Rev Function but Not the Function of the Hepatitis B Virus Posttranscriptional Regulatory Element
Authors: Otero Glen C. , Harris Matthew E. , Donello John E. , Hope Thomas J.
Published: December 2019

Human immunodeficiency virus type 1 Rev export depends upon the presence of the nuclear export signal (NES), a leucine-rich stretch of hydrophobic amino acids. Recently, the nuclear NES-binding receptor has been identified as CRM1 or exportin 1. Rev export has been shown to be CRM1 dependent. The function of the atypical NES-containing Rev-like proteins of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) is inhibited by leptomycin B, a drug that specifically blocks NES-CRM1 interactions. These data suggest that the function of atypical NES-containing proteins is CRM1 dependent. In contrast to the inhibition of EIAV Rev and FIV Rev, the cytoplasmic accumulation of hepatitis B virus (HBV) posttranscriptional regulatory element (PRE)-containing and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-containing RNAs is not inhibited by leptomycin B treatment. We conclude that the HBV PRE, like the MPMV CTE, functions independently of an NES receptor-exportin 1 interaction.
Continued Utilization of CCR5 Coreceptor by a Newly Derived T-Cell Line-Adapted Isolate of Human Immunodeficiency Virus Type 1
Authors: Follis Kathryn E. , Trahey Meg , LaCasse Rachel A. , Nunberg Jack H.
Published: December 2019

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5. This observation had been suggested as an explanation for the sensitivity of TCLA, but not PI, viruses to neutralization by recombinant gp120 antisera and V3-directed monoclonal antibodies, but recent studies have shown coreceptor utilization to be independent of neutralization sensitivity. Here we describe a newly isolated TCLA virus that is sensitive to neutralization but continues to utilize both CXCR4 and CCR5 for infection. This finding further divorces coreceptor specificity from neutralization sensitivity and from certain changes in cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of CCR5 expression in the FDA/H9 T-cell line suggests that the interaction between envelope protein and coreceptor may be mediated by multiple weak interactions along a diffuse surface.
Crystallization and Preliminary X-Ray Analysis of Rotavirus Protein VP6
Authors: Petitpas Isabelle , Lepault Jean , Vachette Patrice , Charpilienne Annie , Mathieu Magali , Kohli Evelyne , Pothier Pierre , Cohen Jean , Rey Félix A.
Published: December 2019

As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca
2+
at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 Å resolution and belong to the cubic space group P4
1
32 with a cell dimension
a
of 160 Å. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.
Attachment but Not Penetration of Bovine Herpesvirus 1 Is Necessary To Induce Apoptosis in Target Cells
Authors: Hanon Emmanuel , Meyer Gilles , Vanderplasschen Alain , Dessy-Doizé Cécile , Thiry Etienne , Pastoret Paul-Pierre
Published: December 2019

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.
Mutations in the Primer Grip of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Impair Proviral DNA Synthesis and Virion Maturation
Authors: Yu Qiang , Ottmann Michele , Pechoux Christine , Le Grice Stuart , Darlix Jean-Luc
Published: December 2019

This report describes the effects of mutating highly conserved residues in the primer grip domain of human immunodeficiency virus type 1 reverse transcriptase (RT) on virus formation and infectivity. Among a series of RT mutant viruses, three (M230A, L234D, and W239A) were found to be noninfectious or very poorly infectious. Our data indicate that these mutations in RT caused severe defects in proviral DNA synthesis. Interestingly, assembly and maturation of mutant virus M230A were similar to those of the wild type, while mutants L234D and W239A showed impaired maturation. The immature morphology of RT mutants L234D and W239A is due at least in part to premature cleavage of the
gag-pol
precursor, prior to virion budding, indicating that intracellular stability of Pr160

gag-pol

is of key importance during virus assembly.
Identification of a Domain Containing B-Cell Epitopes in Hepatitis C Virus E2 Glycoprotein by Using Mouse Monoclonal Antibodies
Authors: Lee Jae Woo , Kim Kwang-mi , Jung Seung-Hye , Lee Ki Jeong , Choi Eung-Chil , Sung Young-Chul , Kang Chang-Yuil
Published: December 2019

Evidence from clinical and experimental studies of human and chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were able to inhibit the binding of CET MAbs to the former fusion protein. Inhibitory activity was observed in most sera tested, which indicated that CET-1 to -6 were similar to anti-E2 antibodies in human sera with respect to the epitope specificity. The spacial relationship of epitopes on E2 recognized by CET MAbs was determined by surface plasmon resonance analysis and competitive enzyme-linked immunosorbent assay. The data indicated that three overlapping epitopes were recognized by CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we analyzed the reactivities of CET MAbs to six truncated forms and two chimeric forms of recombinant E2 proteins. The data suggest that the epitopes recognized by CET-1 to -6 are located in a small domain of E2 spanning amino acid residues 528 to 546.
Transactivation of a Ribosomal Gene by Simian Virus 40 Large-T Antigen Requires at Least Three Activities of the Protein
Authors: Cavender Jane F. , Mummert Christine , Tevethia Mary Judith
Published: December 2019

Simian virus 40 large-T antigen transactivates the ribosomal genes which are transcribed by RNA polymerase (pol I), as well as genes that are dependent on either pol II or pol III. This report identifies regions and activities of T antigen that are required to transactivate a pol I-dependent rat ribosomal gene promoter. By using the rat ribosomal gene (rDNA) promoter linked to a chloramphenicol acetyltransferase gene, we show that at least three separable T-antigen regions are necessary to achieve wild-type levels of transactivation of rDNA in transiently transfected monkey cells. One activity depends on the region of T antigen shared with small-t antigen (T/t common region). A second activity maps to amino acids 109 to 626 and is highly sensitive to mutational inactivation. Complementation analyses suggest that at least one activity in this region is independent of and must be in
cis
with the activity within the T/t common region. In addition, a functional nuclear localization signal is required for maximal T-antigen-mediated transactivation of rat rDNA. The three activities work in concert to override cellular species-specific controls and transactivate the rat ribosomal gene promoter. Finally, we provide evidence that although the tumor suppressor protein Rb has been shown to repress a pol I-dependent promoter, transactivation of the rat rDNA promoter does not depend on T antigen’s ability to bind the tumor suppressor product Rb.
Sequence-Specific and/or Stereospecific Constraints of the U3 Enhancer Elements of MCF 247-W Are Important for Pathogenicity
Authors: DiFronzo Nancy L. , Holland Christie A.
Published: December 2019

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3′ (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-
myc
locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-
myc
contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.