bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Functional Antigenomic Promoter for the Paramyxovirus Simian Virus 5 Requires Proper Spacing between an Essential Internal Segment and the 3′ Terminus
Authors: Murphy Susan K. , Ito Yasuhiko , Parks Griffith D. 
Published: December 2019

ABSTRACT

A previous analysis of naturally occurring defective interfering (DI) RNA genomes of the prototypic paramyxovirus simian virus 5 (SV5) indicated that 113 bases at the 3′ terminus of the antigenome were sufficient to direct RNA encapsidation and replication. A nucleotide sequence alignment of the antigenomic 3′-terminal 113 bases of members of the
Rubulavirus
genus of the
Paramyxoviridae
family identified two regions of sequence identity: bases 1 to 19 at the 3′ terminus (conserved region I [CRI]) and a more distal region consisting of antigenome bases 73 to 90 (CRII) that was contained within the 3′ coding region of the L protein gene. To determine whether these regions of the antigenome were essential for SV5 RNA replication, a reverse genetics system was used to analyze the replication of copyback DI RNA analogs that contained a foreign gene (GL, encoding green fluorescence protein) flanked by 113 5′-terminal bases and various amounts of SV5 3′-terminal antigenomic sequences. Results from a deletion analysis showed that efficient encapsidation and replication of SV5-GL DI RNA analogs occurred when the 90 3′-terminal bases of the SV5 antigenomic RNA were retained, but replication was reduced ∼5- to 14-fold in the case of truncated antigenomes that lacked the 3′-end CRII sequences. A chimeric copyback DI RNA containing the 3′-terminal 98 bases including the CRI and CRII sequences from the human parainfluenza virus type 2 (HPIV2) antigenome in place of the corresponding SV5 sequences was efficiently replicated by SV5 cDNA-derived components. However, replication was reduced ∼20-fold for a truncated SV5-HPIV2 chimeric RNA that lacked the HPIV2 CRII sequences between antigenome bases 72 and 90. Progressive deletions of 6 to 18 bases in the region located between the SV5 antigenomic CRI and CRII segments (3′-end nucleotides 21 to 38) resulted in a ∼25-fold decrease in SV5-GL RNA synthesis. Surprisingly, replication was restored to wild-type levels when these length alterations between CRI and CRII were corrected by replacing the deleted bases with nonviral sequences. Together, these data suggest that a functional SV5 antigenomic promoter requires proper spacing between an essential internal region and the 3′ terminus. A model is presented for the structure of the 3′ end of the SV5 antigenome which proposes that positioning of CRI and CRII along the same face of the helical nucleocapsid is an essential feature of a functional antigenomic promoter.
Reactivation of the Previously Silenced Cytomegalovirus Major Immediate-Early Promoter in the Mouse Liver: Involvement of NFκB
Authors: Löser Peter , Jennings Gary S. , Strauss Michael , Sandig Volker 
Published: December 2019

ABSTRACTThe cytomegalovirus (CMV) major immediate-early promoter/enhancer is active in many cell culture systems and is considered to be one of the strongest promoters in vitro. However, when this promoter was used in in vivo approaches to gene therapy, it was silenced within a few weeks in several organs including the liver. In this study, we demonstrated transcriptional inactivation of the CMV promoter in mouse liver. In contrast to the CMV promoter, a hybrid promoter consisting of a minimal CMV promoter and the enhancer II of hepatitis B virus was active for at least 11 weeks in mouse liver. While investigating the reason for the shutdown of the CMV promoter, we did not find evidence for methylation of adenovirus DNA in the region of transgene insertion, but we could show that the silenced CMV promoter was reactivated after lipopolysaccharide treatment of mice or partial hepatectomy. Both stimuli are known to activate the transcription factor NFκB, which binds to four sites in the CMV promoter/enhancer. We show that expression from the CMV promoter in hepatocyte-derived cell lines in vitro depends on NFκB. In vivo experiments demonstrate that NFκB, which is not present in mouse hepatocytes in vivo, is activated after infection with recombinant adenoviruses and that the time course of NFκB activation parallels that of CMV promoter-dependent expression. Moreover, adenovirus infection of transgenic mice carrying a CMV promoter-drivenlacZgene leads to strong activation of the expression of this gene in the liver. Thus, NFκB is involved in the activation of the CMV promoter in the liver.
DNA Immunization with Japanese Encephalitis Virus Nonstructural Protein NS1 Elicits Protective Immunity in Mice
Authors: Lin Yi-Ling , Chen Li-Kuang , Liao Ching-Len , Yeh Chia-Tsui , Ma Shiou-Hwa , Chen Jin-Ling , Huang Yue-Ling , Chen Shih-Shun , Chiang Hsien-Yuan 
Published: December 2019

ABSTRACTJapanese encephalitis virus (JEV), a mosquito-borne flavivirus, is a zoonotic pathogen that is prevalent in some Southeast Asian countries and causes acute encephalitis in humans. To evaluate the potential application of gene immunization to JEV infection, we characterized the immune responses from mice intramuscularly injected with plasmid DNA encoding JEV glycoproteins, including the precursor membrane (prM) plus envelope (E) proteins and the nonstructural protein NS1. When injected with the plasmid expressing prM plus E, 70% of the immunized mice survived after a lethal JEV challenge, whereas when immunized with the plasmid expressing NS1, 90% of the mice survived after a lethal challenge. As a control, the mice immunized with the DNA vector pcDNA3 showed a low level (40%) of protection, suggesting a nonspecific adjuvant effect of the plasmid DNA. Despite having no detectable neutralizing activity, the NS1 immunization elicited a strong antibody response exhibiting cytolytic activity against JEV-infected cells in a complement-dependent manner. By contrast, immunization with a construct expressing a longer NS1 protein (NS1′), containing an extra 60-amino-acid portion from the N terminus of NS2A, failed to protect mice against a lethal challenge. Biochemical analyses revealed that when individually expressed, NS1 but not NS1′ could be readily secreted as a homodimer in large quantity and could also be efficiently expressed on the cell surface. Interestingly, when NS1 and NS1′ coexisted in cells, the level of NS1 cell surface expression was much lower than that in cells expressing NS1 alone. These data imply that the presence of partial NS2A might have a negative influence on an NS1-based DNA vaccine. The results herein clearly illustrate that immunization with DNA expressing NS1 alone is sufficient to protect mice against a lethal JEV challenge.
Infectious Cellular Load in Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals and Susceptibility of Peripheral Blood Mononuclear Cells from Their Exposed Partners to Non-Syncytium-Inducing HIV-1 as Major Determinants for HIV-1 Transmission in Homosexual Couples
Authors: Blaak Hetty , van’t Wout Angélique B. , Brouwer Margreet , Cornelissen Marion , Kootstra Neeltje A. , Albrecht-van Lent Nel , Keet René P. M. , Goudsmit Jaap , Coutinho Roel A. , Schuitemaker Hanneke 
Published: December 2019

ABSTRACT

To study risk factors for homosexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared 10 monogamous homosexual couples between whom transmission of HIV-1 had occurred with 10 monogamous homosexual couples between whom HIV-1 transmission had not occurred despite high-risk sexual behavior. In the group of individuals who did not transmit virus, peripheral cellular infectious load was lower and the CD4
+
T-cell counts were higher than in the group of transmitters. HIV-1 RNA levels in serum did not differ between transmitters and nontransmitters. Compared with peripheral blood mononuclear cells (PBMC) from normal healthy blood donors, 8 of 10 nonrecipients and only 3 of 8 recipients had PBMC with reduced susceptibility to in vitro infection with non-syncytium-inducing (NSI) HIV-1 variants isolated from either their respective partners or an unrelated individual. No difference in susceptibility was observed for infection with a syncytium-inducing variant. Among the individuals who had PBMC with reduced susceptibility, five nonrecipients and one recipient had PBMC that were equally or even less susceptible to NSI variants than PBMC that had low susceptibility and that were derived from healthy blood donors that were heterozygous for a 32-bp deletion in the CCR5 gene (CCR5 Δ32). Three of these individuals (all nonrecipients) had a CCR5 Δ32 heterozygous genotype themselves, confirming an association between low susceptibility to NSI variants and CCR5 Δ32 heterozygosity. All three recipients with less susceptible PBMC had partners with a high infectious cellular load; inversely, both nonrecipients with normally susceptible PBMC had partners with a very low infectious cellular load. These results suggest that a combination of susceptibility of target cells and inoculum size upon homosexual exposure largely determines whether HIV-1 infection is established.
Antibody-Secreting Cell Responses and Protective Immunity Assessed in Gnotobiotic Pigs Inoculated Orally or Intramuscularly with Inactivated Human Rotavirus
Authors: Yuan Lijuan , Kang S.-Y. , Ward Lucy A. , To Thanh L. , Saif Linda J. 
Published: December 2019

ABSTRACTNewborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075–3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.
Mucosal and Parenteral Vaccination against Acute and Latent Murine Cytomegalovirus (MCMV) Infection by Using an Attenuated MCMV Mutant
Authors: MacDonald Margaret R. , Li Xi-Yang , Stenberg Richard M. , Campbell Ann E. , Virgin Herbert W. 
Published: December 2019

ABSTRACTWe used a live attenuated murine cytomegalovirus (MCMV) mutant to analyze mechanisms of vaccination against acute and latent CMV infection. We selected MCMV mutant RV7 as a vaccine candidate since this virus grows well in tissue culture but is profoundly attenuated for growth in normal and severe combined immunodeficient (SCID) mice (V. J. Cavanaugh et al., J. Virol. 70:1365–1374, 1996). BALB/c mice were immunized twice (0 and 14 days) subcutaneously (s.c.) with tissue culture-passaged RV7 and then challenged with salivary gland-passaged wild-type MCMV (sgMCMV) intraperitoneally (i.p.) on day 28. RV7 vaccination protected mice against challenge with 105PFU of sgMCMV, a dose that killed 100% of mock-vaccinated mice. RV7 vaccination reduced MCMV replication 100- to 500-fold in the spleen between 1 and 8 days after challenge. We used the capacity to control replication of MCMV in the spleen 4 days after challenge as a surrogate for protection. Protection was antigen specific and required both live RV7 and antigen-specific lymphocytes. Interestingly, RV7 was effective when administered s.c., i.p., perorally, intranasally, and intragastrically, demonstrating that attenuated CMV applied to mucosal surfaces can elicit protection against parenteral virus challenge. B cells and immunoglobulin G were not essential for RV7-induced immunity since B-cell-deficient mice were effectively vaccinated by RV7. CD8 T cells, but not CD4 T cells, were critical for RV7-induced protection. Depletion of CD8 T cells by passive transfer of monoclonal anti-CD8 (but not anti-CD4) antibody abrogated RV7-mediated protection, and RV7 vaccination was less efficient in CD8 T-cell-deficient mice with a targeted mutation in the β2-microglobulin gene. Although gamma interferon is important for innate resistance to MCMV, it was not essential for RV7 vaccination since gamma interferon receptor-deficient mice were protected by RV7 vaccination. Establishment of and/or reactivation from latency by sgMCMV was decreased by RV7 vaccination, as measured by diminished reactivation of MCMV from splenic explants. We found no evidence for establishment of splenic latency by RV7 after s.c. vaccination. We conclude that RV7 administered through both systemic and mucosal routes is an effective vaccine against MCMV infection. It may be possible to design human CMV vaccines with similar properties.
Characterization of a Murine Cytomegalovirus Class I Major Histocompatibility Complex (MHC) Homolog: Comparison to MHC Molecules and to the Human Cytomegalovirus MHC Homolog
Authors: Chapman Tara L. , Bjorkman Pamela J. 
Published: December 2019

ABSTRACTBoth human and murine cytomegaloviruses (HCMV and MCMV) down-regulate expression of conventional class I major histocompatibility complex (MHC) molecules at the surfaces of infected cells. This allows the infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to natural killer cells, which lyse cells that lack class I molecules. Both HCMV and MCMV encode class I MHC heavy-chain homologs that may function in immune response evasion. We previously showed that a soluble form of the HCMV class I homolog (UL18) expressed in Chinese hamster ovary cells binds the class I MHC light-chain β2-microglobulin and a mixture of endogenous peptides (M. L. Fahnestock, J. L. Johnson, R. M. R. Feldman, J. M. Neveu, W. S. Lane, and P. J. Bjorkman, Immunity 3:583–590, 1995). Consistent with this observation, sequence comparisons suggest that UL18 contains the well-characterized groove that serves as the binding site in MHC molecules for peptides derived from endogenous and foreign proteins. By contrast, the MCMV homolog (m144) contains a substantial deletion within the counterpart of its α2 domain and might not be expected to contain a groove capable of binding peptides. We have now expressed a soluble version of m144 and verified that it forms a heavy chain–β2-microglobulin complex. By contrast to UL18 and classical class I MHC molecules, m144 does not associate with endogenous peptides yet is thermally stable. These results suggest that UL18 and m144 differ structurally and might therefore serve different functions for their respective viruses.
The Viral Spike Protein Is Not Involved in the Polarized Sorting of Coronaviruses in Epithelial Cells
Authors: Rossen J. W. A. , de Beer R. , Godeke G.-J. , Raamsman M. J. B. , Horzinek M. C. , Vennema H. , Rottier P. J. M. 
Published: December 2019

ABSTRACT

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK
MHVR
cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK
MHVR
cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020–2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.
The Coronavirus Transmissible Gastroenteritis Virus Causes Infection after Receptor-Mediated Endocytosis and Acid-Dependent Fusion with an Intracellular Compartment
Authors: Hansen G. H. , Delmas B. , Besnardeau L. , Vogel L. K. , Laude H. , Sjöström H. , Norén O. 
Published: December 2019

ABSTRACT

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive infection, or whether they fuse directly with the plasma membrane. We have studied the interaction between TGEV and a cell line (MDCK) stably expressing recombinant pig aminopeptidase N (pAPN). By electron microscopy and flow cytometry, TGEV was found to be associated with the plasma membrane after adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38°C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride and bafilomycin A
1
markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site of fusion for TGEV.
Serine 257 Phosphorylation Regulates Association of Polyomavirus Middle T Antigen with 14-3-3 Proteins
Authors: Culleré Xavier , Rose Paul , Thathamangalam Usha , Chatterjee Alakananda , Mullane Karen P. , Pallas David C. , Benjamin Thomas L. , Roberts Thomas M. , Schaffhausen Brian S. 
Published: December 2019

ABSTRACT
Polyomavirus middle T antigen (MT) is phosphorylated on serine residues. Partial proteolytic mapping and Edman degradation identified serine 257 as a major site of phosphorylation. This was confirmed by site-directed mutagenesis. Isoelectric focusing of immunoprecipitated MT from transfected 293T cells showed that phosphorylation on wild-type MT occurred at near molar stoichiometry at S257. MT was previously shown to be associated with 14-3-3 proteins, which have been connected to cell cycle regulation and signaling. The association of 14-3-3 proteins with MT depended on the serine 257 phosphorylation site. This has been demonstrated by comparing wild-type and S257A mutant MTs expressed with transfected 293T cells or with Sf9 cells infected with recombinant baculoviruses. The 257 site is not critical for transformation of fibroblasts in vitro, since S257A and S257C mutant MTs retained the ability to form foci or colonies in agar. The tumor profile of a virus expressing S257C MT showed a striking deficiency in the induction of salivary gland tumors. The basis for this defect is uncertain. However, differences in activity for the wild type and mutant MT lacking the 14-3-3 binding site have been observed in transient reporter assays.
A Defective Interference-Like Phenomenon of Human Hepatitis B Virus in Chronic Carriers
Authors: Yuan Thomas Ta-Tung , Lin Min-Hui , Chen Ding-Shinn , Shih Chiaho 
Published: December 2019

ABSTRACTDefective interfering (DI) particles have been found in many RNA and DNA viruses of bacteria, plants, and animals since their first discovery in influenza virus. However, this fundamental phenomenon has not been demonstrated in human natural infections. Using a new approach, here we provide the first experimental evidence for the existence of DI-like viruses in human chronic carriers of hepatitis B virus (HBV). Functional characterization of naturally occurring core internal deletion (CID) variants of HBV revealed all of the features of DI particles. When equal amounts of wild-type and CID variant DNAs were cotransfected into a human hepatoma cell line, Huh7, a three- to fivefold enrichment of CID variants was most often observed. The fluctuations of the virus populations between CID variants and helper HBV in three chronic carriers are reminiscent of the cycling phenomenon in other DI viral systems. This finding has important implications for chronic viral hepatitis and other chronic progressive viral diseases.