bluuebunny/crossref_metadata_2025 huggingface dataset:
Epstein-Barr Virus Contributes to the Malignant Phenotype and to Apoptosis Resistance in Burkitt’s Lymphoma Cell Line Akata
Authors: Komano Jun , Sugiura Makoto , Takada Kenzo
Published: December 2019

In the present study, we established an in vitro system representing the Burkitt’s lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.
Mutations in the N Terminus of the Brome Mosaic Virus Polymerase Affect Genetic RNA-RNA Recombination
Authors: Figlerowicz M. , Nagy P. D. , Tang N. , Kao C. C. , Bujarski J. J.
Published: December 2019

Previously, we have observed that mutations in proteins 1a and 2a, the two virally encoded components of the brome mosaic virus (BMV) replicase, can affect the frequency of recombination and the locations of RNA recombination sites (P. D. Nagy, A. Dzianott, P. Ahlquist, and J. J. Bujarski, J. Virol. 69:2547–2556, 1995; M. Figlerowicz, P. D. Nagy, and J. J. Bujarski, Proc. Natl. Acad. Sci. USA 94:2073–2078, 1997). Also, it was found before that the N-terminal domain of 2a, the putative RNA polymerase protein, participates in the interactions between 1a and 2a (C. C. Kao, R. Quadt, R. P. Hershberger, and P. Ahlquist, J. Virol. 66:6322–6329, 1992; E. O’Reilly, J. Paul, and C. C. Kao, J. Virol. 71:7526–7532, 1997). In this work, we examine how mutations within the N terminus of 2a influence RNA recombination in BMV. Because of the likely electrostatic character of 1a-2a interactions, five 2a mutants, MF1 to MF5, were generated by replacing clusters of acidic amino acids with their neutral counterparts. MF2 and MF5 retained nearly wild-type levels of 1a-2a interaction and were infectious inChenopodium quinoa. However, compared to that in wild-type virus, the frequency of nonhomologous recombination in both MF2 and MF5 was markedly decreased. Only in MF2 was the frequency of homologous recombination reduced and the occurrence of imprecise homologous recombination increased. In MF5 there was also a 3′ shift in the positions of homologous crossovers. The observed effects of MF2 and MF5 reveal that the 2a N-terminal domain participates in different ways in homologous and in nonhomologous BMV RNA recombination. This work maps specific locations within the N terminus involved in 1a-2a interaction and in recombination and further suggests that the mechanisms of the two types of crossovers in BMV are different.
Adenovirus Induction of an Interferon-Regulatory Factor during Entry into the Late Phase of Infection
Authors: Feigenblum David , Walker Robert , Schneider Robert J.
Published: December 2019

Virus infection of animal cells can induce intracellular antiviral responses mediated by the induction of interferon-regulatory transcription factors (IRFs), which bind to and control genes directed by the interferon-stimulated response element (ISRE). The purpose of this study was to determine whether adenovirus (Ad) induces IRFs during infection, because they might play a role in promoting viral pathogenesis. Here we show that after the late phase of infection, Ad induces a transcription factor related to the IRF family of factors. The IRF is induced shortly after Ad entry into late phase and is shown to stimulate ISRE-directed transcription, to require activation by protein tyrosine kinase signalling, and to be induced several hours prior to the inhibition of cell protein synthesis. Inhibition of tyrosine kinase activity blocks Ad induction and activation of the IRF. Attempts to identify the Ad-induced factor immunologically and by photo-UV cross-linking indicate that it is likely a novel member of the IRF family. Finally, several independent lines of evidence also suggest that Ad induction of the IRF might correlate with the ability of the virus to block host cell protein synthesis later during infection.
CD4+ T-Lymphocyte Depletion in Human Lymphoid Tissue Ex Vivo Is Not Induced by Noninfectious Human Immunodeficiency Virus Type 1 Virions
Authors: Sylwester A. W. , Grivel J.-C. , Fitzgerald W. , Rossio J. L. , Lifson J. D. , Margolis L. B.
Published: December 2019

We tested infectious human immunodeficiency virus type 1 (HIV-1), noninfectious but conformationally authentic inactivated whole HIV-1 virions, and purified gp120 for the ability to induce depletion of CD4+ T cells in human lymphoid tissues ex vivo. Infectious CXCR4-tropic HIV-1, but not matched inactivated virions or gp120, mediated CD4+ T-cell depletion, consistent with mechanisms requiring productive infection.
Identification and Characterization of an RNA-Dependent RNA Polymerase Activity within the Nonstructural Protein 5B Region of Bovine Viral Diarrhea Virus
Authors: Zhong Weidong , Gutshall Lester L. , Del Vecchio Alfred M.
Published: December 2019

Nonstructural protein 5B (NS5B) of bovine viral diarrhea virus (BVDV) contains sequence motifs that are predictive of an RNA-dependent RNA polymerase activity. We describe the expression and purification of the BVDV NS5B protein derived from an infectious cDNA clone of BVDV (NADL strain). BVDV NS5B protein was active in an in vitro RNA polymerase assay using homopolymeric RNA or BVDV minigenomic RNA templates. The major product was a covalently linked double-stranded molecule generated by a “copy-back” mechanism from the input template RNA. In addition, a nucleotide-nonspecific and template-independent terminal nucleotidyl transferase activity was observed with the BVDV NS5B preparation.
Mapping of Epitopes Exposed on Intact Human Immunodeficiency Virus Type 1 (HIV-1) Virions: a New Strategy for Studying the Immunologic Relatedness of HIV-1
Authors: Nyambi Phillipe N. , Gorny Miroslaw K. , Bastiani Lisa , van der Groen Guido , Williams Constance , Zolla-Pazner Susan
Published: December 2019

To study the antigenic conservation of epitopes of human immunodeficiency virus type 1 (HIV-1) isolates of different clades, the abilities of human anti-HIV-1 gp120 and gp41 monoclonal antibodies (MAbs) to bind to intact HIV-1 virions were determined by a newly developed virus-binding assay. Eighteen human anti-HIV MAbs, which were directed at the V2, V3 loop, CD4-binding domain (CD4bd), C5, or gp41 regions, were used. Nine HIV-1 isolates from clades A, B, D, F, G, and H were used. Microtiter wells were coated with the MAbs, after which virus was added. Bound virus was detected after lysis by testing for p24 antigen with a noncommercial p24 enzyme-linked immunosorbent assay. The anti-V3 MAbs strongly bound the four clade B viruses and viruses from the non-B clades, although binding was weaker and more sporadic with the latter. The degrees of binding by the anti-V3 MAbs to CXCR4- and CCR5-tropic viruses were similar, suggesting that the V3 loops of these two categories of viruses are similarly exposed. The anti-C5 MAbs bound isolates of clades A, B, and D. Only weak and sporadic binding of all the viruses tested with anti-CD4bd, anti-V2, and anti-gp41 MAbs was detected. These results suggest that V3 and C5 structures are shared and well exposed on intact virions of different clades compared to the CD4bd, V2, and gp41 regions.
Requirement for CD4+ T Cells in the Friend Murine Retrovirus Neutralizing Antibody Response: Evidence for Functional T Cells in Genetic Low-Recovery Mice
Authors: Super Heidi J. , Brooks Diane , Hasenkrug Kim , Chesebro Bruce
Published: December 2019

Recovery from infection with the Friend murine leukemia retrovirus complex (FV) requires T-helper cells and cytotoxic T cells as well as neutralizing antibodies. Several host genes, including genes of the major histocompatibility complex (H-2) and anH-2-unlinked gene,Rfv-3, influence these FV-specific immune responses. (B10.A × A/Wy)F1mice, which have theH-2a/aRfv-3r/sgenotype, fail to mount a detectable FV-specific T-cell proliferative response but nevertheless produce FV-specific neutralizing immunoglobulin M (IgM) antibodies and can eliminate FV viremia. Thus, this IgM response, primarily influenced by theRfv-3gene, may be T-cell independent. To test this idea, mice were depleted of either CD4+or CD8+T-cell populations in vivo and were monitored for the effect on the neutralizing antibody response following FV infection. Surprisingly, mice in which CD4+cells were depleted showed undetectable FV-neutralizing antibody responses and high viremia levels compared to nondepleted or CD8-depleted animals. In addition to knocking out the FV antibody response, CD4+T-cell depletion reduced survival time significantly, further indicating the importance of CD4+T cells. These studies revealed the first evidence for a functional T-cell response following FV infection in these low-recovery mice and showed that CD4+T-helper cells are required for theRfv-3-controlled FV antibody response.
Distinct Biology of Kaposi’s Sarcoma-Associated Herpesvirus from Primary Lesions and Body Cavity Lymphomas
Authors: Friborg Jacques , Kong Wing-Pui , Flowers C. Clay , Flowers Scarlett L. , Sun Yongnian , Foreman Kimberly E. , Nickoloff Brian J. , Nabel Gary J.
Published: December 2019

The DNA sequence for Kaposi’s sarcoma-associated herpesvirus was originally detected in Kaposi’s sarcoma biopsy specimens. Since its discovery, it has been possible to detect virus in cell lines established from AIDS-associated body cavity-based B-cell lymphoma and to propagate virus from primary Kaposi’s sarcoma lesions in a human renal embryonic cell line, 293. In this study, we analyzed the infectivity of Kaposi’s sarcoma-associated herpesvirus produced from these two sources. Viral isolates from cultured cutaneous primary KS cells was transmitted to an Epstein-Barr virus-negative Burkitt’s B-lymphoma cell line, Louckes, and compared to virus induced from a body cavity-based B-cell lymphoma cell line. While propagation of body cavity-based B-cell lymphoma-derived virus was not observed in 293 cell cultures, infection with viral isolates obtained from primary Kaposi’s sarcoma lesions induced injury in 293 cells typical of herpesvirus infection and was associated with apoptotic cell death. Interestingly, transient overexpression of the Kaposi’s sarcoma-associated herpesvirus v-Bcl-2 homolog delayed the process of apoptosis and prolonged the survival of infected 293 cells. In contrast, the broad-spectrum caspase inhibitors Z-VAD-fmk and Z-DEVD-fmk failed to protect infected cell cultures, suggesting that Kaposi’s sarcoma-associated herpesvirus-induced apoptosis occurs through a Bcl-2-dependent pathway. Kaposi’s sarcoma-associated herpesvirus isolates from primary Kaposi’s sarcoma lesions and body cavity-based lymphomas therefore may differ and are likely to have distinct contributions to the pathophysiology of Kaposi’s sarcoma.
A Novel YY1-Independent Silencer Represses the Activity of the Human Papillomavirus Type 16 Enhancer
Authors: O’Connor Mark J. , Stünkel Walter , Zimmermann Holger , Koh Choon-Heng , Bernard Hans-Ulrich
Published: December 2019

Regulation of the human papillomavirus type 16 (HPV-16) E6 promoter is a complex process in which transcriptional repression as well as activation plays an important role. Here, we identify a negative regulatory element that in the context of a continuous long control region fragment overcomes the activation of the HPV-16 enhancer. This silencing element, which we have termed a PSM (papillomavirus silencing motif), consists of two copies of the sequence 5′-TAYAATAAT-3′ that overlap the origin of replication. Each copy of this 9-bp sequence binds the same unknown cellular factor, which we refer to as PSM-BP (PSM binding protein). Both copies of the binding sequence are required for transcriptional repression, and we provide evidence that suggests that this particular organization results in the stabilization of a PSM-BP dimer. The silencing motif, while functioning in either orientation, showed a positional requirement between the enhancer and the promoter. Experiments with both a heterologous enhancer and a promoter also demonstrated a general ability of this element to function as a transcriptional silencer in non-HPV systems. Our findings provide an important addition to our understanding of HPV-16 gene regulation and an interesting model for the study of transcriptional repression.
The Origin DNA-Binding and Single-Stranded DNA-Binding Domains of Simian Virus 40 Large T Antigen Are Distinct
Authors: Wu Chunxiao , Edgil Dianna , Simmons Daniel T.
Published: December 2019

Little is known about the ability of simian virus 40 (SV40) T antigen to bind single-stranded DNA. We demonstrate here that a mutant (259-708) missing the first 258 amino acids of T antigen and its origin-binding domain bound single-stranded DNA at close to normal levels, whereas a mutant containing only the first 259 amino acids failed to bind any single-stranded DNA. The 259-708 mutant also assembled into high-molecular-weight oligomers in the presence of single-stranded DNA. Its ATPase activity was stimulated by single-stranded DNA similarly to the wild type (WT). Furthermore, WT T antigen’s ability to bind to single-stranded DNA was inhibited by the binding of two monoclonal antibodies that recognize a region after residue 362. These results show that the domain responsible for binding to single-stranded DNA is completely separate from the origin-binding domain.
Morbillivirus Downregulation of CD46
Authors: Galbraith Sareen E. , Tiwari Ashok , Baron Michael D. , Lund Brett T. , Barrett Thomas , Cosby S. Louise
Published: December 2019

There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.
Simian T-Cell Lymphotropic Virus Type 1 from
Mandrillus sphinx
as a Simian Counterpart of Human T-Cell Lymphotropic Virus Type 1 Subtype D
Authors: Mahieux Renaud , Chappey Colombe , Georges-Courbot Marie-Claude , Dubreuil Guy , Mauclere Philippe , Georges Alain , Gessain Antoine
Published: December 2019

A recent serological and molecular survey of a semifree-ranging colony of mandrills (
Mandrillus sphinx
) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR),
env
, and
rex
regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the
env
gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.
Simian Sarcoma-Associated Virus Fails To Infect Chinese Hamster Cells despite the Presence of Functional Gibbon Ape Leukemia Virus Receptors
Authors: Ting Yuan-Tsang , Wilson Carolyn A. , Farrell Karen B. , Chaudry G. Jilani , Eiden Maribeth V.
Published: December 2019

We have sequenced the envelope genes from each of the five members of the gibbon ape leukemia virus (GALV) family of type C retroviruses. Four of the GALVs, including GALV strain SEATO (GALV-S), were originally isolated from gibbon apes, whereas the fifth member of this family, simian sarcoma-associated virus (SSAV), was isolated from a woolly monkey and shares 78% amino acid identity with GALV-S. To determine whether these viruses have identical host ranges, we evaluated the susceptibility of several cell lines to either GALV-S or SSAV infection. GALV-S and SSAV have the same host range with the exception of Chinese hamster lung E36 cells, which are susceptible to GALV-S but not SSAV. We used retroviral vectors that differ only in their envelope composition (e.g., they contain either SSAV or GALV-S envelope protein) to show that the envelope of SSAV restricts entry into E36 cells. Although unable to infect E36 cells, SSAV infects GALV-resistant murine cells expressing the E36-derived viral receptor, HaPit2. These results suggest that the receptors present on E36 cells function for SSAV. We have constructed several vectors containing GALV-S/SSAV chimeric envelope proteins to map the region of the SSAV envelope that blocks infection of E36 cells. Vectors bearing chimeric envelopes comprised of the N-terminal region of the GALV-S SU protein and the C-terminal region of SSAV infect E36 cells, whereas vectors containing the N-terminal portion of the SSAV SU protein and C-terminal portion of GALV-S fail to infect E36 cells. This finding indicates that the region of the SSAV envelope protein responsible for restricting SSAV infection of E36 cells lies within its amino-terminal region.
p53 Status Does Not Determine Outcome of E1B 55-Kilodalton Mutant Adenovirus Lytic Infection
Authors: Goodrum Felicia D. , Ornelles David A.
Published: December 2019

The ability of the adenovirus type 5 E1B 55-kDa mutants
dl
1520 and
dl
338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G
1
. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.
Recombinant, Replication-Defective Adenovirus Gene Transfer Vectors Induce Cell Cycle Dysregulation and Inappropriate Expression of Cyclin Proteins
Authors: Wersto Robert P. , Rosenthal Eugene R. , Seth Prem K. , Eissa N. Tony , Donahue Robert E.
Published: December 2019

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (ΔE1) or lacking the Ad E3 region in addition to E1 sequences (ΔE1ΔE3) induced G
2
cell cycle arrest and inhibited traverse across G
1
/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34

cdc2

protein levels. In some instances, infection with ΔE1 or ΔE1ΔE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation ΔE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in ΔE1 or ΔE1ΔE3 Ad vectors induce G
2
growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.
Interaction of Theiler’s Virus with Intermediate Filaments of Infected Cells
Authors: Nédellec Patrick , Vicart Patrick , Laurent-Winter Christine , Martinat Cécile , Prévost Marie-Christine , Brahic Michel
Published: December 2019

Theiler’s murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler’s virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.
Effect of Bafilomycin A1 and Nocodazole on Endocytic Transport in HeLa Cells: Implications for Viral Uncoating and Infection
Authors: Bayer Nora , Schober Daniela , Prchla Elisabeth , Murphy Robert F. , Blaas Dieter , Fuchs Renate
Published: December 2019

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used
35
S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713–3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.
Human Immunodeficiency Virus Induces a Dual Regulation of Bcl-2, Resulting in Persistent Infection of CD4+T- or Monocytic Cell Lines
Authors: Aillet Fabienne , Masutani Hiroshi , Elbim Carole , Raoul Hervé , Chêne Laurent , Nugeyre Marie-Thérèse , Paya Carlos , Barré-Sinoussi Françoise , Gougerot-Pocidalo Marie-Anne , Israël Nicole
Published: December 2019

This work aims at characterizing the interplay between human immunodeficiency virus type 1 (HIV-1) and the antiapoptotic cellular protein Bcl-2 responsible for a persistent infection in lymphoblastoid T (J.Jhan) or monocytic (U937) cells. We report that the kinetics of Bcl-2 protein level during the establishment of a chronic infection is biphasic, characterized by a transient decrease followed by restoration to the initial level. The extent and duration of this transient decrease were inversely correlated with the basal level of Bcl-2 as shown by kinetics of Bcl-2 levels in J.Jhan or U937 clones exhibiting different levels of Bcl-2. Using these clones, we also showed that Bcl-2 downregulates HIV-1 replication. Therefore, the cells overexpressing Bcl-2 are characterized by a low viral burden which, in turn, has little effect on the level of this protein. The observed bipasic kinetics is the result of a dual regulation of Bcl-2 induced by HIV-1 infection itself: an upregulation at the transcriptional level of thebcl-2gene concomitant with a downregulation at the protein level. Convergent data suggest that this downregulation is caused by the oxidative stress induced by the infection itself as shown by the associated modulations of glutathione and thioredoxin levels and by the prevention of these dysregulations byN-acetylcysteine. Altogether, these data indicate that infection first results in a decrease of Bcl-2, permitting an initial boost of replication. Then, as the synthesis at the transcriptional level proceeds, the replication is negatively controlled by Bcl-2 to reach a balance characterized by low virus production and a level of Bcl-2 compatible with cell survival. We suggest that the basal level of Bcl-2, together with infection-inducible transcription factors able to activatebcl-2gene transcription, is a critical cellular determinant in the tendency toward an acute or a persistent infection.
Ectopic Expression of Hepatitis C Virus Core Protein Differentially Regulates Nuclear Transcription Factors
Authors: Shrivastava Anju , Manna Sunil K. , Ray Ranjit , Aggarwal Bharat B.
Published: December 2019

The putative core protein of hepatitis C virus (HCV) regulates cellular growth and a number of cellular promoters. To further understand its effect, we investigated the role of the core protein in the endogenous regulation of two distinct transcription factors, nuclear factor-κB (NF-κB) and activating protein-1 (AP-1), and the related mitogen-activated protein kinase kinase (MAPKK) and c-Jun N-terminal kinase (JNK). Stable cell transfectants expressing the HCV core protein suppressed tumor necrosis factor (TNF)-induced NF-κB activation. Supershift analysis revealed that NF-κB consists of p50 and p65 subunits. This correlated with inhibition of the degradation of IκBα, the inhibitory subunit of NF-κB. The effect was not specific to TNF, as suppression in core protein-expressing cells was also observed in response to a number of other inflammatory agents known to activate NF-κB. In contrast to the effect on NF-κB, the HCV core protein constitutively activated AP-1, which correlated with the activation of JNK and MAPKK, which are known to regulate AP-1. These observations indicated that the core protein targets transcription factors known to be involved in the regulation of inflammatory responses and the immune system.
Study of Dengue Virus Infection in SCID Mice Engrafted with Human K562 Cells
Authors: Lin Yi-Ling , Liao Ching-Len , Chen Li-Kuang , Yeh Chia-Tsui , Liu Chiu-I , Ma Shiou-Hwa , Huang Yu-Ying , Huang Yue-Ling , Kao Chuan-Liang , King Chwan-Chuen
Published: December 2019

Here we report that severe combined immunodeficient (SCID) mice engrafted with human K562 cells (K562-SCID mice) can be used as an animal model to study dengue virus (DEN) infection. After intratumor injection into K562 cell masses of PL046, a Taiwanese DEN-2 human isolate, the K562-SCID mice showed neurological signs of paralysis and died at approximately 2 weeks postinfection. In addition to being detected in the tumor masses, high virus titers were detected in the peripheral blood and the brain tissues, indicating that DEN had replicated in the infected K562-SCID mice. In contrast, the SCID mice were resistant to DEN infection and the mock-infected K562-SCID mice survived for over 3 months. These data illustrate that DEN infection contributed directly to the deaths of the infected K562-SCID mice. Other serotypes of DEN were also used to infect the K562-SCID mice, and the mortality rates of the infected mice varied with different challenge strains, suggesting the existence of diverse degrees of virulence among DENs. To determine whether a neutralizing antibody against DEN in vitro was also protective in vivo, the K562-SCID mice were challenged with DEN-2 and received antibody administration at the same time or 1 day earlier. Our results revealed that the antibody-treated mice exhibited a reduction in mortality and a delay of paralysis onset after DEN infection. In contrast to K562-SCID, the persistently DEN-infected K562 cells generated in vitro invariably failed to be implanted in the mice. It seems that in the early stage of implantation, a gamma interferon activated, nitric oxide-mediated anti-DEN effect might play a role in the innate immunity against DEN-infected cells. The system described herein offers an opportunity to explore DEN replication in vivo and to test various antiviral protocols in infected hosts.
Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery
Authors: Zufferey Romain , Dull Thomas , Mandel Ronald J. , Bukovsky Anatoly , Quiroz Dulce , Naldini Luigi , Trono Didier
Published: December 2019

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3′ long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.
Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C
Authors: Nüesch Jürg P. F. , Dettwiler Sabine , Corbau Romuald , Rommelaere Jean
Published: December 2019

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1
P
) supported RCR under these conditions, dephosphorylated NS1 (NS1
O
) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1
O
, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1
O
for replication. One of these components, containing atypical PKC, was sufficient to restore NS1
O
helicase activity. The requirement of NS1
O
reactivation for characteristic PKC cofactors such as Ca
2+
/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro.
A New System for Stringent, High-Titer Vesicular Stomatitis Virus G Protein-Pseudotyped Retrovirus Vector Induction by Introduction of Cre Recombinase into Stable Prepackaging Cell Lines
Authors: Arai Tohru , Matsumoto Kazuyuki , Saitoh Kanako , Ui Motoyasu , Ito Taiji , Murakami Masao , Kanegae Yumi , Saito Izumu , Cosset François-Loïc , Takeuchi Yasuhiro , Iba Hideo
Published: December 2019

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the
gag-pol
genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neo
r
) gene and a poly(A) signal flanked by a tandem pair of
loxP
sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neo
r
expression. By inserting an mRNA-destabilizing signal into the 3′ untranslated region of the Neo
r
gene to reduce the amount of Neo
r
transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding β-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 × 10
6
infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.
Overexpression of Nonconvertible PrP

c

Δ114–121 in Scrapie-Infected Mouse Neuroblastoma Cells Leads to
trans
-Dominant Inhibition of Wild-Type PrP

Sc

Accumulation
Authors: Hölscher Christina , Delius Hajo , Bürkle Alexander
Published: December 2019

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP

Sc

of a normal cellular glycoprotein, PrP

c

, which is characterized by a high content of β-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109–122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrP

c

molecule, should play a role in promoting the conversion into PrP

Sc

. By using persistently scrapie-infected mouse neuroblastoma (Sc
+
-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrP

c

that are capable of blocking the conversion of endogenous, wild-type PrP

c

into PrP

Sc

. We constructed a deletion mutant (PrP

c

Δ114–121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109–122). Transient transfections of mammalian expression vectors encoding either wild-type PrP

c

or PrP

c

Δ114–121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrP

c

versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc
+
-MNB cells revealed that PrP

c

Δ114–121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP

Sc

derived from endogenous PrP

c

. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrP

c

plays an important role in the conversion process of PrP

c

into PrP

Sc

and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP

Sc

accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.
The Application of a Homologous Recombination Assay Revealed Amino Acid Residues in an LTR-Retrotransposon That Were Critical for Integration
Authors: Atwood Angela , Choi Jeannie , Levin Henry L.
Published: December 2019

Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells.
Schizosaccharomyces pombe
is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics. In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition. In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration. By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts. These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition. Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain. These results suggested that each of the three IN domains was required for Tf1 transposition. The potential role of these five amino acid residues in the function of IN is discussed. Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase. The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis. These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts.
Development and Characterization of an In Vivo Pathogenic Molecular Clone of Equine Infectious Anemia Virus
Authors: Cook R. Frank , Leroux Caroline , Cook Sheila J. , Berger Sandra L. , Lichtenstein Drew L. , Ghabrial Nadia N. , Montelaro Ronald C. , Issel Charles J.
Published: December 2019

An infectious nonpathogenic molecular clone (19-2-6A) of equine infectious anemia virus (EIAV) was modified by substitution of a 3.3-kbp fragment amplified by PCR techniques from a pathogenic variant (EIAV
PV
) of the cell culture-adapted strain of EIAV (EIAV
PR
). This substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the S1 (encoding the second exon of
tat
), S2, and S3 (encoding the second exon of
rev
) open reading frames, the complete
env
gene (including the first exon of
rev
), and the 3′ long terminal repeat (LTR). Modified 19-2-6A molecular clones were designated EIAV
PV3.3
, and infection of a single pony (678) with viruses derived from a mixture of five of these molecular clones induced clinical signs of acute equine infectious anemia (EIA) at 23 days postinfection (dpi). As a consequence of this initial study, a single molecular clone, EIAV
PV3.3#3
(redesignated EIAV
UK
), was selected for further study and inoculated into two ponies (613 and 614) and two horses (700 and 764). Pony 614 and the two horses developed febrile responses by 12 dpi, which was accompanied by a 48 to 64% reduction in platelet number, whereas pony 613 did not develop fever (40.6°C) until 76 dpi. EIAV could be isolated from the plasma of these animals by 5 to 7 dpi, and all became seropositive for antibodies to this virus by 21 dpi. Analysis of the complete nucleotide sequence demonstrated that the 3.3-kbp 3′ fragment of EIAV
UK
differed from the consensus sequence of EIAV
PV
by just a single amino acid residue in the second exon of the rev gene. Complete homology with the EIAV
PV
consensus sequence was observed in the hypervariable region of the LTR. However, EIAV
UK
was found to contain an unusual 68-bp nucleotide insertion/duplication in a normally conserved region of the LTR sequence. These results demonstrate that substitution of a 3.3-kbp fragment from the EIAV
PV
strain into the infectious nonpathogenic molecular clone 19-2-6A leads to the production of progeny virus particles with the ability to induce clinical signs of EIA. Therefore, EIAV
UK
, which is the first pathogenic, cell culture-adapted molecular clone of EIAV to be described, should be of value in identifying viral determinants of pathogenicity.
A Vaccinia Virus Late Transcription Factor Copurifies with a Factor That Binds to a Viral Late Promoter and Is Complemented by Extracts from Uninfected HeLa Cells
Authors: Wright Cynthia F. , Hubbs Alan E. , Gunasinghe Sajeevani K. , Oswald Betty W.
Published: December 2019

We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.
Moesin Is Not a Receptor for Measles Virus Entry into Mouse Embryonic Stem Cells
Authors: Doi Yoshinori , Kurita Mitsue , Matsumoto Misako , Kondo Takahisa , Noda Tetsuo , Tsukita Sachiko , Tsukita Shoichiro , Seya Tsukasa
Published: December 2019

The involvement of moesin in measles virus (MV) entry was investigated with moesin-positive and -negative mouse embryonic stem (ES) cells. MV infection of these cells was very ineffective and was independent of moesin expression. Furthermore, when these cells were transfected to express human CD46, a 100-fold increase in syncytium formation was observed with these cells and was independent of the expression of moesin. The only obvious difference between moesin-positive and -negative ES cells was the shape of the syncytia formed. Moesin-negative ES cells expressing or not expressing human CD46 formed separate pieces of fragmented syncytia which were torn apart during spreading, whereas ES cells expressing moesin exhibited typical syncytia. In addition, moesin was not detected on the surface of any murine cells or cell lines that we have tested by a flow cytometric assay with moesin-specific antibodies. These findings indicate that murine moesin is neither a receptor nor a CD46 coreceptor for MV entry into mouse ES cells. Moesin is involved in actin filament-plasma membrane interactions as a cross-linker, and it affects only the spreading and shape of MV-mediated syncytia.