bluuebunny/crossref_metadata_2025 huggingface dataset:
Localized sequence heterogeneity in the long terminal repeats of in vivo isolates of equine infectious anemia virus
Authors: Maury W , Perryman S , Oaks J L , Seid B K , Crawford T , McGuire T , Carpenter S
Published: January 2020

The role of in vivo long terminal repeat (LTR) sequence variation of the lentivirus equine infectious anemia virus (EIAV) has not been explored. In this study, we investigated the heterogeneity found in the LTR sequences from seven EIAV-seropositive horses: three horses with clinical disease and four horses without any detectable signs of disease. LTR sequences were targeted in this study because the LTR U3 enhancer region of tissue culture-derived isolates has been identified as one of the few hypervariable regions of the EIAV genome. Furthermore, LTR variation may regulate EIAV expression in vivo. Both intra- and interanimal sequence variations were investigated. The intra-animal variation was low in seropositive, healthy horses (on average 0.44%). Intra-animal variation was consistently higher in clinically ill horses (0.99%), suggesting that greater numbers of quasispecies of EIAV are present when active virus replication is ongoing. Interanimal comparisons of consensus sequences generated from each horse demonstrated that the enhancer region is a hotspot of sequence variation in vivo. Thirty-seven of the 83 nucleotides that compose the U3 enhancer region were variable between the different in vivo-derived LTRs. The remainder of the LTR that was analyzed was more conserved, 8 of 195 nucleotide positions being variable. Results of electrophoretic mobility shift assays demonstrated that some nucleotide substitutions that occurred in the enhancer region eliminated or altered transcription factor binding motifs that are known to be important for EIAV LTR expression. These data suggested that the selective pressures exerted on the EIAV LTR enhancer sequences are different from those exerted on the remainder of the LTR. Our findings are consistent with the possibility that enhancer sequence hypervariability can alter expression of the virus in tissue macrophages and therefore contribute to clinical disease in infected horses.
Protection of human immunodeficiency virus type 2-exposed seronegative macaques from mucosal simian immunodeficiency virus transmission
Authors: Putkonen P , Mäkitalo B , Böttiger D , Biberfeld G , Thorstensson R
Published: January 2020

At present it is not known which form of immunity would be most effective against infection with human immunodeficiency virus (HIV). To evaluate the possible role of cellular immunity, we examined whether four HIV type 2-exposed but seronegative macaques developed cellular immune responses and determined whether these exposed macaques were resistant to mucosal transmission of simian immunodeficiency virus (SIV). Following intrarectal challenge with SIV, 2 monkeys were protected against detectable SIV replication and another showed suppressed viral replication compared to 14 persistently infected controls. The two protected monkeys demonstrated SIV-specific cytotoxic T lymphocytes before as well as after SIV challenge. Here we provide evidence that activation of the cell-mediated arm of the immune system only, without antibody formation, can control SIV replication in macaques. The results imply that vaccines that stimulate a strong and broad cellular immune response could prevent mucosal HIV transmission.
Identification and characterization of a baculovirus structural protein, VP1054, required for nucleocapsid formation
Authors: Olszewski J , Miller L K
Published: January 2020

The defect in a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus, tsN1054, was mapped and characterized. At the nonpermissive temperature of 33 degrees C, this mutant fails to form plaques upon infection of Sf-21 cultured insect cells; infection is limited to a single cell, even though the infection proceeds through the very late phase. Marker rescue mapping and DNA sequencing identified the gene, ORF 54, which was altered by a single nucleotide substitution in tsN1054. Transcriptional analysis of the ORF 54 region identified multicistronic RNAs, from early to very late times of infection, that potentially encode the ORF 54 gene product. Polyclonal antiserum raised to a TrpE-VP1054 fusion protein recognized a 42-kDa late protein, VP1054, in infected-cell lysates. VP1054 was found to be a component of both budded virus and occlusion-derived virions. The level of VP1054 was dramatically reduced in tsN1054-infected Sf-21 cells propagated at 33 degrees C, and electron microscopic analysis of these cells showed that nucleocapsids failed to form in the nuclei of these infected cells. Instead, novel round, electron-dense bodies were found associated with the virogenic stroma in tsN1054-infected cells. Therefore, VP1054 is a virus structural protein required for nucleocapsid assembly.
Specific targeting to CD4+ cells of recombinant vesicular stomatitis viruses encoding human immunodeficiency virus envelope proteins
Authors: Johnson J E , Schnell M J , Buonocore L , Rose J K
Published: January 2020

We generated replication-competent, recombinant vesicular stomatitis viruses (VSVs) expressing the human immunodeficiency virus (HIV) envelope protein or an HIV-VSV chimeric envelope protein in which the cytoplasmic domain of the HIV envelope protein was replaced with that from the VSV glycoprotein (G). These recombinants were generated with HIV type 1 (HIV-1) envelopes from both laboratory and primary isolates of HIV-1. The replication-competent recombinant viruses were stable and expressed the foreign proteins at high levels from extra transcription units in VSV. The foreign proteins were processed appropriately and transported to the cell surface. The incorporation of HIV gp120 into VSV particles was demonstrated biochemically only for the construct expressing the chimeric envelopes containing the VSV G cytoplasmic domain. The incorporation of the chimeric HIV envelope protein into the membrane of the recombinant VSV was also demonstrated by electron microscopy with gold-conjugated antibodies. To determine whether specific infection of CD4-positive cells could be demonstrated for these recombinants, we neutralized VSV infectivity due to VSV glycoprotein with anti-VSV serum. The neutralized recombinants expressing the chimeric envelope were able to infect only HeLa cells expressing CD4, and this CD4-specific infectivity was neutralized with anti-HIV serum. This assay also detected a 100-fold-lower titer of CD4-specific infectivity for the VSV recombinant expressing the wild-type HIV envelope. Our results illustrate that it is possible to express functional HIV envelopes from the VSV genome and target the recombinant virus to an alternative receptor. The recombinants may also prove useful as HIV vaccines.
Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus
Authors: Pyper J M , Gartner A E
Published: January 2020

Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA.
The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells
Authors: Simon J H , Fouchier R A , Southerling T E , Guerra C B , Grant C K , Malim M H
Published: January 2020

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.
Characterization of glycosylated Gag expressed by a neurovirulent murine leukemia virus: identification of differences in processing in vitro and in vivo
Authors: Fujisawa R , McAtee F J , Zirbel J H , Portis J L
Published: January 2020

The neuroinvasiveness of a chimeric murine retrovirus, CasFrKP (KP), is dependent on the expression of glycosylated Gag (gp85gag). This viral protein is the product of alternate translation initiation 88 codons upstream of and in frame with the initiation codon of pr65gag, the precursor of the viral core proteins. Although expression of glycosylated Gag affects virus spread in the spleen, it appears not to affect virus spread in vitro in fibroblast cell lines (J. L. Portis et al., J. Virol. 68:3879-3887, 1994). The differential effects of this protein in vitro and in vivo have not been explained, and its function is unknown. We have here compared the in vitro processing of this molecule with that expressed in spleens of infected mice. In vitro, gp85gag was cleaved near the middle of the molecule, releasing the C-terminal half (containing capsid and nucleocapsid domains of pr65gag) as a secreted glycoprotein. The N-terminal half of the protein was associated with the plasma membrane as a approximately 55-kDa glycoprotein bearing the matrix domain of pr65gag as well as the N-terminal 88 residue L domain. This processing scheme was also observed in vivo, although two differences were seen. There were differences in N-linked glycosylation of the secreted form of the protein expressed in the spleen. In addition, whereas the membrane-associated species assumed the orientation of a type II integral membrane protein (N(cyto) C(exo)) in fibroblasts in vitro, a subpopulation of spleen cells was detected in which the N terminus of the protein was exposed at the cell surface. These results suggest that the differential effects of glycosylated Gag expression in vivo and in vitro may be related to differences in posttranslational processing of the protein.
Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair
Authors: Petersen J , Dandri M , Bürkle A , Zhang L , Rogler C E
Published: January 2020

Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency.
Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes
Authors: Hinkula J , Svanholm C , Schwartz S , Lundholm P , Brytting M , Engström G , Benthin R , Glaser H , Sutter G , Kohleisen B , Erfle V , Okuda K , Wigzell H , Wahren B
Published: January 2020

Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3- to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative B- and T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.
Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity
Authors: Ruesch M N , Laimins L A
Published: January 2020

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.
The pseudorabies virus UL51 gene product is a 30-kilodalton virion component
Authors: Lenk M , Visser N , Mettenleiter T C
Published: January 2020

Positional homologs to the UL51 open reading frame of herpes simplex virus type 1 have been identified throughout the herpesvirus family. However, no respective protein has so far been described for any of the herpesviruses. With rabbit antisera directed against oligopeptides predicted to comprise antigenic regions of the deduced pseudorabies virus (PrV) UL51 protein, a polypeptide with a size of 30 kDa was identified in PrV-infected cell lysates and in purified virions. This molecular mass correlates reasonably well with the predicted mass of 25 kDa of the 236-amino-acid deduced UL51 protein. Antisera raised against peptides derived from different predicted antigenic regions all detected the 30-kDa protein in Western blot (immunoblot) analyses. Specificity was ascertained by peptide competition. Subcellular fractionation showed the presence of the UL51 protein mainly in the nucleus of infected cells. After separation of purified virion preparations into envelope and capsid, the PrV UL51 protein was detected in the capsid fraction. In summary, we identified the first herpesvirus UL51 protein and demonstrate that it represents a structural component of PrV virions.
Parvovirus initiation factor PIF: a novel human DNA-binding factor which coordinately recognizes two ACGT motifs
Authors: Christensen J , Cotmore S F , Tattersall P
Published: January 2020

A novel human site-specific DNA-binding factor has been partially purified from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA replication at the 3' end of the minute virus of mice (MVM) genome and functions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the origin sequence in a reaction which requires ATP hydrolysis. DNase I and copper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the entire region in the minimal left-end origin not already known to be occupied by NS1. Methylation and carboxy-ethylation interference analysis identified two ACGT motifs, spaced by five nucleotides, as the sequences responsible for this binding. A series of mutant oligonucleotides was then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demonstrate that the two motifs comprise a single binding site rather than two separate sites. Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cellular factor is responsible for both mobility shift and nicking activities. UV cross-linking and relative mobility assays suggest that PIF binds DNA as a heterodimer or higher-order multimer with subunits in the 80- to 100-kDa range.
Functional management of an antiviral cytotoxic T-cell response
Authors: Bachmann M F , Speiser D E , Ohashi P S
Published: January 2020

Lymphocytic choriomeningitis virus (LCMV) is known to induce strong, polyclonal cytotoxic T-lymphocyte (CTL) responses. Using a set of variant peptides derived from the major CTL epitope of LCMV, we analyzed the functional fine specificity of the LCMV-specific CTL response. During the primary response, almost all the tested peptides were recognized. In contrast, the secondary response was purged of all minor cross-reactivities and very few peptides were significantly recognized. This study is the first demonstration of the functional maturation of a T-cell response and has important clinical and biological implications.
A viral function represses accumulation of transcripts from productive-cycle genes in mouse ganglia latently infected with herpes simplex virus
Authors: Chen S H , Kramer M F , Schaffer P A , Coen D M
Published: January 2020

Latent infections of neurons by herpes simplex virus form reservoirs of recurrent viral infections that resist cure. In latently infected neurons, viral gene expression is severely repressed; only the latency-associated transcripts (LATs) are expressed abundantly. Using sensitive reverse transcriptase PCR assays, we analyzed the effects of a deletion mutation in the LAT locus on viral gene expression in latently infected mouse trigeminal ganglia. The deletion mutation, which reduced expression of the major LATs 10(5)-fold, resulted in a approximately 5-fold increase in accumulation of transcripts from the immediate-early gene encoding ICP4, an essential transactivator of viral gene expression. The LAT deletion also resulted in a >10-fold increase in the accumulation of transcripts from the early gene encoding thymidine kinase, whose expression during productive infection stringently depends on ICP4, and positively affected the correlation of the levels of these transcripts with the levels of ICP4 transcripts. We also detected transcripts antisense to ICP4 RNA, which were in substantial excess to ICP4 transcripts in ganglia latently infected with wild-type virus. In contrast to its effects on productive-cycle transcripts, the LAT deletion reduced the accumulation of these antisense transcripts approximately 15-fold. Thus, a viral function associated with the LAT locus represses the accumulation of transcripts from at least two productive-cycle genes in latently infected mouse ganglia. We discuss possible mechanisms and consequences of this repression.
VBP and RelA regulate avian leukosis virus long terminal repeat-enhanced transcription in B cells
Authors: Curristin S M , Bird K J , Tubbs R J , Ruddell A
Published: January 2020

The avian leukosis virus (ALV) long terminal repeat (LTR) contains a compact transcription enhancer that is active in many cell types. A major feature of the enhancer is multiple CCAAT/enhancer element motifs that could be important for the strong transcriptional activity of this unit. The contributions of the three CCAAT/enhancer elements to LTR function were examined in B cells, as this cell type is targeted for ALV tumor induction following integration of LTR sequences next to the c-myc proto-oncogene. One CCAAT/enhancer element, termed a3, was found to be the most critical for LTR enhancement in transiently transfected B lymphoma cells, while in chicken embryo fibroblasts all three elements contributed equally to enhancement. Gel shift assays demonstrated that vitellogenin gene-binding protein (VBP), a member of the PAR subfamily of C/EBP factors, is a major component of the nuclear proteins binding to the a3 CCAAT/enhancer element. VBP activated transcription through the a3 CCAAT/enhancer element, supporting the idea that VBP is important for LTR enhancement in B cells. A member of the Rel family of proteins was also identified as a component of the a3 protein binding complex in B cells. Gel shift and immunoprecipitation assays indicated that this factor is RelA. Gel shift assays demonstrated that while RelA does not bind directly to the LTR CCAAT/enhancer elements, it does interact with VBP to potentiate VBP DNA binding activity. The synergistic interaction of VBP and RelA increased CCAAT/enhancer element-mediated transcription, indicating that both factors may be important for viral LTR regulation and also for expression of many cellular genes.
Upregulation of human immunodeficiency virus (HIV) replication by CD4 cross-linking in peripheral blood mononuclear cells of HIV-infected adults
Authors: Than S , Oyaizu N , Tetali S , Romano J , Kaplan M , Pahwa S
Published: January 2020

This study was conducted with peripheral blood mononuclear cells from 67 human immunodeficiency virus (HIV)-infected adults. It supports the hypothesis that cross-linking of CD4 molecules by HIV gp120 can result in HIV upregulation and spread of infection. Underlying mechanisms include activation of latent infection by factors in addition to, or other than, tumor necrosis factor alpha.
Type 1 and type 2 cytokine gene expression by viral gp135 surface protein-activated T lymphocytes in caprine arthritis-encephalitis lentivirus infection
Authors: Cheevers W P , Beyer J C , Knowles D P
Published: January 2020

Peripheral blood mononuclear cells (PBMC) from Saanen goats experimentally infected with the lentivirus caprine arthritis-encephalitis virus (CAEV) were evaluated by semiquantitative reverse transcriptase PCR for gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-2 gene expression following in vitro stimulation with purified CAEV gp135 surface protein (SU). Studies examined three goats with chronic arthritis and four clinically asymptomatic goats at 5 years postinfection. SU-responsive IFN-gamma mRNA-positive cells and IL-4 mRNA-positive cells in PBMC from infected goats reflected differences in lymphokine balance associated with disease status. IFN-gamma mRNA-positive cells were dominant in PBMC from asymptomatic goats, whereas SU-responsive IL-4 mRNA-positive cells were dominant in PBMC from goats with arthritis. IL-2 gene expression was not responsive to SU stimulation of PBMC from either asymptomatic or arthritic goats. Lymphokine mRNA profiles in SU-stimulated PBMC were dependent on the presence of CD4+ T lymphocytes. The results indicate that asymptomatic goats have a dominant population of CAEV SU-reactive T-helper 1 (Th1)-like lymphocytes in PBMC whereas goats with clinical arthritis have a dominant population of SU-reactive Th2-like lymphocytes.
A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus-cell fusion
Authors: Kumar M , Hassan M Q , Tyagi S K , Sarkar D P
Published: January 2020

Sendai virus envelopes devoid of hemagglutinin-neuraminidase but containing the fusion protein (F-virosomes) were prepared. F-virosomes exhibited discernible serine protease activity at neutral pH. Electrophoretic analysis of the protein profile of the F-virosomes under nonreducing conditions, by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, led to the identification of a previously unknown glycoprotein with a relative molecular weight of 45,000 (45K protein) associated with the F protein. The identity of the 45K protein, as distinct from F protein, was established by Western blot analysis with F- and 45K-specific antibodies. This 45K protein forms a nexus with the F protein through noncovalent hydrophobic interactions, as proved by its sensitivity to urea treatment, and it is essential for the proteolytic activity of the F-virosomes as well as for the fusion of the viral envelope with host cell membrane. N-terminal sequence analysis (first 11 amino acids) of this protein showed strong homology (> 90%) to flavivirus NS3 serine proteases but no similarity to any of the Sendai viral proteins. On the basis of the N-terminal sequence, oligonucleotides were designed corresponding to the sense and antisense DNA sequences. Dot blot hybridization and primer extension with these oligonucleotides with the viral and the host genome confirmed the host origin of this protein. Further, the limited proteolytic digestion of the target membrane resulted in significant inhibition of viral fusion with it. On the basis of these results, we postulate a model for the molecular mechanism of F protein-induced membrane fusion, which may provide a rationale for other paramyxoviruses.
Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences
Authors: Muerhoff A S , Smith D B , Leary T P , Erker J C , Desai S M , Mushahwar I K
Published: January 2020

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.
Activation domain requirements for disruption of Epstein-Barr virus latency by ZEBRA
Authors: Asković S , Baumann R
Published: January 2020

Latent infection of B lymphocytes by Epstein-Barr virus (EBV) can be disrupted by expression of the EBV ZEBRA protein. ZEBRA, a transcriptional activator, initiates the EBV lytic cascade by activating viral gene expression. ZEBRA is also indispensable for viral replication and binds directly to the EBV lytic origin of replication. The studies described herein demonstrate that the activation domain. ZEBRA activation can be replaced by a heterologous acidic, proline-rich, or glutamine-rich activation domain. ZEBRA activation domain swap constructs retain ZEBRA's native abilities to activate specific EBV promoters, to disrupt EBV latency, and to stimulate replication at the EBV lytic origin. Additional work, employing sequential and internal deletions of ZEBRA's N-terminal activation domain, indicates that its separate activities are not attributable to specific subdomains but are spread throughout its N terminus and therefore cannot be inactivated by deleting localized regions.
Characterization of the minute virus of mice P38 core promoter elements
Authors: Lorson C , Pintel D J
Published: January 2020

While the minute virus of mice (MVM) P4 promoter, which drives the viral nonstructural genes, is highly active in the absence of viral proteins, P38, the capsid gene promoter, is strictly dependent on the viral nonstructural protein NS1. Once fully transactivated, however, P38 mediates twice the steady-state level of expression achieved by P4. In this report, we address the discrepancy between the ability of P38 to mediate very high levels of activated transcription yet only low levels of basal expression, and we investigate the determinants that govern P38 basal expression. The isolated P38 core promoter elements (the P38 Sp1-binding site and TATA element) are at least as transcriptionally competent as the analogous P4 promoter elements. Proximally positioning P4 enhancer factor-binding sequences (nucleotides [nt] 57 to 157) upstream of isolated P38 core transcription regulatory elements or upstream of a native, though abbreviated, P38 cassette (MVM nt 1938 to 2072) confers significant levels of expression to P38 in the absence of NS1, while the full left-end hairpin sequences (nt 1 to 133) elevate basal P38 activity to levels equivalent to P4 basal levels. In the context of the complete viral genome, however, proximally positioned enhancer sequences are unable to confer significant levels of expression to P38, suggesting that low P38 basal levels are a consequence not only of a lack of proximal enhancer elements but also of additional positional regulatory constraints which can be overcome by NS1.
A 451-nucleotide circular RNA from cherry with hammerhead ribozymes in its strands of both polarities
Authors: Di Serio F , Daròs J A , Ragozzino A , Flores R
Published: January 2020

The sequence of 451 nucleotides of a cherry small circular RNA (csc RNA1) associated with a cherry disease has been determined. Both csc RNA1 and its complementary strand can form hammerhead structures similar to those found previously in other plant and animal small RNAs. In the branched secondary structure of lowest free energy of csc RNA1, the sequences involved in the hammerhead structures, which comprise approximately one-fourth of this RNA, are found opposite each other, forming part of a rod-like segment. Plus- and minus-strand full-length transcripts of csc RNA1 self-cleaved during transcription and after purification, as predicted by the hammerhead structures, which are stable and very probably act as single hammerhead structures. The minus-strand hammerhead structure of csc RNA1 is exceptional in having a central loop with only 11 conserved nucleotides, a situation previously observed in only one other natural hammerhead structure. Both hammerhead structures of csc RNA1 are also peculiar in having an A instead of a C preceding the self-cleavage sites. The in vivo concentration of the plus strand of csc RNA1 is only slightly higher than that of its complementary strand, and significant fractions of both strands are extracted from the tissue in the form of a complex. csc RNA1 has sequence similarities to viroids and especially to some viroid-like satellite RNAs; they also share some characteristics of their corresponding hammerhead structures with these satellite RNAs. These observations, together with the association in symptomatic tissue of csc RNA1 with a set of presumably viral double-stranded RNAs, suggest that csc RNA1 is a new viroid-like satellite RNA.
Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures
Authors: Westaway E G , Mackenzie J M , Kenney M T , Jones M K , Khromykh A A
Published: January 2020

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.
Bovine papillomavirus type 1 DNA replication: the transcriptional activator E2 acts in vitro as a specificity factor
Authors: Bonne-Andréa C , Tillier F , McShan G D , Wilson V G , Clertant P
Published: January 2020

We previously devised cell-free conditions supporting efficient replication of bovine papillomavirus type 1 (BPV1) DNA (C. Bonne-Andréa, S. Santucci, and P. Clertant, J. Virol. 69:3201-3205, 1995): the use of highly active preparations of viral initiator protein E1, together with extract from a particular cell source, allowed the synthesis of complete DNA circles through successive rounds of replication; this occurred in the absence of the viral transcriptional activator E2, required in vivo for the replication of viral genomes. We now report that adding E2 to cell-free assays produced only slight effects both on the yield of E1-dependent DNA synthesis and on the quality of newly made DNA molecules when a template carrying a wild-type BPV1 replication origin (ori) was used. The performance of mouse cell extracts, unable to sustain efficient BPV1 DNA replication in the presence of E1 only, was likewise not improved by the addition of E2. In a proper in vitro environment, E1 is thus fully capable of efficiently initiating viral DNA synthesis by itself, an activity which is not enhanced by interaction with E2. An important effect, however, was detected: E2 totally suppressed the nonspecific replication of ori-defective DNA templates, otherwise observed in high E1 concentrations. We examined the requirements for building a minimal DNA sequence behaving in vitro as a specific ori sequence under stringent recognition conditions, i.e., in the presence of both E1 and E2. Only two elements, the 18-bp E1 binding palindrome and an AT-rich sequence, were required in cis to allow specific cell-free DNA replication; there seemed to be no need for an E2 binding site to ensure discrimination between specific ori templates and other DNA molecules, even in the presence of E2. This suggests that during the initiation of BPV1 DNA replication, at least in vitro, E2 acts as a specificity factor restricting the action of E1 to a defined ori sequence; this function, likely not demanding the direct binding of E2 to cognate DNA sites, might primarily involve protein-protein interactions.
Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off
Authors: Mo H , Stamatatos L , Ip J E , Barbas C F , Parren P W , Burton D R , Moore J P , Ho D D
Published: January 2020

IgG1b12, a human monoclonal antibody (MAb) to an epitope overlapping the CD4-binding site on gp120, has broad and potent neutralizing activity against most primary human immunodeficiency virus type 1 (HIV-1) isolates. To assess whether and how escape mutants resistant to IgG1b12 can be generated, we cultured primary HIV-1 strain JRCSF in its presence. An escape mutant emerged which was approximately 100-fold more resistant to neutralization by IgG1b12. Both virion-associated and solubilized gp120 from this variant had a reduced affinity for IgG1b12, and sequencing of its env gene showed that amino acid substitutions had occurred at three positions within gp120. Two (D164N and D182N) were located in V2, and one (P365L) was in C3. By site-directed mutagenesis, we demonstrated that the D182N and P365L mutations, but not D164N, contribute to the IgG1b12-resistant phenotype. However, the former two substitutions, individually or in combination, hinder the replication of the neutralization-resistant virus. Introduction of the D164N substitution into the P365L variant results in a nonviable virus (D164N/P365L). In contrast, addition of D164N to the D182N or D182N/P365L mutant partially restored replicative function to near wild-type levels. Furthermore, we found that all of the IgG1b12-resistant mutant viruses remained sensitive to other human MAbs, such as 2G12 and 2F5, and to the CD4-IgG molecule, except that the P365L-containing mutant was slightly resistant to CD4-IgG. These results suggest that escape from IgG1b12 neutralization is due to a local rather than a global modification of the gp120 structure. Our findings have implications for the therapeutic and prophylactic applications of antibodies for HIV-1 infection.
Placement of tRNA primer on the primer-binding site requires pol gene expression in avian but not murine retroviruses
Authors: Fu W , Ortiz-Conde B A , Gorelick R J , Hughes S H , Rein A
Published: January 2020

In an early step in the retroviral infectious process, reverse transcriptase copies the genomic RNA of the virus into complementary minus-strand DNA. The primer for this synthetic event is a molecule of cellular tRNA, which is annealed by its 3' 18 nucleotides to a region of the genomic RNA termed the primer-binding site (PBS); the sequence of the PBS and hence the identity of the tRNA depend upon the retrovirus species. In addition to the primer tRNA, retrovirus particles contain a substantial number of other tRNA molecules. The latter tRNA population is enriched for the tRNA species which serves as primer for the virus. While there is considerable evidence that the enrichment for the primer species can be attributed to the pol gene product, nothing is known regarding mechanisms of annealing the primer to the PBS. We have analyzed pol- mutants of avian leukosis virus (ALV) and murine leukemia virus (MuLV) for the presence of primer at the PBS in virion genomic RNA. Remarkably, the results were different for the two viruses: the PBS was substantially occupied by primer in MuLV but not in ALV. Previous data indicates that the Pol-dependent enrichment of the primer within the virion is much greater in ALV than in MuLV. We therefore propose that the absence of primer at the PBS in pol- ALV is due to the deficiency of the primer species within the particle. The results suggest that, at least in MuLV, the tRNA is unwound by either the Gag protein or a cellular protein for annealing to the PBS. Further, the C-terminal 17 amino acids of Gag are unnecessary for this function in MuLV.
Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis
Authors: Berthoux L , Péchoux C , Ottmann M , Morel G , Darlix J L
Published: January 2020

Nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein containing two zinc fingers of the form (CX2CX4HX4C) and is present at about 2,000 copies inside the viral core. NCp7 molecules are tightly associated with the genomic RNA dimer to form the nucleocapsid, which also includes reverse transcriptase and integrase proteins. In vitro, NCp7 has been shown to bind specifically to HIV-1 RNA, inducing NCp7-NCp7 interactions. In the viral context, mutagenesis of amino acid residues in the zinc finger domains showed that NCp7 is responsible for the specific incorporation of genomic RNA into virions and is necessary for correct virion assembly and maturation. In this work, we investigated the consequences of mutating conserved basic residues in the N-terminal region that precedes the first zinc finger. Two of the mutants were poorly infectious and showed only limited, though significant, defects in RNA encapsidation and viral protein maturation. Electron microscopy, together with sucrose gradient analysis, revealed defects in particle core structure and heterogeneity among mutant virions. These defects were associated with strong reduction of proviral DNA synthesis and stability in newly infected cells. Taken together, these data show multiple and probably interdependent implications for the NCp7 protein in both early and late phases of the HIV-1 replicative cycle and emphasize it as a target for antiviral drug development.
Enhanced downregulation of Lck-mediated signal transduction by a Y114 mutation of herpesvirus Saimiri tip
Authors: Guo J , Duboise M , Lee H , Li M , Choi J K , Rosenzweig M , Jung J U
Published: January 2020

Tip of herpesvirus saimiri associates with Lck and downregulates Lck function in cellular signal transduction. In this report, we demonstrate that mutation of tyrosine 114 of Tip significantly increases Lck-binding activity. This mutant exhibits a dramatic increase in the suppression of cellular tyrosine phosphorylation and surface expression of lymphocyte antigens in comparison with wild-type Tip. In addition, the expression of TipY114 converted the transforming morphology of fibroblasts induced by oncogenic F505 Lck to a normal cellular morphology. These results further support a mechanism by which the association of Tip with Lck negatively regulates Lck-mediated signal transduction.