bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a region of a murine leukemia virus long terminal repeat with novel transcriptional regulatory activities
Authors: Chen H , Yoshimura F K
Published: January 2020

The 93-bp region downstream of the enhancer (DEN) in the long terminal repeat (LTR) of the mink cell focus-forming virus (MCF13) has been shown to be important for transcriptional activation and viral lymphomagenicity (J. C. Tupper, H. Chen, E. F. Hays, G. C. Bristol, and F. K. Yoshimura, J. Virol. 66:7080-7088, 1992). In this report, we have further explored the role of the DEN region in transcriptional activation. We observed that it has enhancer-like abilities as well as some unique LTR properties. Transcriptional activation by the DEN region involved interactions with enhancer sequences that were either synergistic or additive, depending on the cell type. The most intriguing property of the DEN region is its ability to induce transcription in activated T cells. This activity is unique for the LTR in that no other LTR region can do this. We also examined the role of the DEN region in retroviral lymphomagenesis. We cloned and sequenced proviral LTRs integrated upstream of the cellular c-myc gene from DNA obtained from thymic tumors induced by DEN region deletion mutant viruses in AKR mice. We determined that for transcriptional activation of the c-myc proto-oncogene, enhancer sequences can substitute for the DEN region. This study identifies the significance of non-enhancer sequences in the LTR for the oncogenesis of the MCF13 retrovirus.
VCAM-1 is a receptor for encephalomyocarditis virus on murine vascular endothelial cells
Authors: Huber S A
Published: January 2020

Murine VCAM-1 has been identified as a receptor for the D variant of encephalomyocarditis (EMC-D) virus on vascular endothelial cells from the heart. Monoclonal antibodies to VCAM-1 inhibited infection and lysis of endothelial cells with EMC-D virus. CHO cells transfected with the VCAM-1 gene were susceptible to EMC-D virus lysis, while control CHO cells transfected with the ELAM-1 gene were resistant. Similarly, 35S-labeled EMC-D virus bound to CHO-VCAM cells, and binding was inhibited with anti-VCAM-1 antibodies. Little or no radiolabeled virus bound to CHO-ELAM-1 cells.
Alteration of V3 loop context within the envelope of human immunodeficiency virus type 1 enhances neutralization
Authors: Robert-Guroff M , Louie A , Myagkikh M , Michaels F , Kieny M P , White-Scharf M E , Potts B , Grogg D , Reitz M S
Published: January 2020

Neutralization of a chimeric human immunodeficiency virus (HIV) type 1, containing the V3 loop of the MN isolate substituted within the HXB2 envelope, was enhanced up to 20-fold compared with the HXB2 or MN parental isolates by human HIV-positive sera. MN V3 loop-specific monoclonal antibodies were better able to recognize the chimeric virus compared with MN, staining a greater percentage of infected cells and exhibiting slight increases in relative affinity with a concomitant increase in neutralization titer. Competition analysis revealed that enhanced neutralization by human HIV-positive sera of the chimera was attributable in some cases to better reactivity with the linear V3 loop epitope but in others to conformational loop epitopes or previously cryptic or poorly recognized epitopes outside the loop region. Mice primed with a vaccinia virus-chimeric envelope recombinant and boosted with gp160 developed a spectrum of antibodies different from that of mice similarly immunized with HXB2 or MN recombinants or that of naturally infected humans. The chimeric envelope elicited antibodies with enhanced binding to the native MN V3 loop; however, the sites seen by the BALB/c mice were not neutralizing epitopes. Nevertheless, similar to the observations made with use of human sera, the chimeric virus was more readily neutralized by all of the immune mouse sera, an effect apparently mediated by non-V3 loop epitopes. These studies illustrate that not only the V3 loop sequence and conformation but also its context within the viral envelope influence neutralization.
Identification of an immediate-early gene in the Marek's disease virus long internal repeat region which encodes a unique 14-kilodalton polypeptide
Authors: Hong Y , Coussens P M
Published: January 2020

Marek's disease virus (MDV) is an oncogenic avian herpesvirus whose genomic structure is similar to those of herpes simplex virus and varicella-zoster virus. Repeat regions of the MDV genome have been intensively investigated because of a potential relationship to MDV oncogenicity and abundant expression of immediate-early transcripts. In this study, a 1.6-kb immediate-early transcript was localized to the BamHI-I2 region by Northern (RNA) hybridization analysis. With cDNA cloning and sequencing, two cDNAs of 1.4 kb (C1) and 1.35 kb (C2) were identified. Both cDNAs are derived from spliced mRNAs spanning the BamHI-H and -I2 fragments. C1 and C2 use the same splice acceptors and 3' ends, but they differ at their 5' ends and utilize different splice donors. The upstream promoter-enhancer region of C1 cDNA has been defined as a bidirectional regulatory region shared by the MDV pp38 gene. Sequencing analysis shows two small open reading frames (ORFs) within each cDNA (ORF1a and ORF2 in C1, ORF1b and ORF2 in C2). Potential ORFs of the sequence have no significant homology with any known protein in the Swiss-Protein data base. DNA fragments encoding ORF1a and ORF1b were cloned into pGEX-3X vectors to produce glutathione S-transferase fusion proteins and induce antisera. In Western blot (immunoblot) analysis of MDV-infected-cell lysates, a 14-kDa polypeptide was identified by antisera against both ORF1a and ORF1b. This 14-kDa protein is expressed in cells which are lytically infected with MDV strains GA, Md11 passage 14 (oncogenic), and Md11 passage 83 (attenuated), as well as in the latently MDV-infected and transformed MSB-1 cell line.
Neuron-specific restriction of a herpes simplex virus recombinant maps to the UL5 gene
Authors: Bloom D C , Stevens J G
Published: January 2020

We have previously shown that, when compared with either parent, a herpes simplex virus type 1/herpes simplex virus type 2 intertypic recombinant (R13-1) is attenuated by 10,000-fold with respect to neurovirulence in mice. Despite this, after intracranial inoculation, R13-1 replicated to titers of 10(5) PFU per brain. We present evidence that the restriction is specific for replication in neurons and have taken a three-step approach in determining the basis of the attenuation by (i) characterizing cellular tropism of the virus in both central and peripheral nervous systems, (ii) defining where in the viral replication cycle the restriction is manifest, and (iii) identifying the genetic basis of the restriction through marker rescue analysis. Following inoculation into the animal, R13-1 viral antigens predominate in nonneuronal cells, and the block to replication in neurons was found to be beyond the level of adsorption and penetration. Despite the restricted replication within neurons, the virus established a latent infection in spinal ganglia and could be reactivated by in vitro cocultivation of the ganglia. In studies carried out in cell culture, R13-1 was found to replicate normally in mouse embryo fibroblasts and primary mouse glial cells but was restricted by 1,000-fold in primary mouse neurons and PC12 cells. R13-1 appeared to produce normal levels of early RNA in these cells, but production of DNA and late RNA was less than that of the wild type. Marker rescue analysis localized the fragment responsible for restoring neurovirulence to UL5, a component of the origin-binding complex implicated in replication of the viral genome. Our results with this virus, with a cell-specific restriction, suggest that a neuron-specific component is involved in viral replication.
Localization of a 34-amino-acid segment implicated in dimerization of the herpes simplex virus type 1 ICP4 polypeptide by a dimerization trap
Authors: Gallinari P , Wiebauer K , Nardi M C , Jiricny J
Published: January 2020

The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes. It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription. ICP4 is a sequence-specific DNA-binding protein and has always been purified in a dimeric form. The part of the protein that consists of the entire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form homodimers in solution. In an attempt to map the dimerization domain of ICP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could confer dimerization properties on a heterologous cellular transcription factor (LFB1), which binds to its cognate DNA sequence only as a dimer. The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still confer DNA-binding activity on the LFB1 reporter protein and thus apparently contained the ICP4 dimerization motif. Consistent with this result, a truncated ICP4 protein containing amino acids 343 to 490, in spite of the complete loss of DNA-binding activity, appeared to retain the capacity to form a heterodimer with a longer ICP4 peptide after coexpression in an in vitro translation system.
Expression of the varicella-zoster virus origin-binding protein and analysis of its site-specific DNA-binding properties
Authors: Chen D , Olivo P D
Published: January 2020

The varicella-zoster virus (VZV) genome contains homologs to each of the seven herpes simplex virus (HSV) genes that are required for viral DNA synthesis. VZV gene 51 is homologous to HSV UL9, which encodes an origin of DNA replication binding protein (OBP). It was previously shown, by using a protein A fusion protein, that the product of gene 51 is a site-specific DNA-binding protein which binds to sequences within the VZV origin (Stow et al., Virology 177:570-577, 1990). In this report, gene 51 was expressed in an in vitro translation system. Rabbit antiserum raised against the carboxyl-terminal 20 amino acids was used to confirm expression of the full-length gene 51 protein, and site-specific DNA-binding activity was demonstrated in a gel retardation assay. The origin-binding domain was located within a 263-amino-acid region of the carboxyl terminus by using a series of deletion mutants. The affinity of binding of the VZV OBP to the three binding sites in the VZV origin was found to be similar. In addition, as with UL9, a CGC triplet within a 10-bp consensus sequence is critical to the interaction between the OBP and the origin. The HSV and VZV OBPs, therefore, appear to have virtually identical recognition sequences despite only 33% identity and 44% similarity in the primary structure of their site-specific DNA-binding domains.
A nonstructural gag-encoded glycoprotein precursor is necessary for efficient spreading and pathogenesis of murine leukemia viruses
Authors: Corbin A , Prats A C , Darlix J L , Sitbon M
Published: January 2020

In addition to the Gag-Pol and Env precursors whose translation initiates at AUG codons, murine, feline, and simian type C oncoviruses also express glycosylated Gag-Pol precursors (glycoGag), glycoGag translation is initiated at CUG codons located upstream of the Gag AUG initiation codon. In contrast to Gag, glycoGag is translocated into the endoplasmic reticulum and is absent from virions. Since glycoGag has been described to be dispensable ex vivo, we investigated the in vivo effects of a glycoGag- mutation in the Friend murine leukemia virus (F-MuLV). F-MuLV induces severe early hemolytic anemia and subsequent erythroleukemia within 2 months after inoculation of newborn mice. We obtained a glycoGag- F-MuLV, strain H5, by inserting an octanucleotide linker downstream of the CUG codon leading to the reading of a stop codon in all reading frames upstream of the Gag AUG. F-MuLV H5 did not induce severe early hemolytic anemia, and latency of erythroleukemia was significantly increased most likely because of an approximately 1-week delay in the in vivo spreading. Accordingly, induction of recombinant polytropic viruses was also significantly delayed. Close examination of ex vivo spreading kinetics also showed a slower dissemination of F-MuLV H5. Western blot (immunoblot) performed after inoculation of newborn mice with this glycoGag- virus indicated the emergence of new glycoGag+ viruses. PCR analyses with F-MuLV-specific primers demonstrated in vivo pseudoreversions restoring the glycoGag reading frame. Our results demonstrated that glycoGag expression is positively selected and essential for full spreading and pathogenic abilities.
Copackaging of different-sized retroviral genomic RNAs: little effect on retroviral replication or recombination
Authors: Jones J S , Allan R W , Seufzer B , Temin H M
Published: January 2020

We tested the effect of copackaging retroviral vectors of different sizes on retroviral replication and recombination. Our results indicate little or no difference in replication or in the rate or pattern of the strand transfers leading to the formation of recombinant proviruses with size. The size difference of the vectors also allowed us to extend our previous analysis of the linkage of markers in the recombinant proviruses. We conclude that the observed linkage is inconsistent with the strand displacement/assimilation model of retroviral recombination.
Complex functional interactions at the early enhancer of the PQ strain of BK virus
Authors: Ferguson A T , Subramani S
Published: January 2020

BK virus is a human papovavirus that latently infects a majority of the world's population. There are more than 30 strains of the virus, most of which differ in the structure of the early enhancer region. The enhancer of the progenitor strain, WW, from which the other strains can be derived, consists of four conserved DNA domains, P, Q, R, and S. Rearrangement of the enhancer occurs upon passage in tissue culture and is thought to occur during virus replication. The strain under study, PQ, was selected upon passage of the Gardner strain (PPPQS) in the permissive cell line, Vero. Mutational analysis of the entire enhancer region demonstrates the importance of five cis-acting sequences: DNA sites B, C, and F, which have homology to the NF-1 protein binding sequence; one purine-rich motif designated A; and site D, which is similar to an SP-1 protein binding site. Two sites, B and C, appear to have a negative influence on gene activity. To study the functional interactions in more detail, promoter-enhancer constructions that contain different combinations of the five DNA sites linked to the chloramphenicol acetyltransferase gene were tested for early gene activity. The results reveal that the proteins binding to the enhancer functionally cooperate with each other. The effects of making mutations at the DNA sites are very similar to the effects of using excess enhancer DNA sequences to titrate the proteins that bind to the cis-acting DNA sites (in vivo competition). Moreover, the effects of changing the spacing between the DNA sites also demonstrate that there are cooperative interactions among the proteins that bind to the PQ strain enhancer. DNA sites B, C, and F are clearly protected from DNase I digestion by Vero cell nuclear proteins. In addition, mutation of each DNA site alters its sensitivity to DNase I in the presence of Vero cell proteins. Interestingly, mutation of site B affects protein binding to site B as well as to sites A, C, D, and F. These results suggest that cooperative functional and physical interactions occur at the early enhancer of the PQ strain.
Progressive squamous epithelial neoplasia in K14-human papillomavirus type 16 transgenic mice
Authors: Arbeit J M , Münger K , Howley P M , Hanahan D
Published: January 2020

To model human papillomavirus-induced neoplastic progression, expression of the early region of human papillomavirus type 16 (HPV16) was targeted to the basal cells of the squamous epithelium in transgenic mice, using a human keratin 14 (K14) enhancer/promoter. Twenty-one transgenic founder mice were produced, and eight lines carrying either wild-type or mutant HPV16 early regions that did not express the E1 or E2 genes were established. As is characteristic of human cancers, the E6 and E7 genes remained intact in these mutants. The absence of E1 or E2 function did not influence the severity of the phenotype that eventually developed in the transgenic mice. Hyperplasia, papillomatosis, and dysplasia appeared at multiple epidermal and squamous mucosal sites, including ear and truncal skin, face, snout and eyelids, and anus. The ears were the most consistently affected site, with pathology being present in all lines with 100% penetrance. This phenotype also progressed through discernible stages. An initial mild hyperplasia was followed by hyperplasia, which further progressed to dysplasia and papillomatosis. During histopathological progression, there was an incremental increase in cellular DNA synthesis, determined by 5-bromo-2'-deoxyuridine incorporation, and a profound perturbation in keratinocyte terminal differentiation, as revealed by immunohistochemistry to K5, K14, and K10 and filaggrin. These K14-HPV16 transgenic mice present an opportunity to study the role of the HPV16 oncogenes in the neoplastic progression of squamous epithelium and provide a model with which to identify genetic and epigenetic factors necessary for carcinogenesis.
Mutations in v-Src SH3 and catalytic domains that jointly confer temperature-sensitive transformation with minimal temperature-dependent changes in cellular tyrosine phosphorylation
Authors: Catling A D , Fincham V J , Frame M C , Haefner B , Wyke J A
Published: January 2020

We have analyzed two functionally significant amino acid alterations encoded by the temperature-sensitive (ts) v-src mutant of Rous sarcoma virus, LA32. The G-to-V change at residue 300 in the catalytic domain nonconditionally impairs morphological transformation, in vitro kinase activity, in vivo tyrosine phosphorylation, and the cytoskeletal association of v-Src while rendering anchorage- and serum-independent growth ts. The R-to-P mutation in the SH3 domain subtly enhances morphological transformation but has no phenotype if the catalytic domain is inactivated. In the presence of the G-300-to-V mutation, this SH3 domain lesion does not affect v-Src in vitro kinase activity and cytoskeletal association, but it nonconditionally enhances cellular tyrosine phosphorylation and restores morphological transformation at the permissive temperature only. This ability to induce a ts transformed morphology, in concert with nonconditional elevations of cellular phosphotyrosine, suggest that a subset of v-Src targets that are crucial to transformation may be affected in ts fashion by the SH3 mutation. Consistent with this, we find that the R-107-to-P mutation confers ts activity and tyrosine phosphorylation on the SH3-binding enzyme phosphatidylinositol 3'-kinase. Thus, both the SH3 and catalytic domain mutations in LA32 have some ts attributes and they cooperate in determining the mutant's behavior. The ts SH3 mutation is unique and offers the potential for deeper understanding of the function of this domain.
Mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir
Authors: Lurain N S , Spafford L E , Thompson K D
Published: January 2020

The same point mutation in the human cytomegalovirus UL97 open reading frame was found in three independently isolated ganciclovir-resistant mutants of strain AD169. Point mutations in the DNA polymerase genes of these strains have been previously identified (N.S. Lurain, K.D. Thompson, E.W. Holmes, and G.S. Read, J. Virol. 66:7146-7152, 1992). All three strains are, therefore, double mutants. To determine the contribution of the UL97 mutation to the high ganciclovir resistance of these mutants, the mutation from the ganciclovir-resistant strain D6/3/1 was transferred to the wild-type strain AD169 to produce the recombinant R6HS. The ganciclovir resistance of R6HS is 4-fold lower than that of D6/3/1 but 10-fold higher than that of AD169. R6HS, like AD169, is sensitive to the nucleotide analogs (S)-1-[(3-hydroxy-2-phosphonylmethoxy) propyl]adenine and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine. Ganciclovir phosphorylation in R6HS-infected cells was at the same reduced level as that found in cells infected with the parental mutant D6/3/1. The same G-to-T transversion at nucleotide 1380 in the UL97 coding sequence is present in both R6HS and D6/3/1. This mutation results in the substitution of isoleucine for methionine at amino acid residue 460. In an alignment of the R6HS UL97 amino acid sequence with the amino acid sequences of a wide range of protein kinase family members, methionine 460 lies within a highly conserved region which may function in nucleotide binding and phosphate transfer.
Reconstitution of Flock House provirions: a model system for studying structure and assembly
Authors: Schneemann A , Gallagher T M , Rueckert R R
Published: January 2020

Assembly of Flock House virus in infected Drosophila cells proceeds through an intermediate, the provirion, which lacks infectivity until the coat precursor protein, alpha, undergoes a spontaneous "maturation" cleavage (A. Schneemann, W. Zhong, T. M. Gallagher, and R. R. Rueckert, J. Virol 6:6728, 1992). We describe here methods for purifying provirions in a state which permitted dissociation and reassembly. Dissociation, to monomeric alpha protein and free RNA, was accomplished by freezing at pH 9.0 in the presence of 0.5 M salt and 0.1 M urea. When dialyzed at low ionic strength and pH 6.5, the dissociation products reassembled spontaneously to form homogeneous provirions with a normal complement of RNA as judged by cosedimentation with authentic virions and by ability to undergo maturation cleavage with acquisition of substantial, though subnormal, infectivity. Reconstitution experiments, i.e., remixing components after separating RNA from capsid protein, generated abnormal particles, suggesting the presence in the unfractionated dissociation products of an unidentified "nucleating" component.
Stimulation of virus production and induction of self-syncytium formation in human T-cell leukemia virus type I- and type II-infected T cells by 12-O-tetradecanoylphorbol-13-acetate
Authors: Wolfson M , Lev M , Avinoah I , Malik Z , Löchelt M , Flügel R M , Dombrovski A , Aboud M
Published: January 2020

Treatment of human T-cell leukemia virus type I (HTLV-I)- and HTLV-II-infected T-cell lines with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated virus release. However, this stimulation was mainly detected at 42 to 48 h of treatment, whereas later virus release declined rapidly. During the first 48 h, TPA had no effect on cell growth, but later, the number of viable cells was profoundly lower in the TPA-treated than in the untreated cultures. This shift in virus release and cell number resulted from self-fusion of a large proportion of the virus-producing cells, which seemed to consequently enter into a dying process. This fusion, which resulted in syncytium formation, was strongly inhibited by anti-HTLV-I env monoclonal antibodies. Furthermore, no self-fusion was detected in three different uninfected T-cell lines similarly treated with TPA. On the other hand, stimulation of virus production by 3-methylcholanthrene (3-MC) treatment failed to induce self-fusion in the infected cells. Moreover, no syncytium was detected when these 3-MC-treated infected cells were cocultured with any of the TPA-treated uninfected cells. The effects of TPA on virus production and syncytium formation were both abolished by three different protein kinase C inhibitors. Taken together, these data suggest that the self-fusion observed in these experiments required both enhanced virus production and protein kinase C-phosphorylated viral or/and virally induced cellular component(s).
The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection
Authors: Hardwicke M A , Sandri-Goldin R M
Published: January 2020

We have previously shown that the herpes simplex virus immediate-early regulatory protein ICP27 acts posttranscriptionally to affect mRNA processing (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992). Specifically, in the presence of ICP27, spliced target mRNAs were decreased 5- to 10-fold in transfections with target genes containing a 5' or 3' intron. Here, we have investigated the effect of ICP27 during herpes simplex virus type 1 (HSV-1) infection on accumulation of spliced cellular mRNAs. ICP27 viral mutants have been shown to be defective in host shutoff (W. R. Sacks, C. C. Greene, D. P. Aschman, and P. A. Schaffer, J. Virol. 55:796-805, 1985). Therefore, we examined whether ICP27 could contribute to this complex process by decreasing cellular mRNA levels through its effects on host cell splicing. It was found that in infections with viral mutants defective in ICP27, the accumulated levels of three spliced host mRNAs were higher than those seen with wild-type HSV-1. The differences occurred posttranscriptionally as shown by nuclear runoff transcription assays. The stabilities of the spliced products during infection with wild-type or ICP27 mutant viruses were similar, and unspliced precursor mRNA for a viral spliced gene was detected in infections with wild-type HSV-1 but not in infections in which ICP27 was not expressed. These results suggest that the reduction in cellular mRNA levels and the accumulation of pre-mRNA are related and may be caused by an impairment in host cell splicing. These data further show that ICP27 is required for these effects to occur.
The extreme carboxyl terminus of the equine herpesvirus 1 homolog of herpes simplex virus VP16 is essential for immediate-early gene activation
Authors: Elliott G D
Published: January 2020

Gene 12 of equine herpesvirus 1 (EHV-1), the homolog of herpes simplex virus (HSV) VP16 (alpha TIF, Vmw65), was cloned into a eukaryotic expression vector by PCR and used in transactivation studies of both the EHV-1 and HSV-1 IE1 promoters. Results demonstrated that the product of gene 12 is a potent transactivator of immediate-early gene expression of both viruses, which requires sequences in the upstream HSV-1 promoter for activity. Mutational analysis of the gene 12 open reading frame indicated that removal of the C-terminal 7 amino acids, which contain a short region of homology with the extreme C terminus of VP16, inactivated the protein. Within this region, only a single methionine residue appeared to be essential for activity, implying that gene 12 may have a modular array of organization similar to that of VP16. However, fusion of the gene 12 C terminus to a truncated form of VP16, which contained the complex formation domain, did not restore activity to the HSV-1 protein. These data demonstrate that the EHV-1 immediate-early transactivator may not be functionally colinear with VP16, with transactivation requiring both the C terminus and another region(s) present within the N-terminal portion.
Evidence that the herpes simplex virus type 1 uracil DNA glycosylase is required for efficient viral replication and latency in the murine nervous system
Authors: Pyles R B , Thompson R L
Published: January 2020

Herpes simplex virus (HSV) encodes a uracil DNA glycosylase (UNG; UL2), which has been shown to be dispensable for normal replication of HSV-1 in cultured cells (J. Mullaney, H.W. Moss, and D.J. McGeoch, J. Gen. Virol. 70:449-454, 1989). In adult neurons, UNG activity is undetectable (F. Focher, P. Mazzarello, A. Verri, U. Hubscher, and S. Spadari, Mutat. Res. 237:65-73, 1990), suggesting that the HSV-1 UNG may play an important role in viral replication in neurons acutely and/or following reactivation. To examine the contribution of the HSV-1 UNG in vivo, two independent strain 17 Syn+ Ung- mutants, designated uB1 and uB2, were examined in a mouse model of herpetic disease. Following direct intracranial inoculation, both mutants exhibited a 10-fold reduction in neurovirulence compared with the parental strain 17 Syn+. Inoculations by a peripheral route demonstrated that the Ung- mutants were at least 100,000-fold less neuroinvasive than 17 Syn+. Replication kinetics in vivo demonstrated that uB1 and uB2 replicated less well in both the mouse peripheral and central nervous systems. Latency was established by both of the mutants in 100% of the animals examined. Following transient hyperthermia, however, the frequency of reactivation of the mutants in vivo was dramatically reduced. Restoration of the UNG locus resulted in full neurovirulence, neuroinvasiveness, and the ability to reactivate in vivo. These findings suggest that the HSV-1 UNG plays an important role during acute viral replication in vivo and possibly in the reactivation process.
Introduction of hepatitis delta virus into animal cell lines via cationic liposomes
Authors: Bichko V , Netter H J , Taylor J
Published: January 2020

Cationic liposomes are known to facilitate efficient transfection of animal cells with DNA and even some viruses. As reported here, we have been able to use such a commercially available formulation (Lipofectamine) and introduce human hepatitis delta virus (HDV) into lines of cultured cells and demonstrate replication of the HDV genome both by immunofluorescence and by Northern (RNA) analysis. As much as 10% of the human hepatoma cell line Huh7 was transfected with HDV. Also transfected were the baby hamster kidney cell line BHK-21 and the Morris rat hepatoma line 7777. Two initial applications of HDV transfection have been made. (i) The ribonucleoprotein structure of HDV was isolated from disrupted virions and demonstrated as being sufficient to transfect Huh7 cells. In contrast, naked HDV RNA was not sufficient. (ii) From a study of cells transfected with HDV particles, it was found that, even after as long as 7 weeks and the associated replication of the transfected cells, the HDV RNA genome was still replicating. Apparently, HDV, in the absence of helper virus and in the absence of virus assembly, can maintain persistent replication and expression of the HDV genome. Transfection was also achieved with woodchuck hepatitis virus introduced into Huh7 cells. In summary, this transfection procedure should be of use for the study of these and maybe other recalcitrant animal viruses.
Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus
Authors: Netter H J , Gerin J L , Tennant B C , Taylor J M
Published: January 2020

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.
Oligo(A) sequences of human respiratory syncytial virus G protein gene: assessment of their genetic stability in frameshift mutants
Authors: García-Barreno B , Delgado T , Melero J A
Published: January 2020

We have described previously antibody-resistant mutants of the human respiratory syncytial virus Long strain that contained frameshift changes generated by deletions or insertions of a single adenosine in oligo(A) tracts (mRNA sense) of the G protein gene. Since these mutations introduced drastic structural and antigenic changes in the G protein C-terminal third, we decided to test the mutant stability by passaging the viruses in either the presence or the absence of selective antibody. Two such mutants (R63/1/2/3 and R63/2/4/8), with a single reading frame shift, reverted after a few passages in the absence of antibody to the wild-type genotype, by insertion of an A at the same homopolymeric tract as in the original deletion. In contrast, a double frameshift mutant (R63/2/4/1), generated by deletion of an A after nucleotide 623 and insertion of another A seven triplets later, was stably maintained after passage in either the absence or the presence of antibody. The stability of this mutant was manifested in its capacity to gradually displace the Long strain from mixed infections and by the fact that mutant R63/2/4/8 acquired the genotype of R63/2/4/1 after several passages in the presence of antibody. These results were indicative of genetic instability in the oligo(A) tract length of certain G protein mutants, which resulted in frameshift changes. The frequency of such errors among the viral RNA population obtained from a single infectious cycle was estimated to be lower than 1%. The relevance of these results for respiratory syncytial virus evolution is discussed.
Ectopic expression of gamma interferon in the eye protects transgenic mice from intraocular herpes simplex virus type 1 infections
Authors: Geiger K , Howes E L , Sarvetnick N
Published: January 2020

Transgenic (rho gamma) mice provide a model for studying the influence of gamma interferon (IFN-gamma) produced in the eye on ocular and cerebral viral infection. To establish this model, we injected BALB/c- and C57BL/6-derived transgenic and nontransgenic mice of different ages intravitreally with herpes simplex virus type 1 (HSV-1) strain F. Eye and brain tissues of these mice were assessed for pathological and immunocytochemical changes. HSV-1 infection induced severe retinitis of the injected eyes and infection of the brain in all mice. In transgenic mice inoculated with HSV-1, the left, nontreated eyes were protected from retinitis, whereas nontransgenic mice developed bilateral retinitis. Additional intravitreal injection of IFN-gamma with the virus protected the noninoculated eyes of nontransgenic mice. Three-week-old nontransgenic mice died from HSV-1 infection, whereas transgenic mice of the same age and nontransgenic mice intravitreally treated with IFN-gamma survived. Ocular IFN-gamma production increased the extent of inflammation in transgenic mice but did not have a significant influence on the growth of HSV-1 until day 3 after inoculation and did not influence the neuroinvasion of this virus. Thus, the effects of IFN-gamma were not caused by an early block of viral replication. Possible mechanisms of IFN-gamma action include activation of the immune response, alteration of the properties of the virus, and direct protection of neurons.
Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants
Authors: Harrich D , Hsu C , Race E , Gaynor R B
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.
A cryptic RNA-binding domain in the Pol region of the L-A double-stranded RNA virus Gag-Pol fusion protein
Authors: Ribas J C , Fujimura T , Wickner R B
Published: January 2020

The Pol region of the Gag-Pol fusion protein of the L-A double-stranded (ds) RNA virus of Saccharomyces cerevisiae has (i) a domain essential for packaging viral positive strands, (ii) consensus amino acid sequence patterns typical of RNA-dependent RNA polymerases, and (iii) two single-stranded RNA binding domains. We describe here a third single-stranded RNA binding domain (Pol residues 374 to 432), which is unique in being cryptic. Its activity is revealed only after deletion of an inhibitory region C terminal to the binding domain itself. This cryptic RNA binding domain is necessary for propagation of M1 satellite dsRNA, but it is not necessary for viral particle assembly or for packaging of viral positive-strand single-stranded RNA. The cryptic RNA binding domain includes a sequence pattern common among positive-strand single-stranded RNA and dsRNA viral RNA-dependent RNA polymerases, suggesting that it has a role in RNA polymerase activity.
Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses
Authors: Horimoto T , Nakayama K , Smeekens S P , Kawaoka Y
Published: January 2020

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.
Isolation and characterization of human immunodeficiency virus type 1 variants infectious to brain-derived cells: detection of common point mutations in the V3 region of the env gene of the variants
Authors: Shimizu N S , Shimizu N G , Takeuchi Y , Hoshino H
Published: January 2020

T-cell-line-tropic human immunodeficiency virus type 1 cannot infect CD4-positive, brain-derived cells. We isolated several new variants that readily infected brain-derived cells. Mutation of proline to serine, to alanine, or to threonine in the well-conserved GPGR sequence in the V3 region of the envelope glycoprotein was found in all these variants. This indicates the importance of amino acid sequences at the tip of the V3 region for brain cell tropism of human immunodeficiency virus type 1.
A human monoclonal antibody to a complex epitope in the V3 region of gp120 of human immunodeficiency virus type 1 has broad reactivity within and outside clade B
Authors: Moore J P , Trkola A , Korber B , Boots L J , Kessler J A , McCutchan F E , Mascola J , Ho D D , Robinson J , Conley A J
Published: January 2020

We have used virus neutralization and antibody-binding techniques to define the epitope for a human monoclonal antibody, designated 19b, within the V3 region of the gp120 surface glycoprotein of human immunodeficiency virus type 1. Unusually, the 19b epitope encompasses residues on both flanks of the V3 loop. However, 19b binding to gp120 is independent of sequences at the crown of the V3 loop, provided that they are compatible with the formation of a type II beta turn that is presumably necessary to juxtapose the antigenic residues on the V3 flanks. By comparing the V3 sequences of virus gp120s able and unable to bind 19b, we were able to define the canonical 19b epitope as -I----G--FY-T, where residues at the positions indicated by the gaps do not contribute directly to the 19b-binding site. A few conservative substitutions at the more critical residues are also compatible with 19b binding. Inspection of V3 sequences in the human immunodeficiency virus database indicated that the canonical 19b epitope is well conserved among isolates from the North American-European clade B and also among clade E isolates from Thailand and clade F isolates from Brazil. A minority of gp120s from clades A and C also possess the 19b epitope. Consistent with the theoretical predictions of its cross-clade reactivity, 19b was found to bind to gp120s from clades A, B, C, E, and F in immunoassays. However, 19b was not able to reduce the infectivity of primary viruses from clades A, E, and F that were predicted to possess the 19b epitope and only modestly reduced the infectivity of a clade C virus at low input virus concentrations. Cross-clade neutralization via V3-directed antibodies may, therefore, be difficult, even if the antibodies show broad reactivities in binding assays and the viruses theoretically possess the relevant binding site.
Local expression of tumor necrosis factor alpha and interleukin-2 correlates with protection against corneal scarring after ocular challenge of vaccinated mice with herpes simplex virus type 1
Authors: Ghiasi H , Wechsler S L , Kaiwar R , Nesburn A B , Hofman F M
Published: January 2020

To correlate specific local immune responses with protection from corneal scarring, we examined immune cell infiltrates in the cornea after ocular challenge of vaccinated mice with herpes simplex virus type 1 (HSV-1). This is the first report to examine corneal infiltrates following ocular challenge of a vaccinated mouse rather than following infection of a naive mouse. Mice were vaccinated systemically with vaccines that following ocular challenge with HSV-1 resulted in (i) complete protection against corneal disease (KOS, an avirulent strain of HSV-1); (ii) partial protection, resulting in moderate corneal disease (baculovirus-expressed HSV-1 glycoprotein E [gE]); and (iii) no protection, resulting in severe corneal disease (mock vaccine). Infiltration into the cornea of CD4+ T cells, CD8+ T cells, macrophages, and cells containing various lymphokines was monitored on days 0, 1, 3, 7, and 10 postchallenge by immunocytochemistry of corneal sections. Prior to ocular challenge, no eye disease or corneal infiltrates were detected in any mice. KOS-vaccinated mice developed high HSV-1 neutralizing antibody titers (> 1:640) in serum. After ocular challenge, they were completely protected against death, developed no corneal disease, and had no detectable virus in their tear films at any time examined. In response to the ocular challenge, these mice developed high local levels of infiltrating CD4+ T cells and cells containing interleukin-2 (IL-2), IL-4, IL-6, or tumor necrosis factor alpha (TNF-alpha). In contrast, only low levels of infiltrating CD8+ T cells were found, and gamma interferon (IFN-gamma)-containing cells were not present until day 10. gE-vaccinated mice developed neutralizing antibody titers in serum almost as high as those of the KOS-vaccinated mice (> 1:320). After ocular challenge, they were also completely protected against death. However, the gE-vaccinated mice developed low levels of corneal disease and virus was detected in one-third of their eyes. Compared with KOS-vaccinated mice, the gE-vaccinated mice had a similar pattern of IFN-gamma, but a delay in the appearance of CD4+ T cells, CD8+ T cells, and IL-4-, IL-6-, and TNF-alpha-containing cells. In sharp contrast to those of the KOS-vaccinated mice, no cells containing IL-2 were detected in the eyes of gE-vaccinated mice at any time. Mock-vaccinated mice developed no detectable neutralizing antibody titer and were not protected from lethal HSV-1 challenge.(ABSTRACT TRUNCATED AT 400 WORDS)
Adaptation to persistent growth in the H9 cell line renders a primary isolate of human immunodeficiency virus type 1 sensitive to neutralization by vaccine sera
Authors: Wrin T , Loh T P , Vennari J C , Schuitemaker H , Nunberg J H
Published: January 2020

Seven diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) were examined and found to be refractory to neutralization by antisera to recombinant gp120 (rgp120) protein from HIV-1 MN. This stands in marked contrast to the sensitivity exhibited by certain laboratory-adapted viruses. To understand the difference between primary and laboratory-adapted viruses, we adapted the primary virus ACH 168.10 to growth in the FDA/H9 cell line. ACH 168.10 was chosen because the V3 region of gp120 closely matches that of MN. After 4 weeks, infection became evident. The virus (168A) replicated in FDA/H9 cells with extensive cytopathic effect but was unchanged in sensitivity to antibody-mediated neutralization. Thus, growth in cell lines is not sufficient to render primary virus sensitive to neutralization. The 168A virus was, however, partially sensitive to CD4 immunoadhesin (CD4-Ig). Adaptation was continued to produce a persistently infected FDA/H9 culture that displayed minimal cytopathic effect. The virus (168C) was now sensitive to neutralization by MN rgp120 vaccine sera and by MN-specific monoclonal antibodies and showed increased sensitivity to HIVIG and CD4-Ig. 168C encoded three amino acid changes in gp120, including one within the V3 loop (I-166-->R, I-282-->N, G-318-->R). MN-specific monoclonal antibodies bound equally to the surface of cells infected with either neutralization-resistant or -sensitive virus. The coincidence of changes in neutralization sensitivity with changes in cell tropism and cytopathic effect suggests a common underlying mechanism(s) acting through the whole of the envelope protein complex.