bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific binding of host cellular proteins to multiple sites within the 3' end of mouse hepatitis virus genomic RNA
Authors: Yu W , Leibowitz J L
Published: January 2020

The initial step in mouse hepatitis virus (MHV) RNA replication is the synthesis of negative-strand RNA from a positive-strand genomic RNA template. Our approach to begin studying MHV RNA replication is to identify the cis-acting signals for RNA synthesis and the proteins which recognize these signals at the 3' end of genomic RNA of MHV. To determine whether host cellular and/or viral proteins interact with the 3' end of the coronavirus genome, an RNase T1 protection/gel mobility shift electrophoresis assay was used to examine cytoplasmic extracts from mock- and MHV-JHM-infected 17Cl-1 murine cells for the ability to form complexes with defined regions of the genomic RNA. We demonstrated the specific binding of host cell proteins to multiple sites within the 3' end of MHV-JHM genomic RNA. By using a set of RNA probes with deletions at either the 5' or 3' end or both ends, two distinct binding sites were located. The first protein-binding element was mapped in the 3'-most 42 nucleotides of the genomic RNA [3' (+42) RNA], and the second element was mapped within an 86-nucleotide sequence encompassing nucleotides 171 to 85 from the 3' end of the genome (171-85 RNA). A single potential stem-loop structure is predicted for the 3' (+)42 RNA, and two stem-loop structures are predicted for the 171-85 RNA. Proteins interacting with these two elements were identified by UV-induced covalent cross-linking to labeled RNAs followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The RNA-protein complex formed with the 3'-most 42 nucleotides contains approximately five host polypeptides, a highly labeled protein of 120 kDa and four minor species with sizes of 103, 81, 70, and 55 kDa. The second protein-binding element, contained within a probe representing nucleotides 487 to 85 from the 3' end of the genome, also appears to bind five host polypeptides, 142, 120, 100, 55, and 33 kDa in size, with the 120-kDa protein being the most abundant. The RNA-protein complexes observed with MHV-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were identical to those observed with uninfected cells. The possible involvement of the interaction of host proteins with the viral genome during MHV replication is discussed.
RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1
Authors: Clever J , Sassetti C , Parslow T G
Published: January 2020

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.
Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery
Authors: Huang S , Endo R I , Nemerow G R
Published: January 2020

Entry of human adenovirus into host cells involves interaction of virus particles with two distinct receptors. The initial binding event is mediated by the fiber protein, while subsequent interaction of the penton base protein with alpha v integrins promotes virus internalization and/or penetration. Although these interactions in epithelial and endothelial cells have been well characterized, relatively little is known as to whether these events occur during virus infection of human peripheral blood mononuclear cells. We demonstrate that freshly isolated peripheral blood monocytes and T lymphocytes express very small amounts of alpha v integrins and also are resistant to adenovirus infection. Exposure of monocytes to hematopoietic growth factors granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor induced expression of cell surface alpha v integrins, promoted the binding of penton base protein, and also rendered these cells susceptible to adenovirus-mediated gene delivery. Stimulation of T cells with a mitogen, phytohemagglutinin, or a cell-activating agent, phorbol myristate acetate, induced expression of alpha v integrins and also enhanced adenovirus-mediated gene delivery. These studies further indicate that alpha v integrins play a crucial role in adenovirus infection and also provide a useful strategy for enhancing adenovirus-mediated gene delivery into human peripheral blood mononuclear cells.
Similarity in env and gag genes between genomic RNAs of human immunodeficiency virus type 1 (HIV-1) from mother and infant is unrelated to time of HIV-1 RNA positivity in the child
Authors: Mulder-Kampinga G A , Simonon A , Kuiken C L , Dekker J , Scherpbier H J , van de Perre P , Boer K , Goudsmit J
Published: January 2020

Variation in the env (V3 region) and gag (p17 region) genes of genomic RNA of human immunodeficiency virus type 1 was studied in three mother-child pairs. One infant was human immunodeficiency virus type 1 RNA positive at birth (pair 114), one became positive 6 weeks after birth (pair 127), and one became positive 30 months after birth (pair 564). The first two children were born to seropositive mothers, and the last child was infected by breast-feeding following seroconversion of the mother after delivery. In both V3 and p17gag, intrasample variability was much higher in the maternal samples, including the first seropositive sample of the seroconverted mother, than in the infants' samples. Variability was less in p17gag than in V3, except in the postnatally infected child. In all three cases, infection of the child was established by variants representing a minority of the cell-free virus population in the maternal samples. For the two infants born to seropositive mothers, V3 sequences were more similar to the sequence populations of maternal samples collected during pregnancy than to those of samples collected at delivery or thereafter. However, in pair 114 a V3 variant identical to the child's virus was also detected in the sample collected at delivery. In contrast to the V3 region, p17gag sequences of maternal samples of the first trimester of pregnancy and at delivery had comparable resemblance to the child's sequences in pair 114, while in pair 127, similarity to sequences of the sample collected at delivery was higher than that to sequences of the sample from early in pregnancy. In the last pair, V3 and p17gag sequences from a maternal sample collected 18 months prior to the first RNA-positive sample of the child resembled the infant's sequences as much as the sample collected close to the presumed time of infection. Taken together, the evolutionary characteristics for genomic RNA env and gag genes did not point to a particular time of mother-to-child transmission.
Two murine coronavirus genes suffice for viral RNA synthesis
Authors: Kim K H , Makino S
Published: January 2020

We identified two mouse hepatitis virus (MHV) genes that suffice for MHV RNA synthesis by using an MHV-JHM-derived defective interfering (DI) RNA, DIssA. DIssA is a naturally occurring self-replicating DI RNA with nearly intact genes 1 and 7. DIssA interferes with most MHV-JHM-specific RNA synthesis, except for synthesis of mRNA 7, which encodes N protein; mRNA 7 synthesis is not inhibited by DIssA. Coinfection of MHV-JHM containing DIssA DI particles and an MHV-A59 RNA- temperature-sensitive mutant followed by subsequent passage of virus at the permissive temperature resulted in elimination of most of the MHV-JHM helper virus. Analysis of intracellular RNAs at the nonpermissive temperature demonstrated efficient synthesis of DIssA and mRNA 7 but not of the helper virus mRNAs. Oligonucleotide fingerprinting analysis demonstrated that the structure of mRNA 7 was MHV-JHM specific and therefore must have been synthesized from the DIssA template RNA. Sequence analysis revealed that DIssA lacks a slightly heterogeneous sequence, which is found in wild-type MHV from the 3' one-third of gene 2-1 to the 3' end of gene 6. Northern (RNA) blot analysis of intracellular RNA species and virus-specific protein analysis confirmed the sequence data. Replication and transcription of another MHV DI RNA were supported in DIssA-replicating cells. Because the products of genes 2 and 2-1 are not essential for MHV replication, we concluded that expression of gene 1 proteins and N protein was sufficient for MHV RNA replication and transcription.
Halophage HF2: genome organization and replication strategy
Authors: Nuttall S D , Dyall-Smith M L
Published: January 2020

Halophage HF2 is a lytic, broad-host-range bacteriophage of the extremely halophilic domain Archaea. It has a 79.7-kb double-stranded DNA genome which is linear, contains no modified nucleotides, and is not susceptible to cleavage by many type II restriction endonucleases. This insensitivity is attributed to selection against palindromic restriction sites, a commonly observed feature of broad-host-range phages. Interestingly, enzymes that did cut the genome recognized AT-rich sites, and five such enzymes, DraI, AseI, HpaI, HindIII, and SspI, were used to construct a physical map of the genome. Southern hybridization experiments used to order fragments on the map indicated homologies between the phage termini, and subsequent sequence analysis showed that HF2 possessed 306-bp direct terminal repeats. The presence of such repeats suggested replication through concatameric intermediates, and this was confirmed by analysis of the state of the phage genome in infected cells. This is a replication strategy adopted by many well-studied bacterial phages, for example T3 and T7. Other similarities between the terminal repeats of T3 or T7 and HF2 include a putative nick site at the repeat border and a series of short imperfect repeats. These observations suggest a long evolutionary history for concatamer-based strategies of phage replication, possibly predating the divergence of Archaea/Eucarya and Bacteria, or alternatively, indicate possible lateral transfer of phage genes or modules between the domains Archaea and Bacteria.
Chimeras of receptors for gibbon ape leukemia virus/feline leukemia virus B and amphotropic murine leukemia virus reveal different modes of receptor recognition by retrovirus
Authors: Pedersen L , Johann S V , van Zeijl M , Pedersen F S , O'Hara B
Published: January 2020

Glvr1 encodes the human receptor for gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B), while the related gene Glvr2 encodes the human receptor for amphotropic murine leukemia viruses (A-MLVs). The two proteins are 62% identical in their amino acid sequences and are predicted to have 10 transmembrane domains and five extracellular loops. A stretch of nine amino acids (region A) in the predicted fourth extracellular loop was previously shown to be critical for the function of Glvr1 as receptor for GALV and FeLV-B. Glvr1 and -2 show clusters of amino acid differences in several of their predicted extracellular loops, with the highest degree of divergence in region A. Chimeras were made between the two genes to further investigate the role of Glvr1 region A in defining receptor specificity for GALV and FeLV-B and to map which regions of Glvr2 control receptor specificity for A-MLVs. Region A from Glvr1 was sufficient to confer receptor specificity for GALV upon Glvr2, with the same chimera failing to act as a receptor for FeLV-B. However, introduction of additional N- or C-terminal Glvr1-encoding sequences in addition to Glvr1 region A-encoding sequences resulted in functional FeLV-B receptors. Therefore, FeLV-B is dependent on Glvr1 sequences outside region A for infectivity. The receptor specificity of Glvr2 for A-MLV could not be mapped to a single critical region; rather, N-terminal as well as C-terminal Glvr2-encoding sequences could confer specificity for A-MLV infection upon Glvr1. Surprisingly, though GALV/FeLV-B and A-MLV belong to different interference groups, some chimeras functioned as receptors for all three viruses.
Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs
Authors: de la Luna S , Fortes P , Beloso A , Ortín J
Published: January 2020

The effect of NS1 protein on the efficiency of influenza virus mRNA translation was evaluated by determining the accumulation of nucleoprotein (NP) or M1 mRNAs in the cytoplasm of cells expressing either of these genes alone or in combination with the NS1 gene, as well as the total cell accumulation of NP or M1 protein. Coexpression of NS1, but not of NS2 protein, led to increases in the translation of these mRNAs in the range of 5- to 100-fold. This translation enhancement was specific for viral mRNAs, since the translation of neither cat nor lacZ mRNAs was affected by the coexpression of NS1 protein. The use of chimeric cat genes containing the 5'-extracistronic sequences of the influenza virus mRNAs corresponding to segment 2, 7, or 8 indicated that these sequences can in part account for the observed effect. The enhancement of viral mRNA translation mediated by NS1 protein was due to an increase in the translation initiation rate, since the sizes of NP-specific polysomes, but not those of lacZ-specific polysomes, was significantly higher in cells coexpressing NS1 protein than in those expressing only the NP gene.
The predominant virus antigen burden is present in macrophages in Theiler's murine encephalomyelitis virus-induced demyelinating disease
Authors: Lipton H L , Twaddle G , Jelachich M L
Published: January 2020

Theiler's murine encephalomyelitis virus (TMEV) produces a persistent central nervous system infection and chronic, inflammatory demyelinating disease in susceptible mice. TMEV antigen(s) and RNA genome have been detected in astrocytes, oligodendrocytes, and macrophages during persistence. Whether there is a predominant cell type in which TMEV persists has not been resolved. Since TMEV-induced demyelinating lesions are infiltrated with macrophages and a number of other persistent viruses show near-exclusive tropism for these phagocytic cells, we used two-color immunofluorescent staining with conventional and confocal microscopy to colocalize TMEV to cells that stain with monoclonal antibodies (MOMA-2) [unknown antigen], Mac-1 [CD11b], FA-11 [CD66], and 2F8 [scavenger receptor]) to macrophages in BeAn-infected SJL mice. A predominant virus antigen burden within macrophages infiltrating demyelinating lesions was seen. A dichotomy of cells staining for virus antigen(s) was found with infected cells containing either a large or small virus antigen load. Ninety percent of cells with a large virus antigen load were large phagocytes (20 to 50 microns) that were readily detected at low power (5x objective). Cells with smaller amounts of virus antigen(s) turned out to be either these same large phagocytic cells or much smaller cells, approximately equal to 10 microns in diameter. Forty percent of cells with a small virus antigen load were macrophages. The unidentified approximately equal to 10-microns cells that are virus antigen positive and macrophage negative in this study could still be macrophages, or they may be oligodendrocytes. The fact that virus was detected in the cytoplasm and not phagolysosomes of macrophages and the sheer mass of fluorescently stained virus proteins in some macrophages suggest that TMEV persists in these phagocytic cells by active virus replication.
NS3-4A of hepatitis C virus is a chymotrypsin-like protease
Authors: Hahm B , Han D S , Back S H , Song O K , Cho M J , Kim C J , Shimotohno K , Jang S K
Published: January 2020

The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.
An EBNA-1-dependent enhancer acts from a distance of 10 kilobase pairs to increase expression of the Epstein-Barr virus LMP gene
Authors: Gahn T A , Sugden B
Published: January 2020

Upon infection of human B lymphocytes, the 172-kbp Epstein-Barr virus genome forms a covalently closed circle via its terminal repeats. This event brings all of the promoters that control expression of the latent gene products, and the viral origin of plasmid replication, oriP, within a 20-kbp stretch of contiguous DNA. We have found that the EBNA-1-dependent transcriptional enhancer FR, located in oriP, increased the expression of a tagged viral oncogene encoding the latent membrane protein (LMP) up to 200-fold in normal Epstein-Barr virus-positive cells. The effect of FR was exerted across 10 kbp of viral DNA that spans the circularized ends of the viral genome. Enhancement of the tagged LMP gene by FR/EBNA-1 did not require the EBNA-2-responsive element.
Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells
Authors: Roberts S R , Compans R W , Wertz G W
Published: January 2020

Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.
Molecular characterization of the 3' terminus of the simian hemorrhagic fever virus genome
Authors: Godeny E K , Zeng L , Smith S L , Brinton M A
Published: January 2020

The 3' end of the simian hemorrhagic fever virus (SHFV) single-stranded RNA genome was cloned and sequenced. Adjacent to the 3' poly(A) tract, we identified a 76-nucleotide noncoding region preceded by two overlapping reading frames (ORFs). The ultimate 3' ORF of the viral genome encodes the capsid protein, and the penultimate ORF encodes the smallest SHFV envelope protein. These two ORFs overlap each other by 26 nucleotides. Northern (RNA) blot hybridization analyses of cytoplasmic RNA extracts from SHFV-infected MA-104 cells with gene-specific probes revealed the presence of full-length genomic RNA as well as six subgenomic SHFV-specific mRNA species. The subgenomic mRNAs are 3' coterminal. In its virion morphology and size, genome structure and length, and replication strategy, SHFV is most similar to lactate dehydrogenase-elevating virus, equine arteritis virus, and porcine reproductive and respiratory syndrome virus.
Papilloma formation in human foreskin xenografts after inoculation of human papillomavirus type 16 DNA
Authors: Brandsma J L , Brownstein D G , Xiao W , Longley B J
Published: January 2020

A mouse model of high-risk human papillomavirus infection was developed in which human papillomavirus (HPV) type 16 DNA was inoculated into human foreskin grafted to the skin of severe combined immunodeficient (scid) mice. Grafted skin contained human epidermis and dermis and, like normal human skin, expressed involucrin in differentiating keratinocytes. HPV type 16 DNA, attached to gold particles, was delivered directly into human epidermal cells and induced exophytic papilloma with histologic features of papillomavirus infection, including koilocytosis and expression of papillomavirus capsid antigen. This model should be useful for determining in vivo the functions of viral genes and for developing strategies to prevent and treat HPV-associated disease. It may also be of value in developing animal models of other human skin diseases.
Molecular and functional interactions of transcription factor USF with the long terminal repeat of human immunodeficiency virus type 1
Authors: d'Adda di Fagagna F , Marzio G , Gutierrez M I , Kang L Y , Falaschi A , Giacca M
Published: January 2020

The human transcription factor USF, purified from HeLa cells, and its recombinant 43-kDa component bind to the long terminal repeat (LTR) of human immunodeficiency virus type 1. The proteins footprint over nucleotides from position -173 to -157 upstream of the transcription start site, generating strong DNAse I hypersensitivity sites at the 3' sides on both strands. As detected by methylation protection studies, the factor forms symmetric contacts with the guanines of the palindromic CACGTG core of the recognized sequence. Its binding ability is abolished by the mutation of this core sequence and is strongly reduced by the cytosine methylation of the central CpG dinucleotide. Upon binding, both recombinant and purified USFs bend the LTR DNA template. The role of USF in the control of transcription initiation from the LTR was tested by in vitro transcription assays. Upon addition of the protein, transcription from constructs containing an intact binding site is increased, while the responsiveness in constructs with a mutated sequence is abolished. Furthermore, the addition of a decoy plasmid which contains multiple repeats of the target sequence results in downregulation of transcription from the LTR. These results suggest that USF is a positive regulator of LTR-mediated transcriptional activation.
Size of genetic bottlenecks leading to virus fitness loss is determined by mean initial population fitness
Authors: Novella I S , Elena S F , Moya A , Domingo E , Holland J J
Published: January 2020

Genetic bottlenecks are important events in the genetic diversification of organisms and colonization of new ecological niches. Repeated bottlenecking of RNA viruses often leads to fitness losses due to the operation of Muller's ratchet. Herein we use vesicular stomatitis virus to determine the transmission population size which leads to fitness decreases of virus populations. Remarkably, the effective size of a genetic bottleneck associated with fitness loss is greater when the fitness of the parental population increases. For example, for starting virus populations with low fitness, population transfers of five-clone-to-five-clone passages resulted in a fitness increase. However, when a parental population with high fitness was transferred, 30-clone-to-30-clone passages were required simply to maintain fitness values.
Two overlapping transcription units which extend across the L-S junction of herpes simplex virus type 1
Authors: Bohenzky R A , Lagunoff M , Roizman B , Wagner E K , Silverstein S
Published: January 2020

A region of the herpes simplex virus type 1 genome located upstream of the alpha 0 promoter contains a promoter which regulates transcription in the opposite orientation to that driven by alpha 0. Analyses of mutants from which this promoter, alpha X, was deleted and a mutant in which a fragment that serves as a transcription terminator and polyadenylation signal was inserted upstream of this promoter demonstrate that two distinct transcription units overlap this region of the genome and are transcribed in a direction antisense to the neurovirulence gene gamma (1)34.5. One unit, dependent on the alpha X promoter, is active when cells are infected in the presence of the protein synthesis inhibitor cycloheximide. The second unit, independent of alpha X, is active during the course of productive infection. This transcription unit originates from a promoter upstream of alpha X which is distinct from the latency-associated promoter (LAP). Two polyadenylated transcripts of 0.9 and 4.9 kb accumulate from this region of the genome during productive infection, but no mature transcripts accumulate in infected cells maintained in the presence of cycloheximide. Kinetic analyses demonstrate that the transcripts that accumulate during productive infection fall into the beta class of herpes simplex virus type 1 genes.
Repression in vitro, by human adenovirus E1A protein domains, of basal or Tat-activated transcription of the human immunodeficiency virus type 1 long terminal repeat
Authors: Song C Z , Loewenstein P M , Green M
Published: January 2020

Human adenovirus E1A proteins can repress the expression of several viral and cellular genes. By using a cell-free transcription system, we demonstrated that the gene product of the E1A 12S mRNA, the 243-residue protein E1A243R, inhibits basal transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). The HIV-1 transactivator protein Tat greatly stimulates transcription from the viral promoter in vitro. However, E1A243R can repress Tat-activated transcription in vitro. Strong repression of both basal and Tat-activated transcriptions requires only E1A N-terminal amino acid residues 1 to 80. Deletion analysis showed that E1A N-terminal amino acids 4 to 25 are essential for repression, whereas amino acid residues 30 to 49 and 70 to 80 are dispensable. Transcriptional repression by E1A in the cell-free transcription system is promoter specific, since under identical conditions, transcription of the adenovirus major late promoter and the Rous sarcoma virus LTR promoter was unaffected. The repression of transcription by small E1A peptides in vitro provides an assay for investigation of molecular mechanisms governing E1A-mediated repression of both basal and Tat-activated transcriptions of the HIV-1 LTR promoter.
Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins
Authors: Stillman E A , Rose J K , Whitt M A
Published: January 2020

We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.
Integration of human papillomavirus type 16 into the human genome correlates with a selective growth advantage of cells
Authors: Jeon S , Allen-Hoffmann B L , Lambert P F
Published: January 2020

Integration of human papillomavirus type 16 (HPV-16) DNA into a host chromosome has been hypothesized to result in altered expression of two viral transforming genes, E6 and E7, in cervical cancers. In order to investigate the role that changes in viral genomic state and gene expression play in cervical carcinogenesis, we have derived clonal populations of human cervical epithelial cells which harbor multiple copies of either extrachromosomal or integrated viral DNA. The clonal populations harboring extrachromosomal HPV-16 DNA stably maintained approximately 1,000 viral copies for at least 15 passages (approximately 100 cell doublings), which contrasted with the unstable HPV-16 replicons in the parental counterpart. In the clonal populations harboring integrated viral DNA, 3 to 60 copies of HPV-16 DNA were found integrated in either of two forms: type 1, in which all the copies of HPV-16 DNA were disrupted in the E2 open reading frame upon integration, and type 2, in which intact viral copies were flanked by disrupted viral copies and cellular sequences. Despite the lower HPV-16 DNA copy number, the clonal populations with integrated viral DNA had levels of E7 protein that were in most cases higher than those found in the clonal populations harboring extrachromosomal viral DNA. Irrespective of viral genomic state, the clonal populations were capable of undergoing terminal differentiation and unable to form colonies in soft agar, which is indicative of the nontumorigenic nature of these cells. Importantly, a cell population with integrated viral DNA was found to outgrow another with extrachromosomal DNA when these cells were cocultured over a period of time. Thus, integration of human papillomaviral DNA correlates with increased viral gene expression and cellular growth advantage. These observations are consistent with the hypothesis that integration provides a selective advantage to cervical epithelial precursors of cervical carcinoma.
Replication of Epstein-Barr virus oriLyt: lack of a dedicated virally encoded origin-binding protein and dependence on Zta in cotransfection assays
Authors: Fixman E D , Hayward G S , Hayward S D
Published: January 2020

Using a transient replication assay in which cosmid DNAs were cotransfected into Vero cells, we had previously demonstrated that oriLyt replication required six Epstein-Barr virus (EBV)-encoded replication genes. No oriLyt origin-binding protein was identified in this study, but oriLyt replication in the cotransfection assay was also dependent on the three lytic cycle transactivators Zta, Rta, and Mta and an activity encoded by the EBV Sal/I F fragment. We have now used expression plasmids for the six known replication proteins to further examine the question of the requirement for an oriLyt origin-binding protein. The activity in Sal/I-F was shown to be encoded by BKRF3. The predicted product of this open reading frame is an enzyme, uracyl DNA glycosylase, not an origin-binding protein, and is dispensable for replication in assays using expression plasmids. BBLF2, which is positionally related to the gene for the herpes simplex virus (HSV) UL9 origin-binding protein, was confirmed to be expressed as a spliced transcript with BBLF3 and not as an independent product. Examination of the requirement for the EBV transactivators revealed that Rta, while contributing to replication efficiency, was dispensable. Mta could be substituted by HSV IE63, and in complementation experiments with HSV replication genes, Mta was no longer required for replication of EBV oriLyt, suggesting that the contribution of Mta to replication may be indirect. Zta continued to be required for detectable oriLyt replication both with the EBV replication proteins and in the complementation assays with HSV replication proteins. We conclude that EBV does not encode an equivalent of HSV UL9 and that Zta is the sole virally encoded protein serving an essential origin-binding function.
Molecular cloning and characterization of a cellular protein that interacts with the human immunodeficiency virus type 1 Tat transactivator and encodes a strong transcriptional activation domain
Authors: Yu L , Zhang Z , Loewenstein P M , Desai K , Tang Q , Mao D , Symington J S , Green M
Published: January 2020

The mechanism by which human immunodeficiency virus type 1 Tat transactivates the long terminal repeat promoter is not understood. It is generally believed that Tat has one or more transcription factors as its cellular target. One might expect a cellular target for Tat to possess several properties, including (i) the ability to bind to the Tat activation region, (ii) the possession of a transcriptional activation domain, and (iii) the ability to contact the cellular transcription machinery. Here we describe the cloning, expression, and characterization of a human protein, termed TAP (Tat-associated protein), which possesses some of these properties. TAP is highly conserved in eukaryotes and is expressed in a variety of human tissues. The major intracellular species of TAP is a highly acidic 209-amino-acid protein that likely is formed by removal of a highly basic 70-amino-acid N-terminal segment from a primary translation product. By deletion analysis, we have identified a TAP C-terminal region rich in acidic amino acids and leucine residues which acts as a strong transcriptional activator when bound through GAL4 sites upstream of the core long terminal repeat promoter, as well as flanking sequences that mask the activation function. Amino acid substitution of two leucine residues within the core activation region results in loss of the TAP activation function. Two lines of evidence suggest that Tat interacts with TAP in vivo. First, promoter-bound Tat can recruit a TAP/VP16 fusion protein to the promoter. Second, transiently expressed Tat is found associated with endogenous TAP, as demonstrated by coimmuno-precipitation analysis. As shown in an accompanying report, the TAP activation region binds the Tat core activation region and general transcription factor TFIIB (L. Yu, P.M. Loewenstein, Z. Zhang, and M. Green, J. Virol. 69:3017-3023, 1995). These combined results suggest the hypothesis that TAP may function as a coactivator that bridges Tat to the general transcription machinery of the cell via TFIIB.
Nucleotide sequence of feline immunodeficiency virus: classification of Japanese isolates into two subtypes which are distinct from non-Japanese subtypes
Authors: Kakinuma S , Motokawa K , Hohdatsu T , Yamamoto J K , Koyama H , Hashimoto H
Published: January 2020

Seven isolates of feline immunodeficiency virus (FIV), Shizuoka, Yokohama, Sendai-1, Sendai-2, Fukuoka, Aomori-1, and Aomori-2, were isolated from FIV-seropositive domestic cats in Japan, and their proviral DNAs were amplified by PCR. The nucleotide sequences of their env and gag genes were determined and compared with those of previously described isolates: U.S. and European isolates and one Japanese isolate, TM2. Phylogenetic analyses of complete env gene sequences demonstrate that worldwide isolates are classified into three subtypes: Japanese TM2, Japanese Shizuoka, and non-Japanese subtypes (U.S. and European isolates), with 20% amino acid distances from each other. This pattern indicates that an evolutionary radiation of these three subtypes of FIV occurred at approximately the same time. The sequence data of gag genes also confirmed these results. Furthermore, the Sendai-1 isolate was identified as an imported FIV isolate.
Analysis of envelope sequence variants suggests multiple mechanisms of mother-to-child transmission of human immunodeficiency virus type 1
Authors: Briant L , Wade C M , Puel J , Brown A J , Guyader M
Published: January 2020

In order to elucidate the molecular mechanisms involved in human immunodeficiency virus type 1 (HIV-1) mother-to-child transmission, we have analyzed the genetic variation within the V3 hypervariable domain and flanking regions of the HIV-1 envelope gene in four mother-child transmission pairs. Phylogenetic analysis and amino acid sequence comparison were performed on cell-associated viral sequences derived from maternal samples collected at different time points during pregnancy, after delivery, and from child samples collected from the time of birth until the child was approximately 1 year of age. Heterogeneous sequence populations were observed to be present in all maternal samples collected during pregnancy and postdelivery. In three newborns, viral sequence populations obtained within 2 weeks after birth revealed a high level of V3 sequence variability. In contrast, V3 sequences obtained from the fourth child (diagnosed at the age of 1 month) displayed a more restricted heterogeneity. The phylogenetic analysis performed for each mother-child sequence set suggested that several mechanisms may potentially be involved in HIV-1 vertical transmission. For one pair, child sequences were homogeneous and clustered in a single branch within the phylogenetic tree, consistent with selective transmission of a single maternal variant. For the other three pairs, the child sequences were more heterogeneous and clustered in several separate branches within the tree. In these cases, it appeared likely that more than one maternal variant was responsible for infection of the child. In conclusion, no single mechanism can account for mother-to-child HIV-1 transmission; both the selective transmission of a single maternal variant and multiple transmission events may occur.
Retinoid activation of retinoic acid receptors but not of retinoid X receptors promotes cellular differentiation and replication of human cytomegalovirus in embryonal cells
Authors: Angulo A , Suto C , Boehm M F , Heyman R A , Ghazal P
Published: January 2020

The susceptibility of human embryonal cell line NT-2/D1 to replicate human cytomegalovirus (hCMV) is dependent on retinoic acid (RA) stimulation. Physiological responses to retinoic acid involve two distinct subfamilies of nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs), which function by activating transcription as heterodimeric or RXR homodimeric complexes from cis-acting DNA response elements. At present, it is not clear whether the association between these two classes of receptors can lead to multiple distinct induction pathways by signalling one or both receptor partners. Here we have determined, by selectively activating endogenous receptors with novel synthetic ligands specific for either RARs or RXRs, what ligand interaction is physiological in the retinoid receptor pathways necessary for inducing replication of hCMV in differentiated embryonal cells. We show that ligand binding to RAR alone is sufficient and that exclusive ligand activation of RXR is insufficient for inducing replication of hCMV. We also find that differentiation and inhibition of NT-2/D1 cell growth are promoted by compounds that signal the RAR pathway. These results provide direct evidence that RAR ligand-mediated physiological responses are separable and distinct from RXR ligand activation functions. Moreover, our results provide insight into a hormone response pathway for cellular differentiation that might be coopted by hCMV in the host.
The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules
Authors: Lopez N , Muller R , Prehaud C , Bouloy M
Published: January 2020

Overlapping cDNAs representing the complete L segment of Rift Valley fever virus were assembled, and the L protein was expressed via a recombinant vaccinia virus. The transcriptase activity of the L protein was assayed with two types of templates: natural ribonucleoproteins (RNPs) and artificial genome-like RNAs. RNPs purified in a CsCl gradient did not retain the RNA polymerase function, but the activity was restored when the L cDNA was expressed in mammalian cells via a recombinant vaccinia virus. Indeed, after transfection of transcriptase-depleted RNPs in cells infected with the recombinant vaccinia virus expressing the L protein, the mRNAs coding for the N and NSs proteins and to a lesser extent, those coding for the glycoproteins were synthesized as well as the corresponding proteins. The transcriptase activity of the recombinant L protein was then investigated by using synthetic templates containing the reporter chloramphenicol acetyltransferase gene in the antisense orientation flanked by the 3' and 5' noncoding region of the S genomic segment. Our results indicate that after transfection of the RNA templates, transcription was achieved in cells coexpressing both the L and N proteins. Together, the experiments demonstrate that the two proteins N and L are absolutely required and sufficient to reconstitute the transcriptase activity.
Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 Gag particles produced in baculovirus-infected cells
Authors: Garnier L , Ravallec M , Blanchard P , Chaabihi H , Bossy J P , Devauchelle G , Jestin A , Cerutti M
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) Pr55gag precursors were previously shown to assemble and bud efficiently as noninfectious virus-like particles (VLPs) when expressed in baculovirus-infected insect cells. In this study, we examined the abilities of foreign antigens to be incorporated on the outer surface of HIV-1 Gag particles. We have used a dual recombinant baculovirus, expressing the HIV-1 Gag gene and gD gene under the control of the P10 and polyhedrin promoters, respectively, to obtain hybrid VLPs. Transmission electron microscopy of insect cells infected with the dual recombinant revealed very large aggregates of particles budding from the cell membrane. The release of VLPs into the culture medium was clearly different for a recombinant baculovirus producing solely HIV-1 Gag, for which particles were uniformly distributed all around the cell surface. Biochemical analysis of hybrid particles indicated that glycoprotein gD was packaged into HIV-1 Gag VLPs. Moreover, the carboxy-terminal p6 region of Gag polyprotein and the glycoprotein gD intracytoplasmic domain were not required for gD incorporation. The experiments described here clearly demonstrate that glycoprotein gD can be packaged with HIV-1 Gag particles and released from insect cells.
Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage
Authors: Reed K E , Grakoui A , Rice C M
Published: January 2020

Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be mediated by a virus-encoded protease composed of the region of the polyprotein encoding NS2 and the N-terminal one-third of NS3. This protease is distinct from the NS3 serine protease, which is responsible for downstream cleavages in the nonstructural region. Site-directed mutagenesis of residues surrounding the 2/3 cleavage site showed that cleavage is remarkably resistant to single-amino-acid substitutions from P5 to P3' (GWRLL decreases API). The only mutations which dramatically inhibited cleavage were the ones most likely to alter the conformation of the region, such as Pro substitutions at the P1 or P1' position, deletion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to provide additional information on the polypeptide requirements for bimolecular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-third of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with constructs expressing intact versions of the defective region. Cotransfection experiments also showed that certain defective NS2-3 constructs partially inhibited cleavage of wild-type polypeptides. Although these results show that inefficient cleavage can occur in a bimolecular reaction, they suggest that both molecules must contribute a functional subunit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to occur at the 2/3 site during processing of the wild-type HCV polyprotein.
The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads
Authors: Willems L , Gatot J S , Mammerickx M , Portetelle D , Burny A , Kerkhofs P , Kettmann R
Published: January 2020

The bovine leukemia virus (BLV) transmembrane protein (gp30) contains three YXXL motifs at its carboxyterminal end. Two of these motifs have been implicated in vitro in signal transduction pathways from the external to the intracellular compartment. In order to analyze the biological relevance of these motifs in vivo, recombinant BLV proviruses were constructed. A mutation of the tyrosine residue of the second YXXL motif completely destroyed the infectious potential of the virus in sheep. In contrast, the tyrosine of the first motif appeared to be dispensable for infectivity. However, the propagation of the recombinant virus within the animal was greatly impaired (as demonstrated by PCR and enzyme-linked immunosorbent assay). These recombinant BLVs thus exhibit an attenuated phenotype. Altogether, our data demonstrate the importance of the YXXL motifs of the BLV transmembrane protein for in vivo infection and viral propagation.