bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and analysis of the human cytomegalovirus UL80-encoded protease: identification of autoproteolytic sites
Authors: Baum E Z , Bebernitz G A , Hulmes J D , Muzithras V P , Jones T R , Gluzman Y
Published: January 2020

The 45-kDa assembly protein of human cytomegalovirus is encoded by the C-terminal portion of the UL80 open reading frame (ORF). For herpes simplex virus, packaging of DNA is accompanied by cleavage of its assembly protein precursor at a site near its C terminus, by a protease encoded by the N-terminal region of the same ORF (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). By analogy with herpes simplex virus, we investigated whether a protease is contained within the N-terminal portion of the human cytomegalovirus UL80 ORF. The entire UL80 ORF was expressed in Escherichia coli, under the control of the phage T7 promoter. UL80 should encode a protein of 85 kDa. Instead, the wild-type construct produces a set of proteins with molecular masses of 50, 30, 16, 13, and 5 kDa. In contrast, when mutant UL80 is deleted of the first 14 amino acids, it produces only an 85-kDa protein. These results suggest that the UL80 polyprotein undergoes autoproteolysis. We demonstrate by deletional analysis and by N-terminal sequencing that the 30-kDa protein is the protease and that it originates from the N terminus of UL80. The UL80 polyprotein is cleaved at the following three sites: (i) at the C terminus of the assembly protein domain, (ii) between the 30- and 50-kDa proteins, and (iii) within the 30-kDa protease itself, which yields the 16- and 13-kDa proteins and may be a mechanism to inactivate the protease.
Long-range mapping of Mis-2, a common provirus integration site identified in murine leukemia virus-induced thymomas and located 160 kilobase pairs downstream of Myb
Authors: Villeneuve L , Jiang X , Turmel C , Kozak C A , Jolicoeur P
Published: January 2020

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.
Alternative transcriptional initiation as a novel mechanism for regulating expression of a baculovirus trans activator
Authors: Wu X , Stewart S , Theilmann D A
Published: January 2020

In this report, we show that the Orgyia pseudotsugata nuclear polyhedrosis virus p34 gene, which is homologous to the Autographa californica nuclear polyhedrosis virus PE-38 gene, is a trans activator. The predicted p34 protein contains a number of motifs that are similar to those found in other eukaryotic transcriptional trans activators, including a putative zinc finger DNA-binding domain, a glutamine-rich domain, and a leucine zipper. Northern (RNA) blot analysis showed that the p34 gene is expressed as a 1.1-kb mRNA from 1 to 48 h postinfection and as a 0.7-kb mRNA from 18 to 120 h postinfection. Mapping of these transcripts showed that they were 3' coterminal but initiated at different 5' start sites. The 1.1-kb transcript initiates at a baculovirus early gene motif (CACAGT) and encodes the entire p34 open reading frame (ORF). The 0.7-kb transcript initiates at a baculovirus late gene start site (GTAAG) internal to the p34 ORF. Western blot (immunoblot) analysis using p34 antisera showed that the 0.7-kb transcript is translated as an amino-terminally truncated 20-kDa form of the full length 34-kDa protein. Functional analysis indicated that the 34-kDa protein transcriptionally trans activates the IE-2 promoter whereas the 20-kDa protein does not. Therefore, p34 produces two functionally different proteins from the same ORF, using the novel mechanism of alternative transcriptional initiation.
Rubella virus-specific cytotoxic T-lymphocyte responses: identification of the capsid as a target of major histocompatibility complex class I-restricted lysis and definition of two epitopes
Authors: Lovett A E , Hahn C S , Rice C M , Frey T K , Wolinsky J S
Published: January 2020

The role of major histocompatibility complex (MHC) class I-restricted CD8+ cytotoxic T lymphocytes in immunity to rubella virus (RV) infection is unknown. Lymphocytes of RV-immune individuals were prestimulated on an RV-infected MHC class I-matched (or partially matched) fibroblast monolayer which generated CD8+ lymphoblasts capable of lysing RV-infected fibroblast targets in a class I-restricted manner. Using an infectious Sindbis virus (SV) vector which expressed the RV capsid protein (SV/RubC), lymphocytes from 17 of 22 RV-immune individuals prestimulated on RV-infected fibroblast monolayers lysed SV/RubC-infected fibroblast targets. A sequence within the amino terminus of the capsid protein that was previously shown to contain immunodominant class II-restricted T-cell epitopes was evaluated for class I-restricted epitopes. Fibroblast targets pulsed with synthetic peptides representing subsequences within C1 to C29 (subscripts indicate amino acid positions) were lysed effectively when the targets and effectors matched at multiple class I alleles. By limiting the number of matching class I alleles, an A2-restricted epitope was identified within C9 to C22 and an epitope that could be presented by multiple class I molecules was identified within C11 to C29. A sequence such as C1 to C29 which contains both MHC class I- and MHC class II-restricted epitopes recognized by a heterologous human population may serve as a component of an effective synthetic vaccine.
Possible involvement of microtubule disruption in bipolar budding of a Sendai virus mutant, F1-R, in epithelial MDCK cells
Authors: Tashiro M , Seto J T , Klenk H D , Rott R
Published: January 2020

Envelope glycoproteins F and HN of wild-type Sendai virus are transported to the apical plasma membrane domain of polarized epithelial MDCK cells, where budding of progeny virus occurs. On the other hand, a pantropic mutant, F1-R, buds bipolarly at both the apical and basolateral domains, and the viral glycoproteins have also been shown to be transported to both of these domains (M. Tashiro, M. Yamakawa, K. Tobita, H.-D. Klenk, R. Rott, and J.T. Seto, J. Virol. 64:4672-4677, 1990). MDCK cells were infected with wild-type virus and treated with the microtubule-depolymerizing drugs colchicine and nocodazole. Budding of the virus and surface expression of the glycoproteins were found to occur in a nonpolarized fashion similar to that found in cells infected with F1-R. In uninfected cells, the drugs were shown to interfere with apical transport of a secretory cellular glycoprotein, gp80, and basolateral uptake of [35S]methionine as well as to disrupt microtubule structure, indicating that cellular polarity of MDCK cells depends on the presence of intact microtubules. Infection by the F1-R mutant partially affected the transport of gp80, uptake of [35S]methionine, and the microtubule network, whereas wild-type virus had a marginal effect. These results suggest that apical transport of the glycoproteins of wild-type Sendai virus in MDCK cells depends on intact microtubules and that bipolar budding by F1-R is possibly due, at least in part, to the disruption of microtubules. Nucleotide sequence analyses of the viral genes suggest that the mutated M protein of F1-R might be involved in the alteration of microtubules.
Glycoprotein B is a specific determinant of herpes simplex virus type 1 neuroinvasiveness
Authors: Yuhasz S A , Stevens J G
Published: January 2020

Herpes simplex virus type 1 strains ANG and KOS lack neuroinvasiveness when inoculated on the footpads of mice, and because the strains are able to complement each other, the genes associated with this phenotype differ. In this study, we used marker rescue techniques to show that at least two genes cloned from ANG are required to restore neuroinvasiveness to KOS. One of the two fragments required is the 6.3-kb BamHI-A/EcoRI-D fragment (0.15 to 0.19 map units). The second has been identified as the sequence encoding glycoprotein B (gB) (UL27). Analysis of ANG and KOS DNA sequences in the relevant region of the gB gene revealed two nucleotide differences which result in amino acid differences in the gB protein. One appears to be unique to the strain of KOS used in our laboratory. The second, at codon 523 of the mature gB protein, encodes a valine in KOS and an alanine in ANG. Recombinant KOS viruses which contained ANG sequences in this region were constructed, and two independently selected recombinants demonstrated increased neuroinvasiveness in mice. From these results, we conclude that gB significantly influences neuroinvasiveness. Mechanisms by which this might occur are discussed.
In vitro expression of the human cytomegalovirus DNA polymerase gene: effects of sequence alterations on enzyme activity
Authors: Ye L B , Huang E S
Published: January 2020

Genomic DNA of the Towne strain human cytomegalovirus polymerase (pol) gene (4.4-kb RsrII-NcoI segment of the EcoRI J fragment) was cloned into plasmids containing either the T3 or the T7 promoter for in vitro transcription-translation studies. The translation efficiency of unmodified pol cRNA was poor in this system and could not be improved by capping. However, the efficiency could be enhanced by replacing the leader sequence with a 40-bp AT-rich sequence derived from an alfalfa mosaic virus, R4. pol cRNA directed the synthesis of a 140-kDa polypeptide in a rabbit reticulocyte translation system. The in vitro-translated wild-type enzyme possessed significant polymerization activity which could be stimulated by salt as could that of the authentic enzyme purified from virus-infected cells. To study the critical domains of this enzyme, nine mutations were introduced into the pol gene around the conserved domains of eukaryotic polymerase by oligonucleotide-directed mutagenesis. Two constructs with mutations at amino acid residues 323 to 325 (M32QS) and 725 to 726 (M72II) remained active, with partial loss of enzyme activity, while the enzyme activities of other mutants with alterations at four domains located around amino acid residues 729 to 730 (M73HN), 804 to 807 (M80 and DE80), 910 to 913 (M91 and DE91), and 962 to 964 (M96 and DE96) were abolished. DNA template and triphosphate binding assays indicated that the mutation at 804 to 807 (conserved region III) lost the ability to bind DNA template, and four mutants, M73HN (within conserved region II), M80 (in region III), M91 (in region I), and M96 (around region V [962 to 964; amino acid sequence KKR]), failed to bind deoxyribonucleoside triphosphate. These data suggest that conserved region III is essential for DNA template binding, while residues between conserved region II and V (725 to 964) are involved in triphosphate binding.
Marked, transient inhibition of expression of the Epstein-Barr virus latent membrane protein gene in Burkitt's lymphoma cell lines by electroporation
Authors: Gahn T A , Sugden B
Published: January 2020

The Raji, EB1, and EB2 cell lines are derived from Epstein-Barr virus (EBV)-positive Burkitt's lymphomas. EBV gene products associated with viral latency, including latent membrane protein (LMP) and Epstein-Barr nuclear antigen 2 (EBNA-2), are expressed in these cell lines. We have found that transfection of all three of these cell lines by electroporation in both the presence and the absence of foreign DNA resulted in a marked decrease in expression of the endogenous EBV gene encoding LMP. An analysis of this response in Raji cells revealed that the level of RNA of this gene was decreased transiently and returned to normal levels by 7 days after transfection. The level of LMP protein was also reduced after transfection. No difference in survival was detected in electroporated versus unperturbed Raji cells. The level of mRNA encoding a modulator of the LMP gene, EBNA-2, was unchanged by electroporation. However, the level of another EBV transcript, BHLF-I, was reduced. The effect of electroporation could not be attributed to flux of Ca2+, Na+, K+, or Cl- ions across the plasma membrane. Expression of LMP in several lymphoblastoid cell lines was unaffected by electroporation.
Analysis of simian immunodeficiency virus sequence variation in tissues of rhesus macaques with simian AIDS
Authors: Kodama T , Mori K , Kawahara T , Ringler D J , Desrosiers R C
Published: January 2020

One rhesus macaque displayed severe encephalomyelitis and another displayed severe enterocolitis following infection with molecularly cloned simian immunodeficiency virus (SIV) strain SIVmac239. Little or no free anti-SIV antibody developed in these two macaques, and they died relatively quickly (4 to 6 months) after infection. Manifestation of the tissue-specific disease in these macaques was associated with the emergence of variants with high replicative capacity for macrophages and primary infection of tissue macrophages. The nature of sequence variation in the central region (vif, vpr, and vpx), the env gene, and the nef long terminal repeat (LTR) region in brain, colon, and other tissues was examined to see whether specific genetic changes were associated with SIV replication in brain or gut. Sequence analysis revealed strong conservation of the intergenic central region, nef, and the LTR. However, analysis of env sequences in these two macaques and one other revealed significant, interesting patterns of sequence variation. (i) Changes in env that were found previously to contribute to the replicative ability of SIVmac for macrophages in culture were present in the tissues of these animals. (ii) The greatest variability was located in the regions between V1 and V2 and from "V3" through C3 in gp120, which are different in location from the variable regions observed previously in animals with strong antibody responses and long-term persistent infection. (iii) The predominant sequence change of D-->N at position 385 in C3 is most surprising, since this change in both SIV and human immunodeficiency virus type 1 has been associated with dramatically diminished affinity for CD4 and replication in vitro. (iv) The nature of sequence changes at some positions (146, 178, 345, 385, and "V3") suggests that viral replication in brain and gut may be facilitated by specific sequence changes in env in addition to those that impart a general ability to replicate well in macrophages. These results demonstrate that complex selective pressures, including immune responses and varying cell and tissue specificity, can influence the nature of sequence changes in env.
Alternate pathways of secretion of simian immunodeficiency virus envelope glycoproteins
Authors: Spies C P , Compans R W
Published: January 2020

A biotinylation assay was used to detect the envelope glycoprotein of the simian immunodeficiency virus (SIV) envelope glycoprotein expressed by a recombinant vaccinia virus on the surface of HeLa T4 cells. The relationship between the detection of the envelope glycoprotein on the cell surface and its secretion from the cell was examined. It was found that much more gp120 was released into the culture medium than could be accounted for by shedding of the biotinylated SIV envelope protein from the cell surface. Treatment with the ionophore monensin showed that this drug did not block the secretion of gp120 into the culture medium even though the expression of gp120 on the cell surface was strongly downregulated. Similar results were observed for the secretion of gp120 in HUT78 cells infected with SIVmac251 virus. Brefeldin A, on the other hand, inhibited both the detection of gp120 on the cell surface and its secretion into the culture medium. On the basis of these results, we propose that gp120 can be secreted into the culture medium via at least two pathways. One pathway involves the dissociation of gp120 from membrane-associated gp41-gp120 complexes on the cell surface. However, the major pathway involves the secretion of gp120 without its transitory appearance on the cell surface as part of a gp41-gp120 complex.
Genetic determinants of dengue type 4 virus neurovirulence for mice
Authors: Kawano H , Rostapshov V , Rosen L , Lai C J
Published: January 2020

Mouse-adapted dengue type 4 virus (DEN4) strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes from mouse neurovirulent DEN4 strain H241 proved to be highly neurovirulent in mice, whereas the chimera that contained the corresponding genes from its non-mouse-adapted parent was not neurovirulent. This finding indicates that most of the genetic loci for the neurovirulence of the DEN4 mutant lie within the structural protein genes. A comparison of the amino acid sequences of the parent and its mouse neurovirulent mutant proteins revealed that there were only five amino acid differences in the structural protein region, and three of these were located in the envelope (E) glycoprotein. Analysis of chimeras which contained one or two of the variant amino acids of the mutant E sequence substituting for the corresponding sequence of the parental virus identified two of these amino acid changes as important determinants of mouse neurovirulence. First, the single substitution of Ile for Thr-155 which ablated one of the two conserved glycosylation sites in parental E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant. Thus, the loss of an E glycosylation site appears to play a role in DEN4 neurovirulence. Second, the substitution of Leu for Phe-401 also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. These findings indicate that at least two of the genetic loci responsible for DEN4 mouse neurovirulence map within the structural protein genes.
Replication of a macrophage-tropic strain of human immunodeficiency virus type 1 (HIV-1) in a hybrid cell line, CEMx174, suggests that cellular accessory molecules are required for HIV-1 entry
Authors: Stefano K A , Collman R , Kolson D , Hoxie J , Nathanson N , Gonzalez-Scarano F
Published: January 2020

To investigate the mechanism underlying one aspect of the cellular tropism of human immunodeficiency virus type 1 (HIV-1), we used a macrophage-tropic isolate, 89.6, and screened its ability to infect a number of continuous cell lines. HIV-1 (89.6) was able to replicate robustly in a T-cell/B-cell hybrid line, CEMx174, while it replicated modestly or not at all in either of its parents, one of which is the CD4-positive line CEM.3. Analysis by transfection of a molecular clone, a virus uptake assay, and polymerase chain reaction all provided strong evidence that the block to HIV-1(89.6) replication in the CEM.3 line lies at the level of cellular entry. These results were complemented by preparing a CD4-expressing derivative of the B-cell parent, 721.174, and demonstrating that it is permissive for productive HIV-1(89.6) replication. Given these experimental findings, we speculate that there exist cellular accessory factors which facilitate virus entry and infection in CD4-positive cells. Furthermore, these cellular accessory factors may be quite virus strain specific, since not all macrophage-tropic strains of HIV-1 were able to replicate in the CEMx174 hybrid cell line. This experimental model provides a system for the identification of one or more of these putative cellular accessory factors.
Species Barrier Prevents an Abnormal Isoform of Prion Protein from Accumulating in Follicular Dendritic Cells of Mice with Creutzfeldt-Jakob Disease
Authors: Muramoto Tamaki , Kitamoto Tetsuyuki , Hoque Mohammad Zahirul , Tateishi Jun , Goto Ikuo
Published: January 2020

The accumulation of abnormal prion protein in follicular dendritic cells did not occur in mice inoculated with materials from human Creutzfeldt-Jakob disease, whereas it always occurred in mice inoculated with mouse-adapted agents, suggesting an intense expression of the species barrier in the lymphoreticular system.
Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain
Authors: Garrett E D , He F , Bogerd H P , Cullen B R
Published: January 2020

The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1.
Anoxia-inducible rat VL30 elements and their relationship to ras-containing sarcoma viruses
Authors: Firulli B A , Anderson G R , Stoler D L , Estes S D
Published: January 2020

VL30 elements are associated with cancer by their overexpression in rodent malignancies, their induction in a fibroblast response to anoxia which shares features with the malignant phenotype, and their presence recombined into Harvey murine sarcoma virus (HaSV) and Kirsten murine sarcoma virus. These sarcoma viruses contain ras oncogenes flanked on both sides by retrotransposon VL30 element sequences, in turn flanked by mouse leukemia virus sequences. Three very basic questions have existed about the VL30 element sequences found in sarcoma viruses: (i) how did they become recombined, (ii) what are their exact boundaries, and (iii) why are they there? To help decipher the nature of VL30 elements in sarcoma viruses, we examined VL30 clones isolated from an anoxic fibroblast cDNA library and independently by polymerase chain reaction cloning from rat cell DNA. Sequence comparisons with HaSV revealed that HaSV was formed by the substitution of 0.7 kb of VL30 sequences by 0.9 kb of c-Ha-ras sequences, with this event possibly facilitated by the presence of an identical Alu-like repeat found upstream of the 5' recombination point in both the VL30 element and c-Ha-ras. Recombination occurred 42 bases beyond the Alu-like sequences in VL30 and 1596 bases beyond them in c-Ha-ras, at position 926 of HaSV. The 3' ras-VL30 recombination event in HaSV occurred within a seven-base region of shared sequence identity, between HaSV bases 1825 and 1825 and 1831. Recombination between Moloney leukemia virus (MoLV) and VL30 appears to have occurred at a point corresponding to base 218 or 219 of MoLV and was near a TAR-like VL30 sequence; such recombination at the 3' end was between positions 7445 and 7456 of MoLV (HaSV positions 4694 to 4703). Kirsten murine sarcoma virus was found to be closely analogous to HaSV, and limited similar features were also seen with Rasheed sarcoma virus.
The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates
Authors: Fu T B , Taylor J
Published: January 2020

Human hepatitis delta virus has a single-stranded circular RNA genome that replicates by RNA-directed RNA synthesis. The virus encodes only a single protein, the delta antigen, which both is small (22 kDa) and lacks sequence homology to known RNA polymerases, suggesting that the virus employs a cellular polymerase for replication. Consistent with this suggestion, we have used homogenized nuclei from a human hepatoma cell line, HepG2, to demonstrate RNA-directed RNA synthesis from both genomic hepatitis delta virus RNA and its complement, the antigenomic RNA. RNA polymerase II was responsible for this transcription because the reaction was inhibited both by low doses of alpha-amanitin and by a monoclonal antibody specific for polymerase II. In addition, it was found that the majority of the RNA products were processed, presumably by self-cleavage and self-ligation, to produce covalently closed circular molecules.
5' sequences of rubella virus RNA stimulate translation of chimeric RNAs and specifically interact with two host-encoded proteins
Authors: Pogue G P , Cao X Q , Singh N K , Nakhasi H L
Published: January 2020

Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postulated to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing chimeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'- and 3'-terminal sequence domains from the RV genome or several deletion derivatives of the same sequences. After in vitro transcription of chimeric RNAs, the translational efficiencies of these RNAs were compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured for chimeric RV/CAT RNAs expressed in transfected cells by the adenovirus major late promoter. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chimeric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the primary enhancing effect on translation. To identify host proteins which interact with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host proteins 59 and 52 kDa in size, present in cytosolic extracts from both uninfected and RV-infected cells, specifically interacted with the RV 5'(+)SL RNA. Direct binding comparisons between wild-type and mutant 5'(+)SL RNAs demonstrated that sequences in and around the bulge region of the terminal stem domain of this structure constituted a protein binding determinant. Human serum, qualified for anti-Ro/SS-A antigen specificity, immunoprecipitated 59- and 52-kDa protein-RNA complexes containing the RV 5'(+)SL RNA. However, poly- and monoclonal antisera raised against the recombinant 60- and 52-kDa Ro proteins failed to precipitate complexes containing the 5'(+)SL RNA. The identity of the proteins binding this RV cis-acting element remains to be determined; however, their role in RV translation is discussed.
Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication
Authors: Lund A H , Duch M , Lovmand J , Jørgensen P , Pedersen F S
Published: January 2020

Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching tRNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site.
Viral genomes maintained extrachromosomally in hamster polyomavirus-induced lymphomas display a cell-specific replication in vitro
Authors: de La Roche Saint André C , Mazur S , Feunteun J
Published: January 2020

Hamster polyomavirus causes lymphomas when injected into newborn Syrian hamsters. Large amounts of extrachromosomal viral genomes are accumulated in the lymphoma cells. These genomes are characterized by deletions affecting the late coding region as well as a specific part of the noncoding regulatory region. By contrast with wild-type genomes, lymphoma-associated genomes replicate in a lymphoblastoid cell line but not in a fibroblastic cell line. The deletion acts in a cis-dominant manner and is the primary determinant of this host-range effect on replication. The boundaries of the regulatory region necessary for viral DNA replication in the two cell contexts have been defined. The regulatory region can be functionally divided in two domains: one domain (distal from the origin of replication) is necessary for viral genome replication in fibroblasts, whereas the other domain (proximal to the origin of replication) is functional only in the lymphoblastoid cell context and contains the sequence specifically conserved in the lymphoma-associated genomes. This sequence harbors a motif recognized by a lymphoblastoid cell-specific trans-acting factor.
A novel class of transcripts expressed with late kinetics in the absence of ICP4 spans the junction between the long and short segments of the herpes simplex virus type 1 genome
Authors: Yeh L , Schaffer P A
Published: January 2020

A novel family of transcripts that span the junction between the long and short segments of the herpes simplex virus type 1 genome has been identified. These transcripts, designated L/S junction-spanning transcripts (L/STs), are synthesized in abundance in a variety of cells infected with mutant viruses defective in the gene for ICP4, the major transcriptional regulatory protein of the virus. Transcription of abundant 2.3- and 8.5-kb series of L/STs was shown to initiate within the same sequences as less abundant 4.2-, 7.3-, and > 9.5-kb transcripts by Northern (RNA) blot analysis. S1 nuclease analysis revealed a single 5' terminus 28 bp downstream of a TATA box and 6 bp downstream of a consensus ICP4 binding site. The location of the transcriptional start site indicates that the promoter of the L/STs likely corresponds to the bidirectional promoter described by Bohenzky et al. (R. A. Bohenzky, A. G. Papavassiliou, I. H. Gelman, and S. Silverstein, J. Virol. 67:632-642, 1993). The L/STs accumulate with late kinetics in ICP4 mutant-infected cells and are polyadenylated. Mutant viruses encoding forms of ICP4 unable to bind the consensus site, ATCGTC, exhibited abundant expression of the L/STs, whereas mutants encoding forms of ICP4 able to bind this site expressed no detectable L/STs, suggesting that ICP4 plays a critical role in repressing L/ST expression. Their synthesis in ICP4 mutant-infected cells is inhibited by the protein synthesis inhibitor cycloheximide, indicating that they are induced either by an immediate-early viral protein other than ICP4 or by a virus-induced cellular protein. Preliminary evidence indicates that the L/STs are not present in latently infected ganglia. The abundant expression of the L/STs with late kinetics only in the absence of functional ICP4 and the sensitivity of their synthesis to cycloheximide indicate that they are not members of any of the recognized kinetic classes of herpes simplex virus type 1 transcripts but constitute a new class of viral transcript.
High viral load in lymph nodes and latent human immunodeficiency virus (HIV) in peripheral blood cells of HIV-1-infected chimpanzees
Authors: Saksela K , Muchmore E , Girard M , Fultz P , Baltimore D
Published: January 2020

We have examined human immunodeficiency virus type 1 (HIV-1) infection in chimpanzees by analyzing HIV-1 DNA and RNA in lymph nodes and peripheral mononuclear cells (PBMCs). Like certain asymptomatic HIV-infected persons, these chimpanzees had no detectable viral replication in their PBMCs. However, viral replication and a high viral load were observed in the lymphatic tissue. Despite the absence of viral replication in PBMCs, 1/1,000 to 1/10,000 of the PBMCs contained HIV-1 proviral DNA, and HIV transcription could be rapidly induced in these cells in vitro. These results provide direct evidence of cellular latency of HIV in vivo and suggest that HIV infection in chimpanzees may be a useful model for clinical latency of HIV infection in humans.
Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription
Authors: Diaz-Guerra M , Esteban M
Published: January 2020

We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with a temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the receptor gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way of an auxiliary function which regulates mRNA transcription during the virus growth cycle.
Reverse transcriptase and protease activities of avian leukosis virus Gag-Pol fusion proteins expressed in insect cells
Authors: Stewart L , Vogt V M
Published: January 2020

Protease (PR)-defective avian leukosis virus particles display 300-fold-reduced levels of reverse transcriptase (RT) activity relative to wild-type particles. This observation suggests that during virion assembly RT is activated by proteolytic maturation of the Gag-Pol polyprotein precursor. To study the relationship between proteolytic cleavage and RT activation, we subjected PR-defective virion cores to digestion with purified viral PR and analyzed the structure of the major polypeptides produced as well as RT activity. Under conditions in which Gag precursors were fully matured, the RT domain was only incompletely released from the Gag-Pol precursor, remaining tethered to the upstream Gag domains PR or NC-PR. In the same reaction, RT activity was stimulated only three-fold, or 100-fold less than expected for a fully active RT. The poor activation suggested that the NC or PR domains could repress RT activity. To test this idea, we constructed recombinant baculoviruses expressing 19 different fusion proteins with upstream Gag or downstream Pol sequences attached to RT. Each protein was partially purified and assayed for its inherent RT activity. The results are consistent with the idea that Gag sequences can inhibit RT activity but indicate that the size of the Pol domain as well as the status of the PR domain (wild-type or mutant) also can profoundly influence activity. Several of the constructed Gag-Pol fusion proteins contained a wild-type PR domain. Some of these underwent intracellular PR-mediated processing, while others did not. All proteins in which the PR domain was preceded by upstream Gag sequences showed specific proteolysis. By contrast, all proteins initiated with a methionine placed one residue upstream of the natural N terminus of PR failed to show specific proteolysis. Amino-terminal sequencing of one such protein yielded the correct amino acid sequence and showed that the initiating methionine was not removed. One interpretation of these findings is that activation of PR requires the generation of the precise N terminus of the mature PR.
Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29
Authors: Dewhurst S , Dollard S C , Pellett P E , Dambaugh T R
Published: January 2020

DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal region of approximately 400 bp which lies upstream of the gene that is homologous to herpes simplex virus UL29, a region that carries an origin in other betaherpesviruses and in some alphaherpesviruses.
Molecular mimicry between the human immunodeficiency virus type 1 gp120 V3 loop and human brain proteins
Authors: Trujillo J R , McLane M F , Lee T H , Essex M
Published: January 2020

Immunologically cross-reactive proteins in the human brain that resemble the V3 loop of human immunodeficiency virus type 1 (HIV-1) gp120 have been identified. When several homogenized tissues from normal brains were used, a monoclonal antibody raised against amino acids 308 to 320 of the V3 loop reacted with three prominent human brain proteins (HBP) of 35, 55, and 110 kDa. Among the three, the 55-kDa HBP appears to be specific to the central nervous system. These results indicate that the V3 loop of HIV-1 gp120 shares an epitope with HBP. An immune response to the V3 loop that generates cross-reactive antibodies to cellular proteins may be an autoimmune mechanism by which HIV-1 can damage the central nervous system.
Analysis of multiple mRNAs from pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse reveals a novel protein, Ttm, derived from the carboxy terminus of the EIAV transmembrane protein
Authors: Beisel C E , Edwards J F , Dunn L L , Rice N R
Published: January 2020

Transcription of pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. Four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, EIAV. Nucleotide sequence analysis demonstrated that three of these mRNAs encode known viral proteins: the envelope precursor, the product of the S2 open reading frame, and the regulatory proteins Tat and Rev. The fourth transcript encodes a novel Tat-TM fusion protein, Ttm. Ttm is a 27-kDa protein translated from the putative tat CTG initiation codon and containing the carboxy-terminal portion of TM immediately downstream from the membrane-spanning domain. p27ttm is expressed in EIAV-infected canine cells and was recognized by peptide antisera against both Tat and TM. Cells transfected with ttm cDNA also expressed p27ttm, which appeared to be localized to the endoplasmic reticulum or Golgi apparatus by indirect immunofluorescence. The carboxy terminus of lentiviral TM proteins has previously been shown to influence viral infectivity, growth kinetics, and cytopathology, suggesting that Ttm plays an important role in the EIAV life cycle.
Isolation of a human T-lymphotropic virus type I strain from Australian aboriginals
Authors: Bastian I , Gardner J , Webb D , Gardner I
Published: January 2020

A human T-lymphotropic virus type I (HTLV-I) strain was isolated in a CD4+ T-lymphocyte culture established from a healthy seropositive Australian Aboriginal. This isolate, identified as HTLV-IMSHR-1, was detected by immunofluorescence with monoclonal antibodies, by the presence of gag-encoded protein p24 in the culture supernatant, and by cocultivation leading to infection and transformation of lymphocytes from an HTLV-I-negative donor. By using the polymerase chain reaction technique, the env gene and segments of the pol and pX regions of the proviral genome of HTLV-I(MSHR-1) were amplified and sequenced. Comparison with the envelope sequences of prototype strains revealed up to 7% divergence at the nucleotide level and 3.1 to 4.3% divergence in the predicted amino acid sequence. Phylogenetic analysis showed that the Australian and Melanesian isolates are related. Differential reactivity with monoclonal antibodies suggests that gag protein p19 of HTLV-I(MSHR-1) is also divergent. The potential for antigenic divergence between the prototype HTLV-I isolates and the Austro-Melanesian variants requires further investigation, because it would have implications for serodiagnosis and vaccine development.
CD4 molecules with a diversity of mutations encompassing the CDR3 region efficiently support human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion
Authors: Broder C C , Berger E A
Published: January 2020

The third complementarity-determining region (CDR3) within domain 1 of the human CD4 molecule has been suggested to play a critical role in membrane fusion mediated by the interaction of CD4 with the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein. To analyze in detail the role of CDR3 and adjacent regions in the fusion process, we used cassette mutagenesis to construct a panel of 30 site-directed mutations between residues 79 and 96 of the full-length CD4 molecule. The mutant proteins were transiently expressed by using recombinant vaccinia virus vectors and were analyzed for cell surface expression, recombinant gp120-binding activity, and overall structural integrity as assessed by reactivity with a battery of anti-CD4 monoclonal antibodies. Cells expressing the CD4 mutants were assayed for their ability to form syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. Surprisingly in view of published data from others, most of the mutations had little effect on syncytium-forming activity. Normal fusion was observed in 21 mutants, including substitution of human residues 85 to 95 with the corresponding sequences from either chimpanzee, rhesus, or mouse CD4; a panel of Ser-Arg double insertions after each residue from 86 to 91; and a number of other charge, hydrophobic, and proline substitutions and insertions within this region. The nine mutants that showed impaired fusion all displayed defective gp120 binding and disruption of overall structural integrity. In further contrast with results of other workers, we observed that transformant human cell lines expressing native chimpanzee or rhesus CD4 efficiently formed syncytia when mixed with cells expressing the HIV-1 envelope glycoprotein. These data refute the conclusion that certain mutations in the CDR3 region of CD4 abolish cell fusion activity, and they suggest that a wide variety of sequences can be functionally tolerated in this region, including those from highly divergent mammalian species. Syncytium formation mediated by several of the CDR3 mutants was partially or completely resistant to inhibition by the CDR3-directed monoclonal antibody L71, suggesting that the corresponding epitope is not directly involved in the fusion process.(ABSTRACT TRUNCATED AT 400 WORDS)
Immunological responses of mice and cattle to baculovirus-expressed F and H proteins of rinderpest virus: lack of protection in the presence of neutralizing antibody
Authors: Bassiri M , Ahmad S , Giavedoni L , Jones L , Saliki J T , Mebus C , Yilma T
Published: January 2020

Rinderpest is a highly contagious viral disease of ruminants and has greater than 95% morbidity and mortality. The etiological agent, rinderpest virus (RPV), is a member of the family Paramyxoviridae and the genus Morbillivirus. Immune responses to both the hemagglutinin (H) and the fusion (F) antigens of morbilliviruses play an important role in the prevention of infection, and only attenuated live vaccines have been shown to provide protective immunity against the group. The lack of protection with inactivated vaccines has been attributed to the denaturation of the F glycoprotein of the virus. Our previous study, however, demonstrated complete protection of cattle vaccinated with infectious vaccinia virus recombinants expressing the H (vRVH) or F (vRVF) protein alone, even in the presence of only 4 U of serum-neutralizing (SN) antibody to RPV (T. Yilma, D. Hsu, L. Jones, S. Owens, M. Grubman, C. Mebus, M. Yamanaka, and B. Dale, Science 242:1058-1061, 1988). We have constructed recombinant baculoviruses that express the F (Fb) and H (Hb) glycoproteins of RPV. Furthermore, we have analyzed the immune responses of mice and cattle to these antigens. Cattle vaccinated with Fb or Hb or a mixture of both antigens were not protected from challenge inoculation with RPV, even when the SN titer was greater than in cattle vaccinated with vRVF alone. This lack of protection, in the presence of SN antibody, would indicate that live attenuated and recombinant vaccines induce immune responses necessary for protection (e.g., cell-mediated immunity) that are not generated by subunit or inactivated whole-virus vaccines.
Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein
Authors: Kinney R M , Chang G J , Tsuchiya K R , Sneider J M , Roehrig J T , Woodward T M , Trent D W
Published: January 2020

The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.