bluuebunny/crossref_metadata_2025 huggingface dataset:
Virulent Mutants of Bacteriophage P22 I. Isolation and Genetic Analysis
Authors: Bronson Morley J. , Levine Myron
Published: January 2020

Mutants of phage P22 which form plaques on a P22 lysogen have been isolated. These virulent mutants have been classified into three groups. (i)
VirA
mutants arise spontaneously in wild-type stocks and form very small turbid plaques on a P22 lysogen. The single mutation responsible for
VirA
virulence maps near the
mnt
locus, one of the immunity regions of phage P22. (ii)
VirB
mutants do not arise spontaneously and have been isolated only from mutagenized P22 stocks.
VirB
mutants form small, clear plaques on a P22 lysogen. One of the
VirB
mutants,
virB-3
, was analyzed in detail. The
virB-3
mutant is comprised of two mutations:
K5
, which maps within the
c
2
gene, and
Vx
, which maps in the region between the
c
2
and
c
3
genes. Phages carrying either the
K5
or
Vx
mutation are not virulent in themselves but mutate to
VirB
virulence at a frequency of 10
−5
to 10
−6
. It is concluded that
K5
and
Vx
are mutations at specific sites which together confer the ability to undergo phage development in the presence of repressor. (iii)
VirC
mutants are defined by a large clear plaque morphology when plated on a P22 lysogen.
VirC
mutants are comprised of the determinants of both
VirA
and
VirB
virulence.
Australia Antigen: Large-Scale Purification from Human Serum and Biochemical Studies of Its Proteins
Authors: Gerin J. L. , Holland P. V. , Purcell R. H.
Published: January 2020

Biophysical techniques are described for the large-scale isolation of Australia antigen (Au) from unit quantities of human serum by using the batch-type zonal centrifuge rotors. A three-step procedure involving isopycnic banding of the particle in CsCl density gradients and rate-zonal centrifugation on sucrose gradients resulted in a highly purified Au preparation which was used for biochemical studies of Au proteins and as immunizing antigen for the production of reagent antiserum in animals. The spherical form of Au, which was devoid of detectable nucleic acid, was composed of two major proteins (AuP1 and AuP2) and a minor protein (AuP3) of 26,000, 32,000, and 40,000 molecular weight, respectively, as determined by acrylamide gel electrophoresis. The significance of these findings to the possibility of Au subtypes is discussed.
Comparison of Cytocidal and Noncytocidal Strains of Shope Rabbit Fibroma Virus
Authors: Hinze Harry C. , Walker Duard L.
Published: January 2020

Seven strains of Shope fibroma virus were compared for their effect on rabbit cells in vitro. All but one of the naturally occurring strains examined in this study produced a similar response in the infected cultures. This consisted of continued cell multiplication together with changes in cell morphology and growth pattern. In contrast, a recently isolated strain of fibroma virus, the M1 strain, was found to produce a gradual cell destruction under the same cultural conditions. A comparison of the cytocidal M1 strain with a representative noncytocidal strain in vitro showed no differences in the rate of multiplication, plaque type, antigenic composition, or heat lability. Only minor differences were found in the tumors produced in rabbits by these strains.
Phospholipid Composition of Venezuelan Equine Encephalitis Virus
Authors: Heydrick Fred P. , Comer Joann F. , Wachter Ralph F.
Published: January 2020

Phospholipid analyses of Venezuelan equine encephalitis virus showed that virus propagated in L-cell monolayers had a higher sphingomyelin content and a lower phosphatidylcholine content than virus grown in chick fibroblast monolayers. Virus of L-cell origin also was found to possess greater thermal stability than virus derived from the chick fibroblast cell.
Susceptibility of Differentiating Muscle Cells of the Fetal Mouse in Culture to Coxsackievirus A13
Authors: Goldberg Robert J. , Crowell Richard L.
Published: January 2020

The interaction of coxsackievirus A13 with differentiating muscle cells, cultured from tissues of the fetal mouse, was studied. Cultures infected at that stage of myogenic differentiation characterized by the rapid formation of multinucleated myotubes produced maximum virus titers of over 10
7
plaque-forming units. Virus-induced cytopathic effect was characterized by a marked diminution in the number of multinucleated cells. The susceptibility of these cultures decreased appreciably when infection was initiated after the majority of the myotubes had formed. The demonstration of newly synthesized A13 virus antigen by immunofluorescence provided direct evidence that A13 virus replication occurred both in myoblasts and myotubes. The synthesis of A13 virus was markedly depressed in muscle cultures in which the formation of multinucleated cells was inhibited by BUDR or by fusion-inhibiting media. After reversal of this inhibition, the cultures acquired the increased susceptibility to A13 virus characteristic of cells undergoing myogenic differentiation. In contrast to the results obtained with coxsackievirus A13, the primary fetal mouse muscle cultures were resistant to poliovirus T1. It is suggested that changes in the surfaces of developing muscle cells may coincide with the formation and disappearance of specific virus receptors and thereby regulate the cell susceptibility to coxsackievirus A13.
Transfer of Thymidine Kinase to Thymidine Kinaseless L Cells by Infection with Ultraviolet-Irradiated Herpes Simplex Virus
Authors: Munyon William , Kraiselburd Edmundo , Davis Daniel , Mann Judith
Published: January 2020

L cells lacking thymidine kinase (TK) activity (Ltk
−
cells) have been stably transformed to a TK-positive phenotype by infection with ultraviolet-irradiated herpes simplex virus (HSV-UV). The highest frequency of the Ltk
−
to Ltk
+
transformation observed in these experiments was approximately 10
−3
, whereas no measurable transformation was observed (less than 10
−8
) in the absence of HSV-UV infection. Cell lines of HSV-transformed Ltk
+
cell lines contain 7 to 24 times as much TK activity as do the parental Ltk
−
cells, and they have been maintained in culture for a period exceeding 8 months. The kinetics of thermal inactivation of the TK activity derived from an Ltk
+
HSV-transformed cell line and the TK activity from Ltk
−
cells lytically infected with infectious HSV are similar. Both of these TK activities are much more thermolabile than the TK activity present in wild-type L cells. A mutant strain of HSV which does not induce TK activity during lytic infection does not cause the Ltk
−
to Ltk
+
transformation. These data suggest that either an HSV TK gene has been transferred to Ltk
−
cells or that an HSV gene product has caused the expression of a previously repressed cellular enzyme.
Viral replication is required for induction of ocular immunopathology by herpes simplex virus
Authors: Babu J S , Thomas J , Kanangat S , Morrison L A , Knipe D M , Rouse B T
Published: January 2020

Corneal infection of BALB/c mice with herpes simplex virus type 1 results in a chronic inflammatory response in the stroma termed herpetic stromal keratitis (HSK). This disease is considered to be immunopathological and mediated primarily by CD4+ T cells of the type 1 cytokine profile. However, the nature of the antigens, virus or host derived, which drive the inflammatory response remains in doubt. In this study, the relevance of infection with replicating virus for the subsequent development of HSK was evaluated with immunocompetent mice as well as with SCID mice reconstituted with herpes simplex virus-immune CD4+ T cells. In the corneas of immunocompetent mice, infectious virus, viral antigen, and mRNA expression were detectable for only a brief period of time (< or = 7 days postinfection), and all were undetectable by the time clinical lesions were evident (10 to 15 days). Viral replication, however, was necessary for the development of HSK in both models, since infection with UV-inactivated virus or with mutant viruses which were incapable of multiple rounds of replication in vivo failed to induce HSK. The inactivated and mutant viral preparations did, however, stimulate T-cell immune responses in immunocompetent mice. The results are discussed in terms of possible involvement of host antigens exposed in response to transient progeny virion replication in the immune-privileged cornea.
Gene expression and cytopathic effect of vaccinia virus inactivated by psoralen and long-wave UV light
Authors: Tsung K , Yim J H , Marti W , Buller R M , Norton J A
Published: January 2020

Induction of the cytopathic effect (CPE) in cells infected with poxvirus seems ubiquitous in that it has been associated with all different strains and preparations of poxviruses, regardless of the replicating status of these viruses. The study of the mechanisms by which CPE is induced by nonreplicating poxviruses is hampered by the lack of any noncytopathic mutant strains and preparations. In this paper, we report on the patterns of gene expression and induction of CPE by vaccinia viruses treated by limited cross-linking with psoralen and long-wave UV light (PLWUV). We show that treatment of cell-free virus with PLWUV could inactivate viral replication without abolishing the ability of the virus to infect cells. Viral transcription as indicated by reporter genes was generally enhanced and prolonged under early viral promoters and abolished under late promoters. Furthermore, increasing the levels of cross-linking with PLWUV resulted in a decrease and abolishment of viral expression of a large reporter gene and a concomitant loss of the induction of CPE. Cells infected with such a virus were able to express the reporter genes and proliferate. The generation of nonreplicating and noncytopathic recombinant vaccinia viruses may help in studies of the mechanisms of CPE induction by poxvirus and may facilitate the use of poxviral vectors in broader areas of research and clinical applications.
Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells
Authors: Ponniah S , Doherty P C , Eichelberger M
Published: January 2020

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.
Use of recombinant protein to identify a motif-negative human cytotoxic T-cell epitope presented by HLA-A2 in the hepatitis C virus NS3 region
Authors: Kurokohchi K , Akatsuka T , Pendleton C D , Takamizawa A , Nishioka M , Battegay M , Feinstone S M , Berzofsky J A
Published: January 2020

To define cytotoxic T-cell (CTL) epitopes, the common approach involving the use of a series of overlapping synthetic peptides covering the whole protein sequence is impractical for large proteins. Motifs identify only a fraction of epitopes. To identify human CTL epitopes in the NS3 region of hepatitis C virus (HCV), we modified an approach using recombinant protein and the ability of short peptides to bind to class I major histocompatibility complex (MHC) molecules. Peripheral blood mononuclear cells from an HCV-infected patient were stimulated with a proteolytic digest of the recombinant NS3 protein to expand CTL to any active peptides in the digest. The digest was fractionated by reverse-phase high-performance liquid chromatography, and fractions were assessed for the ability to sensitize targets for lysis by CTL. The most active fraction was sequenced, identifying a 15-residue peptide (NS3-1J; TITTGAPVTYSTYGK). This sequence was confirmed to be the source of the activity by synthesis of the corresponding peptide. CTL lines specific for NS3-1J were established from two HCV-infected patients (both HLA-A2 and -B7 positive) by stimulation with the synthetic peptide in vitro. The CTL were HLA-A2 restricted, and the minimal epitope was mapped to a decapeptide NS3-1J (10.4). As this minimal epitope lacks the common HLA-A2-binding motif, this technique is useful for mapping CTL epitopes independent of known motifs and without the requirement for enormous numbers of overlapping peptides. Because this peptide is presented by the most common HLA class I molecule, present in almost half the population, it might be a useful component of a vaccine against HCV.
Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection
Authors: Chen M , Harty R N , Zhao Y , Holden V R , O'Callaghan D J
Published: January 2020

Defective interfering (DI) particles of equine herpesvirus type 1 (EHV-1) are capable of mediating persistent infection (S. A. Dauenhauer, R. A. Robinson, and D. J. O'Callaghan, J. Gen. Virol. 60:1-14, 1982; R. A. Robinson, R. B. Vance, and D. J. O'Callaghan, J. Virol. 36:204-219, 1980). Sequence analysis of cloned DI particle DNA revealed that portions of two regulatory genes, ICP22 (IR4) and ICP27 (UL3), are linked in frame to form a unique hybrid open reading frame (ORF). This hybrid ORF, designated as the IR4/UL3 gene, encodes the amino-terminal 196 amino acids of the IR4 protein (ICP22 homolog) and the carboxy-terminal 68 amino acids of the UL3 protein (ICP27 homolog). Portions of DNA sequences encoding these two regulatory proteins, separated by more than 115 kbp in the standard virus genome, were linked presumably by a homologous recombination event between two identical 8-bp sequences. Reverse transcriptase-PCR and S1 nuclease analyses revealed that this unique ORF is transcribed by utilizing the transcription initiation site of ICP22 and the polyadenylation signal of ICP27 in DI particle-enriched infection. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the ICP22 and ICP27 proteins demonstrated that a 31-kDa hybrid protein was synthesized in the DI particle-enriched infection but not in standard virus infection. This 31-kDa hybrid protein was expressed at the same time as the ICP22 protein in DI particle-enriched infection and migrated at the same location on polyacrylamide gel electrophoresis as the protein expressed from a cloned IR4/UL3 expression vector. These observations suggested that the unique IR4/UL3 hybrid gene is expressed from the DI particle genome and may play a role in DI particle-mediated persistent infection.
The human papillomavirus type 16 E7 protein complements adenovirus type 5 E1A amino-terminus-dependent transactivation of adenovirus type 5 early genes and increases ATF and Oct-1 DNA binding activity
Authors: Wong H K , Ziff E B
Published: January 2020

We have previously shown that conserved region 1 (CR1) of the adenovirus type 5 (Ad5) E1A protein synergizes with CR3 in the transactivation of Ad5 early genes (H.K. Wong and E. B. Ziff, J. Virol. 68:4910-4920, 1994). CR1 lies within the E1A amino terminus and binds host regulatory proteins such as the RB protein, p107, p130, and p300. Since simian virus 40 (SV40) large T antigen and human papillomavirus type 16 (HPV16) E7 protein also bind host regulatory factors, we investigated whether these viral proteins can complement E1A mutants which are defective in early gene activation. We show that the HPV16 E7 protein but not SV40 T antigen can complement mutations in the Ad5 E1A CR1 in the transactivation of viral early promoters. The inability of SV40 T antigen to complement suggests that RB binding on its own is not sufficient for early promoter transactivation by the E1A amino terminus. Nuclear runoff assays show that complementation by HPV16 E7 restores the ability of the E1A mutants to stimulate early gene expression at the level of transcription. Furthermore, nuclear extracts from the E7-transformed cells show increased binding activity of ATF and Oct-1, factors that can recognize the elements of Ad5 early genes, consistent with gene activation by E1A and E7 at the transcriptional level.
Identification of EFIV, a stable factor present in many avian cell types that transactivates sequences in the 5' portion of the Rous sarcoma virus long terminal repeat enhancer
Authors: Houtz E K , Conklin K F
Published: January 2020

We define a protein complex present in avian nuclear extracts that interacts with the Schmidt-Ruppin strain of the Rous sarcoma virus (RSV) long terminal repeat (LTR) between positions -197 and -168 relative to the transcriptional start site. We call this complex EFIV and demonstrate that the EFIV protein(s) is present in several avian cell types examined, including B cells (S13 and DT40), T cells (MSB), and chicken embryo fibroblasts. We also report that the EFIV binding site activates transcription of reporter constructs after transfection into avian B cells and chicken embryo fibroblasts, demonstrating that the EFIV region constitutes a functional transactivator sequence. By chemical interference footprinting and mutational analyses we define the EFIV binding site as including the sequence GCAACATG, which is present in two copies between positions -197 and -168, as well as sequences that lie between the two repeats. Electrophoretic mobility shift competition experiments suggest that the EFIV protein(s) may be related to members of the CCAAT/enhancer-binding protein family of transcription factors that interact with different regions of the RSV and the avian leukosis virus (ALV) LTRs. However, as defined by differences in sensitivity to protein synthesis inhibitors and footprinting patterns, EFIV is clearly distinct from these previously defined LTR binding factors. In addition, the finding that EFIV binding activity is stable in B cells indicates either that the lability of all 5' LTR binding activities is not required for B-cell transformation by the ALV/RSV family of viruses or that nonacute transforming viruses that include an RSV LTR may use a mechanism to effect cellular transformation different from that proposed for ALV.
Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit
Authors: Yeh P , Dedieu J F , Orsini C , Vigne E , Denefle P , Perricaudet M
Published: January 2020

Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated.
Single amino acid substitution in constant region 1 or 4 of gp120 causes the phenotype of a human immunodeficiency virus type 1 variant with mutations in hypervariable regions 1 and 2 to revert
Authors: Wang W K , Essex M , Lee T H
Published: January 2020

The second major cysteine loop of human immunodeficiency virus type 1 envelope glycoprotein gp120 contains 5 to 11 consensus N-linked glycosylation sites, which is disproportionately higher than the number of such sites found in other regions of gp120. Amino acid substitutions introduced at three of six N-linked glycosylation sites in this region of an infectious molecular clone, HXB2, resulted in severe impairment of virus infectivity. Isolation and genetic characterization of a revertant of this mutant revealed an isoleucine-for-valine substitution at position 84 in constant region 1 and an isoleucine-for-methionine substitution at position 434 in constant region 4. Further mutational analysis indicated that either isoleucine substitution was sufficient to confer the revertant phenotype. These findings demonstrate that V1/V2 not only functionally interacts with C4, as previously reported, but also interacts with C1. The observation that compensatory changes do not involve regeneration of N-linked glycosylation sites in the second major cysteine loop suggests that replication of human immunodeficiency virus type 1 in vitro is independent of the presence of a disproportionate number of N-linked glycosylation sites within this loop.
Transcription start sites downstream of the Epstein-Barr virus (EBV) Fp promoter in early-passage Burkitt lymphoma cells define a fourth promoter for expression of the EBV EBNA-1 protein
Authors: Nonkwelo C , Skinner J , Bell A , Rickinson A , Sample J
Published: January 2020

In Epstein-Barr virus (EBV)-transformed B lymphoblastoid and many Burkitt lymphoma cell lines, the EBV EBNA-1 protein is one of six viral nuclear antigens expressed from a common transcription unit under the control of one of two promoters, Wp or Cp. In contrast, EBNA-1 is the only EBV nuclear antigen expressed in Burkitt and other EBV-positive tumors. We previously identified a promoter of EBNA-1 transcription, designated Fp, in early-passage Mutu Burkitt tumor cells, and this promoter is also active in long-term Mutu and Akata Burkitt cell lines which maintain the exclusive expression of EBNA-1 characteristic of the tumor. However, transcription initiation within Fp reporter gene plasmids in EBV-negative cells occurs at positions 100 to 200 bases downstream of the Fp start site in the BamHI-Q restriction fragment. Here we demonstrate that transcription initiation within newly established Burkitt lymphoma cell lines is consistent with the transcription initiation we observed in reporter plasmids. Furthermore, previous observations of transcription from Fp to generate EBNA-1 transcripts can be attributed to lytic-cycle gene expression. These data, in conjunction with our previous characterization of promoter regulatory elements, define a fourth EBNA-1 promoter, Qp, that is active in latently infected Burkitt tumor cells.
Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein
Authors: Stevenson D , Xue M , Hay J , Ruyechan W T
Published: January 2020

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization.
Monoclonal antibodies to reovirus sigma 1 and mu 1 proteins inhibit chromium release from mouse L cells
Authors: Hooper J W , Fields B N
Published: January 2020

Reovirus intermediate subviral particles (ISVPs) but not intact virions or cores have been shown to possess the capacity to permeabilize mouse L cells as determined by a 51Cr release assay. We used monoclonal antibodies (MAbs) directed against proteins exposed on the ISVP surface (sigma 1, mu 1, and lambda 2) to probe the role(s) of these proteins in membrane interaction and penetration. One sigma 1-specific MAb (MAb-G5) and two mu 1-specific MAbs (MAb-10H2 and MAb-8H6) inhibited reovirus-induced 51Cr release when added pre- or post-ISVP attachment to L cells. MAb-G5 inhibits 51Cr release by interfering with ISVP attachment (via sigma 1) to L-cell receptor sites. The mu 1-specific MAbs (MAb-10H2 and MAb-8H6) inhibit 51Cr release by interfering with an undefined post-L-cell-attachment event that involves bivalent binding of the mu 1-specific MAbs to an epitope located in a central region of the mu 1 protein.
Regulation of a human cytomegalovirus immediate-early gene (US3) by a silencer-enhancer combination
Authors: Thrower A R , Bullock G C , Bissell J E , Stinski M F
Published: January 2020

The US3 open reading frame of human cytomegalovirus (HCMV) is transcribed at immediate-early (IE) times after infection. Upstream of the US3 promoter, between -84 and -259 bp relative to the transcription start site, there are five copies of an 18-bp repeat, referred to as 5R2. Between -340 and -560 bp there are seven copies of a 10-bp dyad repeat, referred to as 7R1. We investigated the roles of these repeats in transcription from the US3 promoter in human foreskin fibroblast or HeLa cells. In transient transfection assays, the region containing 5R2 up-regulated transcription and was responsive to the p65 subunit of NF-kappa B. The DNA region containing 7R1 down-regulated transcription from either the US3 promoter or a heterologous promoter in a position- and orientation-independent manner. Mutational analysis and transient transfections indicated that DNA containing the 10-bp dyad or one-half of the dyad was sufficient to cause repression of downstream gene expression. DNA probes containing one or more copies of the pentanucleotide sequence TGTCG specifically bound cellular proteins, as demonstrated by electrophoretic mobility shift assays and cold-competition electrophoretic mobility shift assays. Two different DNA-protein complexes were detected with DNA probes containing one or two copies of the pentanucleotide. In HCMV-infected cell nuclear extracts, one of the DNA-protein complexes was present in amounts inversely proportional to the amount of US3 transcription. Its formation was affected by dephosphorylation of the DNA-binding protein(s). Transient dephosphorylation of the cellular repressor protein may occur during HCMV infection. Repression of US3 transcription may relate to the number of pentanucleotides and the cellular proteins that bind to it. Twenty-one copies of a TRTCG motif (R = purine) were found clustered upstream of the US3 gene and also in the modulator upstream of the HCMV IE1 and IE2 genes.
Autogenous regulation of RNA translation and packaging by Rous sarcoma virus Pr76gag
Authors: Sonstegard T S , Hackett P B
Published: January 2020

Unspliced cytoplasmic retroviral RNA in chronically infected cells either is encapsidated by Gag proteins in the manufacture of virus or is used to direct synthesis of Gag proteins. Several models have been suggested to explain the sorting of viral RNA for these two purposes. Here we present evidence supporting a simple biochemical mechanism that accounts for the routing of retroviral RNA. Our results indicate that ribosomes compete with the Gag proteins to determine the fate of nascent retroviral RNA. Although the integrity of the entire Rous sarcoma virus leader sequence is important for retroviral packaging and translation, the RNA structure around the third small open reading frame, which neighbors the psi site required for packaging of the RNA, is particularly critical for maintenance of the balance between translation and packaging. These results support the hypothesis that Gag proteins autogenously regulate their synthesis and encapsidation of retroviral RNA and that an equilibrium exists between RNA destined for translation and packaging that is based on the intracellular levels of Gag proteins and ribosomes. To test the model, mRNAs with natural or mutated 5' leader sequences from Rous sarcoma virus were expressed in avian cells in the presence and absence of Pr76gag. We demonstrate that Pr76gag acts as a translational repressor of these mRNAs in a dose-dependent manner, supporting the hypothesis that Pr76gag can sort retroviral RNA for translation and encapsidation.
Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1
Authors: Ho C K , Van Etten J L , Shuman S
Published: January 2020

We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.
Adenovirus-pulsed dendritic cells stimulate human virus-specific T-cell responses in vitro
Authors: Smith C A , Woodruff L S , Kitchingman G R , Rooney C M
Published: January 2020

Adenovirus infections cause significant morbidity and mortality in immunocompromised patients, yet little is known about the immune response to adenovirus infections. We established a system for the generation of a cytotoxic immune response to adenovirus in vitro. Cytotoxic T cells (CTLs) were derived from normal donors by using peripheral blood dendritic cells as antigen-presenting cells. The CTLs were found to contain a mixture of effector cells that recognized virus peptides in the context of both class I and class II antigens. Endogenous viral gene expression was not required to sensitize cells to lysis by adenovirus-specific CTLs. CTLs raised against subgroup C adenovirus type 5 can lyse cells infected with subgroup B adenovirus type 11, indicating that viruses of different subgroups have epitopes in common. This system holds promise for defining the human immune response to adenovirus, including characterization of the viral protein(s) against which the response is generated, and the identity of the effector cells. Such studies are in progress.
Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers
Authors: Kao C C , Sun J H
Published: January 2020

Various DNA- and RNA-dependent RNA polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. For the brome mosaic virus RNA-dependent RNA polymerase (RdRp), we determined that in reactions performed with limited GTP concentrations, minus-strand RNA synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. Furthermore, guanylyl-3',5'-guanosine (GpG) was incorporated into minus-strand RNA and increased the rate of minus-strand RNA synthesis. In the presence of GpG, RdRp's Km for GTP decreased from 50 microM to approximately 3 microM while the Kms for other nucleotides were unaffected. These results have implications for the mechanism of initiation by RdRp.
The heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A/E) recombinant of African origin
Authors: Gao F , Robertson D L , Morrison S G , Hui H , Craig S , Decker J , Fultz P N , Girard M , Shaw G M , Hahn B H , Sharp P M
Published: January 2020

Since 1989, human immunodeficiency virus type 1 (HIV-1) has spread explosively through the heterosexual population in Thailand. This epidemic is caused primarily by viruses classified as "subtype E", which, on the basis of limited sequence comparisons, appear to represent hybrids of subtypes A (gag) and E (env). However, the true evolutionary origins of "subtype E" viruses are still obscure since no complete genomes have been analyzed, and only one full-length subtype A sequence has been available for phylogenetic comparison. In this study, we determined full-length proviral sequences for "subtype E" viruses from Thailand (93TH253) and the Central African Republic (90CR402) and for a subtype A virus from Uganda (92UG037). We also sequenced the long terminal repeat (LTR) regions from 16 virus strains representing clades A, C, E, F, and G. Detailed phylogenetic analyses of these sequences indicated that "subtype E" viruses do indeed represent A/E recombinants with multiple points of crossover along their genomes. The extracellular portion of env, parts of vif and vpr, as well as most of the LTR are of subtype E origin, whereas the remainder of the genome is of subtype A origin. The possibility that the discordant phylogenetic positions of "subtype E" viruses in gag- and env-derived trees are the result of unusual rates or patterns of evolution was also considered but was ruled out on the basis of two lines of evidence: (i) phylogenetic trees constructed for synonymous and nonsynonymous substitutions yielded the same discordant branching orders for "subtype E" gag and env gene sequences, thus excluding selection-driven evolution, and (ii) multiple crossovers in the viral genome are most consistent with the copy choice model of recombination and have been observed in other documented examples of HIV-1 intersubtype recombination. Thai and CAR "subtype E" viruses exhibited the same pattern of A/E mosaicism, indicating that the recombination event occurred in Africa prior to the spread of virus to Asia. Finally, all "subtype E" viruses were found to contain a distinctive two-nucleotide bulge in their transactivation response (TAR) elements. This feature was present only in viruses which also contained a subtype A 5' pol region (i.e., subtype A viruses or A/D and A/E recombinants), raising the possibility of a functional linkage between the TAR region and the polymerase. The implications of epidemic spread of a recombinant HIV-1 strain to viral natural history and vaccine development are discussed.
A p6Pol-protease fusion protein is present in mature particles of human immunodeficiency virus type 1
Authors: Almog N , Roller R , Arad G , Passi-Even L , Wainberg M A , Kotler M
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) protease (PR) and p6(Pol) are translated as part of the Gag-Pol polyprotein after a ribosomal frameshift. PR is essential to virus replication and is responsible for cleaving Gag and Gag-Pol precursors, but the role of p6(Pol) in HIV-1 infection is poorly understood. Here, we report that (i) PR is present in mature HIV-1 virions primarily as a p6(Pol)-PR fusion protein; (ii) HIV-1 PR cleaves viral precursor proteins expressed in bacterial cells at the Phe-Leu bond (positions 1639 to 1642) located at the junction of the NC and p6(Pol) proteins, releasing the p6(Pol)-PR fusion protein; and (iii) purified p6(Pol)-PR fusion protein undergoes autocleavage in vitro at at least three sites.
A source of glycosylated human T-cell lymphotropic virus type 1 envelope protein: expression of gp46 by the vaccinia virus/T7 polymerase system
Authors: Arp J , LeVatte M , Rowe J , Perkins S , King E , Leystra-Lantz C , Foung S K , Dekaban G A
Published: January 2020

Heterologous expression of the human T-cell lymphotropic virus type 1 (HTLV-1) envelope surface glycoprotein (gp46) in a vaccinia virus/T7 polymerase system resulted in the production of authentic recombinant gp46. Five differentially glycosylated forms of the surface envelope protein were produced by this mammalian system, as demonstrated by tunicamycin inhibition of N-glycosylation and N-glycan removal with endoglycosidase H and glycopeptidase F. These studies revealed that all four potential N-glycosylation sites in gp46 were used for oligosaccharide modification and that the oligosaccharides were mannose-rich and/or hybrid in composition. Conformational integrity of the recombinant HTLV-1 envelope protein was determined by the ability to bind to various HTLV-1-infected human sera and a panel of conformational-dependent human monoclonal antibodies under nondenaturing conditions. Furthermore, this recombinant gp46 was recognized by a series of HTLV-2-infected human sera and sera from a Pan paniscus chimpanzee infected with the distantly related simian T-cell lymphotropic virus STLVpan-p. Maintenance of highly conserved conformational epitopes in the recombinant HTLV-1 envelope protein structure suggests that it may serve as a useful diagnostic reagent and an effective vaccine candidate.
Identification of amino acids controlling the low-pH-induced conformational change of rabies virus glycoprotein
Authors: Gaudin Y , Raux H , Flamand A , Ruigrok R W
Published: January 2020

The glycoprotein (G) of rabies virus assumes at least three different conformations: the native state detected at the viral surface above pH 7, the activated state involved in the first step of the fusion process, and the fusion-inactive conformation (I). A new category of monoclonal antibodies (MAbs) which recognized specifically the I conformation at the viral surface has recently been described. These MAbs (17A4 and 29EC2) became neutralizing when the virus was preincubated at acidic pH to induce the conformational change toward the I state of G. Mutants escaping neutralization were then selected. In this study, we have investigated the fusion and the low-pH-induced fusion inactivation properties of these mutants. All of these mutants have fusion properties similar to those of the CVS parental strain, but five mutants (E282K, M44I, M44V, V392G, and M396T) were considerably slowed in their conformational change leading to the I state. These mutants allow us to define regions that control this conformational change. These results also reinforce the idea that structural transition toward the I state is irrelevant to the fusion process. Other mutations in amino acids 10, 13, and 15 are probably located in the epitopes of selecting MAbs. Furthermore, in electron microscopy, we observed a hexagonal lattice of glycoproteins at the viral surface of mutants M44I and V392G as well as strong cooperativity in the conformational change toward the I state. This finding demonstrates the existence of lateral interactions between the spikes of a rhabdovirus.
Retroviral insertional activation of the c-myb proto-oncogene in a Marek's disease T-lymphoma cell line
Authors: Le Rouzic E , Perbal B
Published: January 2020

Marek's disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5' long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.
Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication
Authors: van Kuppeveld F J , van den Hurk P J , Zoll J , Galama J M , Melchers W J
Published: January 2020

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.
In vivo transcriptional regulation of the human immunodeficiency virus in the central nervous system in transgenic mice
Authors: Kurth J , Buzy J M , Lindstrom L , Clements J E
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) causes infections of the central nervous system (CNS) and has been implicated as the causative agent of AIDS-associated encephalopathy and the AIDS dementia complex. The development of in vivo models of HIV-1-mediated gene expression has shown that the HIV long terminal repeat (LTR) from the viral isolate HIV(JR-CSF) specifically supports gene expression in adult and developing CNS. To determine the molecular basis for HIV-1 developmental CNS gene expression, in vivo footprinting analysis by the ligation-mediated PCR technique was performed on CNS tissue from the brain stem of a transgenic mouse. The association of cellular proteins in the CNS with sequences in the LTR was found over sequences that defined the TATA region, the Sp-1 and NF-kappaB sites, and two upstream regions (-111 to -150 and -260 to -300). A purine-rich sequence at positions -256 to -296 of the HIV(JR-CSF) LTR but not of the HIV(IIIB) LTR specifically bound protein in nuclear extracts of newborn brain tested in electrophoretic mobility shift assays. No specific protein binding was observed to this region in liver or HeLa cell nuclear extracts. This suggests the presence of a newly identified transcription factor involved in regulation of HIV-1 gene expression in the CNS.
Intracellular expression of human immunodeficiency virus type 1 (HIV-1) protease variants inhibits replication of wild-type and protease inhibitor-resistant HIV-1 strains in human T-cell lines
Authors: Junker U , Escaich S , Plavec I , Baker J , McPhee F , Rose J R , Craik C S , Böhnlein E
Published: January 2020

The enzymatic activity of the human immunodeficiency type 1 (HIV-1) protease (PR) is crucial to render HIV-1 virions mature and infectious. Hence, genetic intervention strategies based on trans-dominant (td) variants of the HIV-1 PR might be an alternative to current pharmacological and gene therapy regimens for AIDS. CD4-positive human CEM-SS T-cell lines were generated which constitutively expressed HIV-1 td PR variants. HIV-1 infection experiments demonstrated severely reduced HIV-1 replication in these td PR CEM-SS cell lines compared with control T cells expressing wild-type PR. Furthermore, replication of an HIV-1 isolate bearing a PR inhibitor-resistant PR was blocked, showing that genetic intervention strategies based on td PRs can be effective against HIV-1 isolates containing PR inhibitor-resistant mutants.