bluuebunny/crossref_metadata_2025 huggingface dataset:
E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis
Authors: Kowalik T F , DeGregori J , Schwarz J K , Nevins J R
Published: January 2020

Various experiments have demonstrated a role for the E2F transcription factor in the regulation of cell growth during the G0/G1/S phase transition. Indeed, overexpression of the E2F1 product, a component of the cellular E2F activity, induces DNA synthesis in quiescent fibroblasts. To provide an approach to a more detailed biochemical analysis of these events, we have made use of a recombinant adenovirus containing the E2F1 cDNA in order to efficiently express the E2F1 product in an entire population of cells. We demonstrate an induction of DNA synthesis when quiescent cells are infected with the E2F1 recombinant virus. However, we also find that the induction does not lead to a complete replication of the cellular genome, as revealed by flow cytometry. The incomplete nature of cellular DNA replication is due, at least in part, to the fact that E2F1 overexpression leads to massive cell death that is characteristic of apoptosis. This E2F1-mediated induction of apoptosis is largely dependent on endogenous wild-type p53 activity and can be subverted by introducing mutant forms of p53 into these cells or by overexpressing E2F1 in fibroblasts derived from p53-null mouse embryos. We conclude that E2F1 can induce events leading to S phase but that the process is not normal and appears to result from the activation of a cell death pathway.
Fgf-8, activated by proviral insertion, cooperates with the Wnt-1 transgene in murine mammary tumorigenesis
Authors: MacArthur C A , Shankar D B , Shackleford G M
Published: January 2020

We have used mouse mammary tumor virus (MMTV) infection of Wnt-1 transgenic mice to accelerate mammary tumorigenesis and to molecularly tag insertionally activated proto-oncogenes that cooperate oncogenically with Wnt-1 (G. M. Shackleford, C. A. MacArthur, H. C. Kwan, and H. E. Varmus, Proc. Natl. Acad. Sci. USA 90:740-744, 1993). Here we report the identification and characterization of a 31-kb genomic locus that contains clonal MMTV integrations in 8 of 80 mammary tumors from MMTV-infected Wnt-1 transgenic mice. Two genes were identified within this locus, one of which was transcriptionally activated by MMTV insertions. This activated gene is identical to androgen-induced growth factor (AIGF/Fgf-8) (A. Tanaka, K. Miyamoto, N. Minamino, M. Takeda, B. Sato, H. Matsuo, and K. Matsumoto, Proc. Natl. Acad. Sci. USA 89:8928-8932, 1992), the eighth member of the fibroblast growth factor (FGF) family. Transcriptional activation of Fgf-8 was found in all tumors with MMTV insertions in this locus. Fgf-8 mRNA was absent in normal mammary glands and was detected only in adult testis and ovary and in midgestational embryos. The sequences of Fgf-8 genomic and cDNA clones revealed five coding exons, in contrast to the three coding exons found in other FGF genes. cDNAs encoding three isoforms of the FGF-8 protein were isolated. The three corresponding mRNAs resulted from the alternative use of two 5' splice sites and two 3' splice sites for the second and third exons, respectively. These results implicate Fgf-8 as the third FGF gene found to cooperate with Wnt-1 in MMTV-induced murine mammary tumorigenesis, suggesting that FGFs and Wnts are strong collaborators in this process.
Constitutive activation of a variant of the env-mpl oncogene product by disulfide-linked homodimerization
Authors: Courtois G , Bénit L , Mikaeloff Y , Pauchard M , Charon M , Varlet P , Gisselbrecht S
Published: January 2020

The myeloproliferative leukemia retrovirus (MPLV) has the v-mpl cellular sequences transduced in frame with the deleted and rearranged Friend murine leukemia virus env gene. The resulting env-mpl fusion oncogene is responsible for an acute myeloproliferative disorder induced in mice by MPLV. v-mpl is a truncated form of the c-mpl gene which encodes the receptor for thrombopoietin. We investigated the contribution of the Env-Mpl extracellular domain in the constitutive activation of this truncated cytokine receptor and found that the rearrangement of the env sequences in the env-mpl fusion gene was not required for oncogenicity. A pathogenic variant, DEL3MPLV, was generated, which differs from MPLV by the deletions of 22 amino acids of the Env signal peptide, all of the mature Env sequences, and 18 N-terminal amino acids of the v-Mpl extracellular domain. The resulting del3-mpl oncogene product conserves in its extracellular region the first 12 amino acids of the Env signal sequence including a cysteine residue, and 25 amino acids of the v-Mpl. We show here that a mutation converting this cysteine to a glycine completely abolishes del3-mpl oncogenicity and that the del3-mpl oncogene product is constitutively activated by disulfide-linked homodimerization.
Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain
Authors: Stevenson S C , Rollence M , White B , Weaver L , McClelland A
Published: January 2020

The adenovirus fiber protein is responsible for attachment of the virion to cell surface receptors. The identity of the cellular receptor which mediates binding is unknown, although there is evidence suggesting that two distinct adenovirus receptors interact with the group C (adenovirus type 5 [Ad5]) and the group B (Ad3) adenoviruses. In order to define the determinants of adenovirus receptor specificity, we have carried out a series of competition binding experiments using recombinant native fiber polypeptides from Ad5 and Ad3 and chimeric fiber proteins in which the head domains of Ad5 and Ad3 were exchanged. Specific binding of fiber to HeLa cell receptors was assessed with radiolabeled protein synthesized in vitro, and by competition analysis with baculovirus-expressed fiber protein. Fiber produced in vitro was found as both monomer and trimer, but only the assembled trimers had receptor binding activity. Competition data support the conclusion that Ad5 and Ad3 interact with different cellular receptors. The Ad5 receptor distribution on several cell lines was assessed with a fiber binding flow cytometric assay. HeLa cells were found to express high levels of receptor, while CHO and human diploid fibroblasts did not. A chimeric fiber containing the Ad5 fiber head domain blocked the binding of Ad5 fiber but not Ad3 fiber. Similarly, a chimeric fiber containing the Ad3 fiber head blocked the binding of labeled Ad3 fiber but not Ad5 fiber. In addition, the isolated Ad3 fiber head domain competed effectively with labeled Ad3 fiber for binding to HeLa cell receptors. These results demonstrate that the determinants of receptor binding are located in the head domain of the fiber and that the isolated head domain is capable of trimerization and binding to cellular receptors. Our results also show that it is possible to change the receptor specificity of the fiber protein by manipulation of sequences contained in the head domain. Modification or replacement of the fiber head domain with novel ligands may permit adenovirus vectors with new receptor specificities which could be useful for targeted gene delivery in vivo to be engineered.
Establishment of a stable, inducible form of human immunodeficiency virus type 1 DNA in quiescent CD4 lymphocytes in vitro
Authors: Spina C A , Guatelli J C , Richman D D
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) possesses the ability to establish a complete infection in nondividing host cells. The capacity of HIV-1 to infect nondividing cells probably contributes significantly to its pathology in vivo, as reflected by infection of peripheral T lymphocytes, tissue macrophages, and microglial cells. However, the in vitro demonstration of the establishment of stable HIV-1 infection in quiescent T cells remains controversial. We have developed a primary T-cell model of acute HIV-1 infection of quiescent CD4 lymphocytes that demonstrates the development of a complete, reverse-transcribed form of virus that is stable for over 10 days in culture. To ensure that our primary cell culture was representative of a quiescent population, the CD4 lymphocyte targets were monitored for membrane expression of activation antigens and for shifts in cell cycle from G0/G1 to S/G2 phase. The presence of viral DNA fragments reflecting progressive reverse transcription was determined by PCR analysis. HIV entered primary CD4 cells rapidly, but viral DNA accumulated slowly in the resting cell cultures. DNA species containing regions of full-length reverse transcription were not detected until 3 to 5 days after infection. In parallel with the appearance of complete viral DNA, spliced RNA transcripts, predominantly of the nef species, were detected by reverse transcriptase PCR amplification. When infected CD4 cells were sorted on the basis of cell cycle analysis of DNA content, the accumulation of a complete viral DNA form was found to occur in both the purified G0/G1-phase cell subset and the cell fraction enriched for the minor S-phase subset. In contrast, spliced viral RNA products could be detected only in the enriched S-phase cell fraction. These results demonstrate that HIV-1 can infect and establish a complete, stable form of viral DNA in primary CD4 lymphocytes in vitro but is blocked from transcription in the absence of cell activation. The findings are consistent with in vivo data from HIV-infected individuals that show the existence of viral DNA predominantly as a stable, extrachromosomal form in T cells of the peripheral circulation.
In vitro interaction of the human immunodeficiency virus type 1 Tat transactivator and the general transcription factor TFIIB with the cellular protein TAP
Authors: Yu L , Loewenstein P M , Zhang Z , Green M
Published: January 2020

We have reported the molecular cloning, expression, and characterization of a human cellular protein, TAP, which possesses a strong transcriptional activation domain and binds the human immunodeficiency virus type 1 Tat transactivator in vitro and in vivo (L. Yu, Z. Zhang, P.M. Loewenstein, K. Desai, Q. Tang, D. Mao, J.S. Symington, and M. Green, J. Virol. 69:3007-3016, 1995). Here we show that TAP binds the general transcription factor TFIIB. Furthermore, we delineate the binding domains of TAP, Tat, and TFIIB, as well as measure the strengths and specificity of these protein-protein interactions. TAP binds strongly to Tat, with a Kd of (approximately 2 to 5) x 10(-7) M. The Tat activation region contains a 17-amino-acid conserved core domain which is the single contact site for TAP. Single-amino-acid substitutions within the Tat core domain inactivate transactivation in vivo and in vitro and greatly reduce binding of Tat to TAP in vitro. TAP binds strongly to TFIIB, with about the same Kd as for Tat. The interaction between TAP and TFIIB requires a sequence near the carboxy terminus of TFIIB which is also required for binding the strong acidic activator VP16. The contact sites for Tat and TFIIB map within the TAP C-terminal region, which contains the TAP activation domain. These combined results are consistent with the hypothesis that TAP is a cellular coactivator that bridges the Tat transactivator to the general transcription machinery via TFIIB.
Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription
Authors: Fallows D A , Goff S P
Published: January 2020

Hepadnaviruses replicate by reverse transcription of an RNA intermediate within subviral core particles in the cytoplasm of infected hepatocytes. Recognition of the epsilon encapsidation signal located on the 5' end of the pregenomic RNA by the viral polymerase occurs early in core particle assembly. The epsilon sequences contain a set of nested inverted repeats which form a stable stem-loop structure shown to play a role in RNA packaging and recently implicated as the site of initiation of minus-strand DNA synthesis. We have introduced a variety of site-directed mutations into the epsilon sequences of human hepatitis B virus to study their effects on viral replication in transfected HuH7 cells. We have identified two classes of mutations: those which adversely affect packaging and those which package RNA but adversely affect DNA synthesis. Analysis of these mutants has allowed us to identify separate features of the epsilon cis-acting signal which function in the processes of RNA packaging and reverse transcription.
Transcriptional trans activation by human immunodeficiency virus type 1 Tat requires specific coactivators that are not basal factors
Authors: Suñé C , García-Blanco M A
Published: January 2020

Expression of human immunodeficiency virus type 1 (HIV-1) genes is regulated by the trans activator Tat. Tat exerts its effects by increasing the rate of transcription, but the mechanism by which it does so is still unknown. To study the cellular factors required for Tat trans activation, we have expressed functional Gst-Tat fusion protein and used it to construct affinity columns. Our findings are as follows. (i) A Gst-Tat affinity matrix depleted HeLa nuclear extracts of a factor(s) required for Tat function. A Tat mutant bearing the missense mutation lysine to alanine at position 41 was incapable of this depletion. (ii) Tat trans activation was recovered by addition of unfractionated nuclear extract, the 0.5 M KCl elution fraction from the Tat affinity column, or sedimentation gradient fractions of HeLa extracts. The activity from the gradients sedimented with an apparent molecular mass of 200 kDa. (iii) Tat trans activation could not be recovered by use of recombinant human TATA-binding protein or partially purified TFIID. (iv) trans activation by Tat was blocked by heating of the nuclear extract under conditions in which basal transcription was not decreased. Our data demonstrate for the first time the existence of unique Tat coactivators distinct from factors required for general basal transcription.
Sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the S, 3, and 3-1 genes
Authors: Vaughn E M , Halbur P G , Paul P S
Published: January 2020

Four new porcine respiratory coronavirus (PRCV) isolates were genetically characterized. Subgenomic mRNA patterns and the nucleotide sequences of the 5' ends of the S genes, the open reading frame (ORF) 3/3a genes, and the ORF 3-1/3b genes of these PRCV isolates were determined and compared with those of other PRCV and transmissible gastroenteritis virus (TGEV) isolates. The S, ORF 3/3a, and ORF 3-1/3b genes are under intense study because of their possible roles in determining tissue tropism and virulence. Northern (RNA) blot analysis of subgenomic mRNAs revealed that mRNA 2, which encodes for the S gene, of the PRCV isolates migrated faster than the mRNA 2 of TGEV. The PRCV isolates AR310 and LEPP produced eight subgenomic mRNA species, the same number as produced by the virulent Miller strain of TGEV. However, the PRCV isolates IA1894 and ISU-1 produced only seven subgenomic mRNA species. All four of the PRCV isolates were found to have a large in-frame deletion in the 5' end of the S gene; however, the size and location of the deletion varied. Analysis of the ORF 3/3a gene nucleotide sequences from the four PRCV isolates also showed a high degree of variability in this area. The ORF 3 gene of the PRCV isolates AR310 and LEPP was preceded by a CTAAAC leader RNA-binding site, and the ORF 3 gene was predicted to yield a protein of 72 amino acids, the same size as that of the virulent Miller strain of TGEV. The PRCV isolates AR310 and LEPP are the first PRCV isolates found to have an intact ORF 3 gene. The ORF 3a gene of the PRCV isolate IA1894 was preceded by a CTAAAC leader RNA-binding site and was predicted to yield a truncated protein of 54 amino acids due to a 23-nucleotide deletion. The CTAAAC leader RNA-binding site and ATG start codon of ORF 3 gene of the PRCV isolate ISU-1 were removed because of a 168-nucleotide deletion. Analysis of the ORF 3-1/3b gene nucleotide sequences from the four PRCV nucleotides isolates also showed variability.
Different roles of bases within the integration signal sequence of human immunodeficiency virus type 1 in vitro
Authors: Yoshinaga T , Fujiwara T
Published: January 2020

To investigate the roles of bases near the tips of each strand of the long terminal repeat of the human immunodeficiency virus type 1 in the integration reaction, we examined the efficiencies of both binding and integration activities of staggered-ended substrates and mismatched mutant substrates by the integration assay and the UV cross-linking assay. Our results suggest that some bases of the human immunodeficiency virus type 1 long terminal repeat are required primarily for binding, whereas others are more critical for later reaction steps in vitro.
Binding and internalization of human papillomavirus type 33 virus-like particles by eukaryotic cells
Authors: Volpers C , Unckell F , Schirmacher P , Streeck R E , Sapp M
Published: January 2020

Infection of cells by human papillomaviruses (HPVs) associated with malignant genital lesions has not been studied because of the lack of an in vitro system and the unavailability of virions. We have now used virus-like particles (VLPs) of HPV type 33 to analyze the initial events in the interaction of the HPV capsid with cell lines. Binding of VLPs to HeLa cells was observed in biochemical assays and by immunofluorescence. VLP binding was inhibited by antisera raised against VLPs but not by monoclonal antibodies recognizing either L1 or L2 epitopes accessible on VLPs. Under saturating conditions, approximately 2 x 10(4) VLPs were bound per cell, with a dissociation constant of about 100 pM. VLPs composed of L1 alone bound as well as VLPs composed of both capsid proteins, indicating that L2 is not required for initial binding. VLPs dissociated into capsomers did not bind, demonstrating that intercapsomer contacts are required. Neither capsomers nor simian virus 40 virions competed with VLP binding. Uptake of VLPs by small and smooth endocytic vesicles was demonstrated by immunoelectron microscopy. Cellular binding of VLPs was sensitive to trypsin but not to sialidase, N-glycosidase, or octyl-beta-D-glycopyranoside treatment, suggesting that a cell surface protein is involved in the VLP binding. Cell lines originating from a variety of tissues and organisms as distantly related as insects and humans bound VLPs with similar efficiency and specificity. Therefore, the putative receptor mediating VLP attachment should be highly conserved and cannot be responsible for the species and tissue specificity of HPVs.
The small envelope glycoprotein (GS) of equine arteritis virus folds into three distinct monomers and a disulfide-linked dimer
Authors: de Vries A A , Raamsman M J , van Dijk H A , Horzinek M C , Rottier P J
Published: January 2020

The small membrane glycoprotein (GS) of equine arteritis virus (EAV) is a minor virion component but is abundantly expressed in EAV-infected cells. In this study, we have analyzed its membrane topology, folding, oligomerization, and intracellular transport. We show that GS is a class I integral membrane protein with one functional N-glycosylation site. Gel electrophoresis under nonreducing conditions revealed that GS occurs in EAV-infected cells in four monomeric conformations and as disulfide-linked homodimers. The slowest-migrating monomeric form corresponded to the fully reduced GS protein; the three faster-migrating monomeric species are probably generated by the formation of alternative intrachain disulfide bonds between the three luminal cysteines in the molecule. The GS monomers were selectively retained in the endoplasmic reticulum, as judged by their permanent susceptibility to endoglycosidase H, whereas the GS dimers were specifically incorporated into virus particles and became endoglycosidase H resistant and sialylated during passage through the Golgi apparatus.
Inducible and conditional inhibition of human immunodeficiency virus proviral expression by vesicular stomatitis virus matrix protein
Authors: Paik S Y , Banerjea A C , Harmison G G , Chen C J , Schubert M
Published: January 2020

Besides its role in viral assembly, the vesicular stomatitis virus (VSV) matrix (M) protein causes cytopathic effects such as cell rounding (D. Blondel, G. G. Harmison, and M. Schubert, J. Virol. 64:1716-1725, 1990). DNA cotransfection assays demonstrated that VSV M protein was able to inhibit the transcription of a reporter gene (B. L. Black and D. S. Lyles, J. Virol. 66:4058-4064, 1992). We have confirmed these observations by using cotransfections with an infectious clone of human immunodeficiency virus type 1 (HIV-1) and found that the amino-terminal 32 amino acids of M protein which are essential for viral assembly were not required for this inhibition. For the study of the potential role of M protein in the shutoff of transcription from chromosomal DNA, we have isolated stable HeLa T4 cell lines which encode either a wild-type or a temperature-sensitive (ts) VSV M gene under control of the HIV-1 long terminal repeat promoter. Transcription of the M mRNA was transactivated after HIV-1 infections. A cell line which encodes the wild-type M protein was nonpermissive for either HIV-1 or HIV-2. A cell line that encodes the ts M gene was transfected with the infectious HIV-1 DNA or was infected with HIV-1 or HIV-2. In all cases, at 32 degrees C, the permissive temperature for M protein, the cells were nonpermissive for HIV replication. At 40 degrees C, the ts M protein was nonfunctional and both HIV-1 and HIV-2 were able to replicate at high levels. A comparison of the amounts of proviral HIV-1 DNAs and HIV-1 mRNAs at 10 and 36 h after HIV-1 infection demonstrated that proviral insertion had not been prevented by M protein and that the block in HIV-1 replication was at the level of proviral expression. The severe reduction of HIV-1 proviral transcripts demonstrates that the VSV M protein alone can inhibit expression from chromosomal DNA. These results strongly support the hypothesis that the VSV M protein is involved in the shutoff of host cell transcription. M protein was able to attenuate HIV-1 infections and protect the cell population from HIV-1 pathogenesis. The temperature-dependent switch from a persistent to a lytic HIV-1 infection in the presence of ts M protein could be useful for studies of HIV-1 replication and pathogenesis.
Overlapping initiator and TATA box functions in the basal core promoter of hepatitis B virus
Authors: Chen I H , Huang C J , Ting L P
Published: January 2020

The regulation of transcription of the hepatitis B virus core promoter is an important event in the viral life cycle. Two messages, precore and pregenomic RNAs, that are initiated 30 nucleotides apart are produced by the core promoter. Precore RNA encodes nucleocapsid protein and pregenomic RNA core and polymerase. The latter transcript also serves as a template for viral genome replication via reverse transcription. We have previously defined a basal core promoter, which contains four TA-rich sequences (TA1 through TA4) but no canonical TATA element, that can direct transcription of both messages. In this study, we demonstrated that a stretch of 15 nucleotides containing TA4 is sufficient to direct precise initiation of both precore and pregenomic transcripts. This sequence can function as both an initiator and a TATA element. Mutational analysis further revealed that sequences essential for either function are colocalized. The significance of this finding with respect to the basal transcription mechanism and regulation of viral gene expression is discussed.
Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus
Authors: van der Most R G , Luytjes W , Rutjes S , Spaan W J
Published: January 2020

The defective interfering (DI) RNA MIDI of mouse hepatitis virus strain A59 (MHV-A59) contains a large open reading frame (ORF) spanning almost its entire genome. This ORF consists of sequences derived from ORF1a, ORF1b, and the nucleocapsid gene. We have previously demonstrated that mutations that disrupt the ORF decrease the fitness of MIDI and its derivatives (R. J. de Groot, R. G. van der Most, and W. J. M. Spaan, J. Virol. 66:5898-5905, 1992). To determine whether translation of the ORF per se is required or whether the encoded polypeptide or a specific sequence is involved, we analyzed sets of related DI RNAs containing different ORFs. After partial deletion of ORF1b and nucleocapsid gene sequences, disruption of the remaining ORF is still lethal; translation of the entire ORF is not essential, however. When a large fragment of the MHV-A59 spike gene, which is not present in any of the MHV-A59 DI RNAs identified so far, was inserted in-frame into a MIDI derivative, translation across this sequence was vital to DI RNA survival. Thus, the translated sequence is irrelevant, indicating that translation per se plays a crucial role in DI virus propagation. Next, it was examined during which step of the viral life cycle translation plays its role. Since the requirement for translation also exists in DI RNA-transfected and MHV-infected cells, it follows that either the synthesis or degradation of DI RNAs is affected by translation.
A subpopulation of normal B cells latently infected with Epstein-Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP1
Authors: Chen F , Zou J Z , di Renzo L , Winberg G , Hu L F , Klein E , Klein G , Ernberg I
Published: January 2020

Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.
Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry
Authors: Zolla-Pazner S , O'Leary J , Burda S , Gorny M K , Kim M , Mascola J , McCutchan F
Published: January 2020

The immunologic relatedness of the various human immunodeficiency virus type 1 (HIV-1) clades was determined with 13 human anti-HIV-1 monoclonal antibodies (MAbs) to six immunogenic regions of the HIV-1 structural proteins. The immunoreactivity of the native, oligomeric viral envelope glycoproteins expressed on the surfaces of human peripheral blood mononuclear cells infected in vitro with primary isolates from clades A through E was determined by flow cytometry. Some epitopes in the immunodominant region of gp41 and the C terminus of gp120 appear to be HIV-1 group specific in that they are expressed on the surfaces of cells in cultures infected with the majority of viruses tested from clades A to E. Epitopes within the V3 region appear to be clade restricted. Surprisingly, one MAb to an epitope in the C terminus of gp120 was entirely clade B specific. Staining with anti-V2 and anti-CD4 binding domain (CD4bd) reagents was infrequently detected. Anti-CD4bd MAbs stained only CD4-negative T cells because the CD4bd of gp120 appeared to be complexed with membrane CD4. When present, the epitopes of V2 and the CD4bd appeared to be expressed on cells infected with various clades. Thus, the results suggest that MAbs to gp41, the C terminus, and the V3 loop of gp120 are most useful in serotyping primary isolates of HIV-1, providing group-specific, clade-restricted, and clade-specific reagents. The use of the immunofluorescent method with the reagents described herein distinguishes infection with clade B from that with all other HIV-1 clades. With additional MAbs, this technique will allow a broadly applicable, reproducible, and practical method for serotyping HIV-1.
Immunization with viruslike particles from cottontail rabbit papillomavirus (CRPV) can protect against experimental CRPV infection
Authors: Breitburd F , Kirnbauer R , Hubbert N L , Nonnenmacher B , Trin-Dinh-Desmarquet C , Orth G , Schiller J T , Lowy D R
Published: January 2020

We tested the ability of vaccination with virus-like particles (VLPs) to protect domestic rabbits against papillomas induced by the cottontail rabbit papillomavirus (CRPV). A recombinant baculovirus system that expressed only the L1 major papillomavirus structural protein or L1 plus the minor L2 protein was used in insect cells as the source of VLPs. Groups of 10 rabbits were immunized with native or denatured VLPs from CRPV or type 1 bovine papillomavirus by using Freund's adjuvant. Alum was used as the adjuvant for an additional group immunized with CRPV L1-L2 VLPs. Animals were challenged with 5 x 10(10) and 2 x 10(11) particles on opposing flanks. No protection was seen in rabbits immunized with native or denatured bovine papillomavirus L1-L2 or with denatured CRPV L1-L2. In these groups, the lower and higher challenge doses resulted in 27 of 30 animals with extensive papillomas, with each of the remaining animals having a smaller number of persistent papillomas. Progression to carcinoma developed in 20 rabbits. Animals inoculated with native CRPV VLPs composed of L1 alone or L1-L2 developed many fewer lesions; the lower and higher challenge doses resulted in 17 of 29 and 5 of 29 rabbits, respectively, with no lesions, and the remainder developed only one to eight papillomas, which all regressed except for those on 1 rabbit. None developed cancer within 1 year of infection. Rabbits vaccinated with native CRPV VLPs developed high-titer antibodies in an enzyme-linked immunosorbent assay based on native VLPs, and passive transfer of serum or immunoglobulin G from rabbits immunized with CRPV VLPs protected against CRPV challenge. We conclude that native VLPs can induce antibody-mediated, type-specific protection against experimental papillomavirus infection.
Complementation of vif-defective human immunodeficiency virus type 1 by primate, but not nonprimate, lentivirus vif genes
Authors: Simon J H , Southerling T E , Peterson J C , Meyer B E , Malim M H
Published: January 2020

The productive infection of many susceptible human cells, including lymphocytes and macrophages derived from peripheral blood, by the pathogenic lentivirus human immunodeficiency virus type 1 requires expression of the virally encoded vif (for virion infectivity factor) gene. Interestingly, this gene appears to have been conserved among all of the lentiviruses of primates and almost all of the lentiviruses of nonprimates. Using T cells constitutively expressing vif genes derived from diverse sources and virus replication assays, we show that the vif gene of a second primate lentivirus, simian immunodeficiency virus from macaques, complements vif-defective human immunodeficiency virus type 1 but that those of three distinct nonprimate lentiviruses do not. Although the molecular basis for Vif function has yet to be defined, the potential implications of this noted restriction of vif complementarity are discussed.
Defective accessory genes in a human immunodeficiency virus type 1-infected long-term survivor lacking recoverable virus
Authors: Michael N L , Chang G , d'Arcy L A , Ehrenberg P K , Mariani R , Busch M P , Birx D L , Schwartz D H
Published: January 2020

We have been studying a patient who acquired human immunodeficiency virus (HIV) infection via a blood transfusion 13 years ago. She has remained asymptomatic since that time. The blood donor and two other recipients have all died of AIDS. Although this patient has shown persistently strong seroreactivity to HIV type 1 (HIV-1) antigens by Western blot (immunoblot), she has been continually HIV culture negative in results from multiple laboratories over the last 6 years and has a very low viral burden. Her CD4+ T-cell count has fluctuated around a mean of 399 cells per microliters, with little change in lymphocyte subset percentages. Strong cellular immune responses to HIV-1 epitopes by this patient have been demonstrated. We now report the results of an intensive molecular genetic analysis of the HIV-1 proviral quasispecies from this patient sampled over 5 years. Long terminal repeat region sequences supported the argument for normal basal and Tat-mediated promoter activities. Sequential sequencing of the nef gene revealed a low frequency (8.3%) of defective genes and a striking lack of sequence evolution. Functional analysis of predominant nef genes by both a cell surface CD4 downregulation and a viral infectivity complementation assay showed wild-type function. In contrast, sequential analysis of an amplicon containing the vif, vpr, vpu, tat1, and rev1 genes revealed the presence of inactivating mutations in 64% of the clones. These data suggest that this patient, initially infected with a virulent swarm of HIV-1, is presently infected with a more-attenuated viral quasispecies as a result of effective host immunity.
Inefficient complementation activity of poliovirus 2C and 3D proteins for rescue of lethal mutations
Authors: Teterina N L , Zhou W D , Cho M W , Ehrenfeld E
Published: January 2020

Poliovirus (PV) 2C protein is a nonstructural polypeptide involved in viral RNA replication, whose biochemical activity(ies) in this process has not been defined. By using site-directed mutagenesis, it was shown previously that disruption of nucleotide-binding motifs present in this protein abolished viral RNA synthesis (C. Mirzayan and E. Wimmer, Virology 189:547-555, 1992; N. L. Teterina, K. M. Kean, E. Gorbalenya, V. I. Agol, and M. Girard, J. Gen. Virol. 73:1977-1986, 1992). We have tested whether PV 2C or 2BC protein provided in trans could rescue the replication of these mutated genomes. Rescuing proteins were provided either by cotransfection with helper chimeric PV-coxsackievirus genomes or by expression in cells with a vaccinia virus-T7 RNA polymerase transient-expression system. We report here that replication of mutated RNAs genomes was poorly supported in trans both by helper genomes and by expressed 2C or 2BC proteins. Similarly, very inefficient complementation was observed for two mutated genomes with lethal lesions in 3D polymerase coding sequence. Our results indicate that poliovirus RNA replication shows marked preference for proteins contributed in cis.
Evidence that measles virus hemagglutinin initiates modulation of leukocyte function-associated antigen 1 expression
Authors: Nagendra A R , Smith C W , Wyde P R
Published: January 2020

Measles virus (MV), human immunodeficiency virus, Epstein-Barr virus, and other leukotropic viruses can modulate the expression of leukocyte function antigen 1 (LFA-1) on the surface of infected and nearby leukocytes. This ability to induce changes in LFA-1 expression may play an important role in the pathogenesis of these viruses. However, the mechanism(s) involved in virus-mediated regulation of LFA-1 is unknown. Evidence is presented in this report that it is the MV hemagglutinin (H) protein that initiates up-regulation of LFA-1 expression in leukocyte cultures infected with this virus. Indeed, comparison of the abilities of different MV strains to modulate LFA-1 expression, examination of published nucleotide sequences for the H proteins of different vaccine strains, and competitive inhibition assays using oligopeptides homologous or heterologous to a region of the H protein gene encompassing amino acid 116 (from the amino terminus) all suggest that it is this portion of the H protein that is responsible for MV-induced alteration of LFA-1. These comparisons also support the hypothesis that there is a relationship between the abilities of different MV strains to alter LFA-1 expression and their pathogenic potentials.
Human cytomegalovirus proteinase: candidate glutamic acid identified as third member of putative active-site triad
Authors: Cox G A , Wakulchik M , Sassmannshausen L M , Gibson W , Villarreal E C
Published: January 2020

The human cytomegalovirus (HCMV) proteinase is synthesized as a 709-amino-acid precursor that undergoes at least three autoproteolytic cleavages. The mature proteinase, called assemblin, is one of the products of autoproteolysis and is composed of the first 256 amino acids of the precursor. HCMV assemblin and its homologs in other herpes group viruses contain five highly conserved domains (CD1 through CD5). An absolutely conserved serine in CD3 has been shown by site-directed mutagenesis of the simian cytomegalovirus (SCMV) and herpes simplex virus type 1 (HSV-1) enzymes and by inhibitor affinity labeling of the HSV-1 and HCMV enzymes to be the active-site nucleophile of assemblin. An absolutely conserved histidine in CD2 has also been demonstrated by site-directed mutagenesis of the SCMV and HSV-1 enzymes to be essential for proteolytic activity and has been proposed to be a second member of the catalytic triad of this serine proteinase. We report here the use of site-directed mutagenesis to investigate the active-site amino acids of HCMV assemblin. Substitutions were made for the CD3 serine and CD2 histidine residues implicated as active-site components, and for other amino acids whose influence on enzyme activity was of interest. The mutant proteinases were tested in a transient transfection assay for their ability to cleave their natural substrate, the assembly protein precursor. Results of these experiments verified that HCMV CD3 serine (Ser-132) and CD2 histidine (His-63) are essential for proteolytic activity and identified a glutamic acid (Glu-122) within CD3 that is also essential for proteolytic activity and may be conserved among all herpesvirus assemblin homologs. We suggest that CD3 Glu-122, CD3 Ser-132, and CD2 His-63 constitute the active-site triad of this serine proteinase.
Prior infection with a nonpathogenic chimeric simian-human immunodeficiency virus does not efficiently protect macaques against challenge with simian immunodeficiency virus
Authors: Letvin N L , Li J , Halloran M , Cranage M P , Rud E W , Sodroski J
Published: January 2020

Prior infection with a nef-deleted simian immunodeficiency virus (SIV) protects macaques not only against a homologous pathogenic SIV challenge but also against challenge with a chimeric SIV expressing a human immunodeficiency virus type 1 env gene (SHIV). Since this SHIV is itself nonpathogenic, we sought to explore the use of a nonpathogenic SHIV as a live, attenuated AIDS virus vaccine. Four cynomolgus monkeys infected for greater than 600 days with a chimeric virus composed of SIVmac 239 expressing the human immunodeficiency virus type 1 HXBc2 env, tat, and rev genes were challenged intravenously with 100 animal infectious doses of the J5 clone of SIVmac 32H, an isolate derived by in vivo passage of SIVmac 251. Three of the four monkeys became infected with SIVmac. This observation underlines the difficulty, even with a live virus vaccine, in protecting against an AIDS virus infection.
Substrate specificity of Ty1 integrase
Authors: Moore S P , Powers M , Garfinkel D J
Published: January 2020

Integration of the Saccharomyces cerevisiae retrotransposon Ty1 requires the element-encoded integrase (IN) protein, which is a component of cytoplasmic virus-like particles (VLPs). Using purified recombinant Ty1 IN and an oligonucleotide integration assay based on Ty1 long terminal repeat sequences, we have compared IN activity on substrates having either wild-type or altered donor ends. IN showed a marked preference for blunt-end substrates terminating in an A:T pair over substrates ending in a G:C pair or a 3' dideoxyadenosine. VLP activity on representative substrates also showed preference for donor strands which have an adenosine terminus. Staggered-end substrates showed little activity when nucleotides were removed from the end of the wild-type donor strand, but removal of one nucleotide from the complementary strand did not significantly diminish activity. Removal of additional nucleotides from the complementary strand reduced activity to minimal detection levels. These results suggest that the sequence specificity of Ty1 IN is not stringent in vitro. The absence of Ty1 IN-mediated 3' dinucleotide cleavage, a characteristic of retroviral integrases, was demonstrated by using selected substrates. In addition to the forward reaction, both recombinant IN and VLP-associated IN carry out the reverse disintegration reaction with long terminal repeat-based dumbbell substrates. Disintegration activity exhibits sequence preferences similar to those observed for the forward reaction.
Stimulation of basal transcription from the mouse mammary tumor virus promoter by Oct proteins
Authors: Kim M H , Peterson D O
Published: January 2020

The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif ATGCAAAT. Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreased by two-to threefold in in vitro transcription assays. Oct-1 protein purified from HeLa cell nuclear extracts, as well as recombinant Oct-1 expressed in bacteria, recognized MMTV octamer-related sequences, as shown by DNase I footprinting. Furthermore, rabbit polyclonal antiserum directed against recombinant Oct-1 completely inhibited the formation of specific complexes between MMTV octamer-related sequences and proteins present in nuclear extracts of HeLa cells, indicating that Oct-1 is the major protein in HeLa nuclear extracts that recognizes octamer-related sequences in the MMTV promoter. In addition, depletion of Oct-1 from the nuclear extract by using Oct-1-specific antiserum or a sequence-specific DNA affinity resin decreased in vitro transcription from the wild-type MMTV promoter to a level identical to that obtained from a promoter in which all three octamer-related sequences were mutated. Addition of purified HeLa Oct-1 or recombinant Oct-1 to the depleted extract selectively increased transcription from the wild-type relative to the mutated promoter, demonstrating that Oct-1 transcription factor stimulates basal transcription from the MMTV promoter. A similar effect was observed when purified recombinant Oct-2 was added to the Oct-1-depleted extract, suggesting that Oct-2 may play an important role in MMTV transcription in B cells.
Characterization of defective RNAs derived from RNA 3 of the Fny strain of cucumber mosaic cucumovirus
Authors: Graves M V , Roossinck M J
Published: January 2020

Two defective RNAs (designated D RNA 3 alpha and D RNA 3 beta) were found to be associated with the Fny strain of cucumber mosaic cucumovirus but not with the Sny strain after serial passages in a tobacco host. The D RNAs were derived from RNA 3 by single, in-frame deletions within the 3a open reading frame. A full-length cDNA clone from which biologically active transcripts can be produced in vitro has been constructed for D RNA 3 beta. This transcript can be replicated in tobacco plants infected with subgroup I and II cucumber mosaic cucumovirus strains and with peanut stunt cucumovirus. Translation of D RNA 3 beta in vitro produced a 20-kDa peptide, which was consistent with the predicted coding capacity of the deleted 3a open reading frame. D RNA 3 beta was also associated with polyribosomes isolated from infected tobacco plants. The presence of the D RNAs had no apparent effect upon helper virus yield or symptom production.
Analysis of the subgroup A avian sarcoma and leukosis virus receptor: the 40-residue, cysteine-rich, low-density lipoprotein receptor repeat motif of Tva is sufficient to mediate viral entry
Authors: Rong L , Bates P
Published: January 2020

The genes encoding the receptor for subgroup A Rous sarcoma viruses (tva) were recently cloned from both chicken and quail cells (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Previous work suggested that only the extracellular domain of Tva interacts with the virus (P. Bates, J. A. T. Young, and H. E. Varmus, Cell 74:1043-1051, 1993). Tva is a small membrane-associated protein containing in its extracellular domain a 40-amino-acid region which is closely related to the low-density lipoprotein receptor (LDLR) repeat motif. To determine the region of the Tva extracellular domain responsible for viral receptor function, we created chimeric proteins containing various regions of the Tva extracellular domain fused with a murine CD8 membrane anchor. Analysis of these proteins demonstrates that any chimera containing the Tva LDLR repeat motif can specifically bind the envelope protein of subgroup A avian sarcoma and leukosis viruses. Furthermore, NIH 3T3 cell lines expressing these chimeric proteins were efficiently infected by subgroup A avian sarcoma and leukosis virus vectors. Our results demonstrate that the 40-residue-long LDLR repeat motif of Tva is responsible for viral receptor function.
Transactivation of the Moloney murine leukemia virus and T-cell receptor beta-chain enhancers by cbf and ets requires intact binding sites for both proteins
Authors: Sun W , Graves B J , Speck N A
Published: January 2020

The Moloney murine leukemia virus (Mo-MLV) enhancer contains binding sites (LVb and LVc) for the ets gene family of proteins and a core site that binds the polyomavirus enhancer-binding protein 2/core-binding factor (cbf) family of proteins. The LVb and core sites in the Mo-MLV enhancer contribute to its constitutive activity in T cells. All three binding sites (LVb, LVc, and core) are required for phorbol ester inducibility of the Mo-MLV enhancer. Adjacent binding sites for the ets and cbf proteins likewise constitute a phorbol ester response element within the human T-cell receptor beta-chain (TCR beta) enhancer and contribute to constitutive transcriptional activity of the TCR beta enhancer in T cells. Here we show that the CBF alpha subunit encoded by the mouse Cbfa2 gene (the murine homolog of human AML1) and three ets proteins, Ets-1, Ets-2, and GA-binding protein (GABP), transactivate both the Mo-MLV and mouse TCR beta enhancer in transient-expression assays. Moreover, we show that transactivation by Cbf alpha 2 requires both intact ets and cbf binding sites. Transactivation by Ets-1, Ets-2, and GABP likewise requires intact binding sites for ets proteins and CBF. Supportive biochemical analyses demonstrate that both proteins can bind simultaneously to a composite enhancer element. These findings suggest that ets and cbf proteins cooperate in vivo to regulate transcription from the Mo-MLV and TCR beta enhancers.