bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly of double-shelled rotaviruslike particles by simultaneous expression of recombinant VP6 and VP7 proteins
Authors: Sabara M , Parker M , Aha P , Cosco C , Gibbons E , Parsons S , Babiuk L A
Published: January 2020

Simultaneous coinfection of Spodoptera frugiperda cells with baculovirus recombinants containing the genes for VP6 and VP7 of bovine rotavirus strain C486 resulted in the production of spherical particles resembling smooth, double-shelled rotavirus. These particles were predominantly located in the cell culture supernatant instead of being cell associated. Pretreatment of infected-cell culture supernatants with CaCl2, prior to particle purification, was found to enhance the smooth appearance of the particles. The authenticity of the proteins making up the particles was demonstrated by their reactivity with antiserum specific for double-shelled C486 rotavirus.
Low-level inversion of the L component of pseudorabies virus is not dependent on sequence homology
Authors: Rall G F , Kupershmidt S , Lu X Q , Mettenleiter T C , Ben-Porat T
Published: January 2020

Pseudorabies virus has a class 2 genome in which the S component is found in two orientations relative to the L component. The L component is bracketed by sequences that are partially homologous; it is found mainly in one orientation, but a small proportion is inverted (J. M. DeMarchi, Z. Lu, G. Rall, S. Kuperschmidt, and T. Ben-Porat, J. Virol. 64:4968-4977, 1990). We have ascertained the role of the patchy homologous sequences bracketing the L component in its inversion. A viral mutant, vYa, from which the sequences at the right end of the L component were deleted was constructed. Despite the absence of homologous sequences bracketing the L component in vYa, its L component inverted to an extent similar to that of the L component in the wild-type virus. These results show the following. (i) The low-frequency inversion of the L component of PrV is not mediated by homologous sequences bracketing this component. (ii) Cleavage of concatemeric DNA at the internal junction between the S and L components is responsible for the appearance of the minority of genomes with an inverted L component in populations of pseudorabies virus. (iii) The signals present near or at the end of the S component are sufficient to allow low-frequency cleavage of concatemeric DNA; the sequences at the end of the L component are not essential for cleavage, although they enhance it considerably.
Tumor immunity generated in the course of regression of v-src-induced sarcomas
Authors: Wisner T W , England J M , Pan D Y , Stoker A W , Halpern M S
Published: January 2020

Previous studies have indicated that the regression versus progression of v-src-DNA-induced sarcomas is dependent on chicken line. As a first step in analyzing the role of tumor immunity as a determinant of this line dependence, experiments were undertaken to ascertain whether an antisarcoma immune response is generated in the course of sarcoma growth in TK chickens, a regressor line. To assay for this response, test TK chickens in which v-src-induced wing web sarcomas had regressed, as well as control TK chickens that had not been exposed to v-src, were challenged in protocols known to yield v-src-dependent sarcoma formation and monitored for challenge sarcoma growth. Compared with the control chickens, the test chickens showed a significant resistance to the sarcomagenic challenge. These results raise the possibility that the antisarcoma response that is inducible in regressor lines, as demonstrated here in terms of a protective effect against a subsequent sarcomagenic challenge, may also underlie the regression of v-src-induced primary sarcomas.
A highly divergent simian immunodeficiency virus (SIVstm) recovered from stored stump-tailed macaque tissues
Authors: Khan A S , Galvin T A , Lowenstine L J , Jennings M B , Gardner M B , Buckler C E
Published: January 2020

We report here the results of molecular analysis of a simian immunodeficiency virus (designated SIVstm) which was isolated from a rhesus monkey inoculated with stored lymph node tissue of an Asian stump-tailed macaque. The latter monkey had died in 1977 during an epidemic of acquired immunodeficiency and lymphoma at the California Regional Primate Research Center (L. J. Lowenstine, N. W. Lerche, P. A. Marx, M. B. Gardner, and N. C. Pedersen, p. 174-176, in M. Girard and L. Valette, ed., Retroviruses of Human AIDS and Related Animal Viruses, 1988). Nucleotide sequence analysis of the gag and env regions indicates that SIVstm is an ancient member of the SIV/human immunodeficiency virus type 2 group; it is quite divergent from known SIVs isolated from African sooty mangabeys as well as from Asian macaques. Furthermore, of all SIV strains described to date, SIVstm is the most closely related to human immunodeficiency virus type 2.
The long terminal repeat is not a major determinant of the cellular tropism of human immunodeficiency virus type 1
Authors: Pomerantz R J , Feinberg M B , Andino R , Baltimore D
Published: January 2020

The long terminal repeats (LTRs) of human immunodeficiency virus type 1 (HIV-1) strains from the central nervous systems of four patients with AIDS and of an HIV-1 isolate which is highly macrophage-tropic were isolated by using the polymerase chain reaction. In transient transfection assays, these LTRs demonstrated no significant difference in basal or stimulated levels of transcription in any of a variety of cell lines tested, compared with expression directed from the LTR of a T-lymphocyte-tropic strain of HIV-1. Chimeric viruses were created with the LTRs of the macrophage-tropic and brain-derived viruses ligated to the viral backbone from a T-lymphocyte-tropic strain. No change in cellular tropism was demonstrated with these chimeric viruses. Thus, unlike the LTRs of some murine retroviruses, the LTR of HIV-1 does not appear to play a major role in determining cellular tropism.
Expression and characterization of the thymidine kinase gene of African swine fever virus
Authors: Martin Hernandez A M , Tabares E
Published: January 2020

The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.
The DNA-binding properties of polyomavirus large T antigen are altered by ATP and other nucleotides
Authors: Lorimer H E , Wang E H , Prives C
Published: January 2020

We have examined the influence of ATP on the DNA-binding properties of polyomavirus large T antigen (Py TAg). Utilizing nitrocellulose filter binding, DNase I footprinting, and gel mobility shift assays, we observed that ATP increased Py TAg binding to DNA fragments containing either all Py TAg-binding sites (whole origin) or those sites within (core origin) or adjacent to (early) the origin of replication. Even nonspecific binding to DNA fragments lacking Py TAg-binding sites was increased somewhat by ATP. Binding to the core origin was increased to a greater extent than binding to other DNA fragments tested. Gel band mobility shift assays revealed that ATP increased the production of core origin-specific Py TAg-DNA complexes of high molecular weight. ATP stimulation depended on the presence of MgCl2. Other nucleotides and nonhydrolyzable ATP analogs also increased Py TAg binding to the core origin but to various degrees: ATP, dATP, 5'-adenylyl imidodiphosphate (AMPPNP) greater than 5'-adenylyl methylenediphosphate (AMPPCP) greater than dCTP greater than UTP greater than TTP. GTP and dGTP did not increase DNA binding by Py TAg. The rates of association and disassociation of Py TAg with all the DNA fragments were altered by the presence of ATP. DNase I footprinting showed that ATP extensively extended the region protected within the core origin and also produced a distinctive DNase I-hypersensitive site on the late strand at nucleotides 5255 to 5262 (TTACTATG).
Solid matrix-antibody-antigen complexes induce antigen-specific CD8+ cells that clear a persistent paramyxovirus infection
Authors: Randall R E , Young D F
Published: January 2020

We have previously shown that the adoptive transfer of splenocytes, isolated from mice immunized by infection with the paramyxovirus simian virus 5 (SV5), enhance the speed of clearance of SV5 from the lungs of immunodeficient mice; clearance is mediated primarily through CD8+ effector cells and not by serum neutralizing antibody (D.F. Young, R.E. Randall, J.A. Hoyle, and B.E. Souberbielle, J. Virol. 64:5403-5411, 1990). In this article we demonstrate that immunization of mice with solid matrix-antibody-antigen (SMAA) complexes also induces CD8+ effector cells that are responsible for clearing persistent SV5 infections in immunodeficient mice. The demonstration that immunization with SMAA complexes (an exogenous antigen) can induce class I-restricted cytotoxic T lymphocytes (CTLs) suggests that that these cells may be responsible for virus clearance in vivo. This premise is supported indirectly by the observation that immunization with SMAA complexes was less efficient in inducing class I-restricted CTLs (as measured in vitro) than was infectious virus and that splenocytes isolated from mice immunized with SMAA complexes were also less efficient in clearing virus from lungs of immunodeficient mice than were splenocytes isolated from mice immunized by infection with virus. This was not because the SMAA complexes were generally less immunogenic than infectious virus, since mice immunized with SMAA complexes (which contained the HN protein of SV5) produced higher levels of neutralizing antibody than mice immunized with infectious virus. In the majority of experiments, fixed and killed suspensions of Staphylococcus aureus Cowan strain A were used as the solid matrix in the construction of SMAA complexes. However, in this article we present evidence that alum-antibody-antigen complexes are as immunogenic as S. aureus A-antibody-antigen complexes. These results suggest that the immunological reactivity of the solid matrix itself does not influence the intensity of the immune response to the antigens of interest in the SMAA complexes. The significance of these results for vaccine design are discussed.
Characterization of the cis elements involved in basal and E2-transactivated expression of the bovine papillomavirus P2443 promoter
Authors: Spalholz B A , Vande Pol S B , Howley P M
Published: January 2020

Transcriptional transactivation and repression by the viral E2 proteins are important regulatory mechanisms for the papillomaviruses. In the bovine papillomavirus type 1 (BPV-1), several viral promoters can be transactivated by E2 through E2-dependent enhancer elements located in the viral long control region (LCR), including promoters involved in E2 expression itself. This report demonstrates that the BPV-1 P2443 promoter is transactivated by E2-responsive elements in the LCR and that this promoter is responsible for a major part of the expression of the E2 and E5 gene products. Characterization of the cis elements involved in P2443 regulation indicated that the single E2-binding site directly upstream of P2443 is not required for either the E2 transactivation or for any E2 repression of the basal or transactivated activity of this promoter. Therefore, cooperative interactions between E2 bound at the LCR and E2 bound near P2443 do not have any role in the regulation of this promoter. Further definition of the cis regulatory elements of this promoter indicated that a binding site for the transcriptional factor Sp1 exists directly upstream of the P2443 TATA box and is critical for the basal level of transcription from this promoter. Disruption of this Sp1 site eliminated P2443 promoter activity in transient expression assays for E2 and E5 and resulted in a loss of transforming activity when introduced into the full viral genome.
Expression of a human immunodeficiency virus type 1 long terminal repeat/simian virus 40 early region fusion gene in transgenic mice
Authors: Skowronski J
Published: January 2020

Three lines of transgenic mice carrying the human immunodeficiency virus type 1 (HIV-1) long terminal repeat fused to the simian virus 40 early region (HIV-1 Tag) were constructed. Expression of the transgenes was reproducibly observed in the lymphoid tissue and skin of all three transgenic lines studied. Interestingly, cell types other than T cells, i.e., B cells and thymic stromal cells, contributed most of the expression detectable in the lymphoid organs. Each transgenic line also displayed a different but consistent pattern of transgene expression in nonlymphoid organs. These individual patterns probably reflect the effects of particular chromosomal integration sites on transcriptional activity of the HIV-1 promoter.
Mechanisms of protection against herpes simplex virus type 1-induced retinal necrosis by in vitro-activated T lymphocytes
Authors: Igietseme J U , Streilein J W , Miranda F , Feinerman S J , Atherton S S
Published: January 2020

In BALB/c mice, acute retinal necrosis occurs in the uninoculated eye 8 to 10 days following uniocular anterior chamber inoculation of the KOS strain of herpes simplex virus type 1 (HSV-1). Retinitis in the uninjected eye can be prevented if HSV-1-specific immune effector cells that have been restimulated with virus in vitro are administered intravenously within 1 day of anterior chamber inoculation of virus. We explored further the mechanism of protection afforded by these activated immune effector cells. The results of our studies revealed that optimal protection from retinitis required in vitro restimulation, since infusion of 50 x 10(6) HSV-1-primed but nonrestimulated cells could not protect as well as 10 x 10(6) activated cells. Analysis of both restimulated and nonrestimulated cells showed that only in vitro-restimulated cells were cytotoxic to HSV-1-infected syngeneic target cells. From these studies, we concluded that the ability to kill virus-infected target cells contributed to optimal protection achieved by intravenous administration of activated immune effector cells. Furthermore, T-cell subset depletion of activated immune effector cells demonstrated that both L3T4+ and Lyt-2+ T cells in the transfer inoculum contributed to protection. Additional studies revealed that although the transferred immune effector cells reached the injected eye within 24 h, virus replication in the injected eye was not affected. In the uninjected eye, virus titers were low, consistent with protection of this eye from retinitis. Taken together, the virus recovery results suggest that the interaction of virus with intravenously administered HSV-1-specific immune effector cells which limits virus spread and/or replication of virus probably occurred within the central nervous system and prevented the second wave of virus from entering the uninoculated eye.
Differential loss of envelope glycoprotein gp120 from virions of human immunodeficiency virus type 1 isolates: effects on infectivity and neutralization
Authors: McKeating J A , McKnight A , Moore J P
Published: January 2020

Several parameters which may affect the infectivity of human immunodeficiency virus type 1 in tissue culture were analyzed. In particular, we used gel exclusion chromatography to investigate how the loss of the surface glycoprotein gp120 from virions of the HTLV-IIIB (IIIB), HTLV-IIIRF (RF), and SF-2 isolates modulates infectivity. In IIIB and RF cultures, a high proportion of the total gp120 was virion bound initially but was gradually lost from the virions over time. In contrast, most of the gp120 (and p24) in SF-2-infected cultures was soluble and the few particles present had a fivefold-lower level of virus-bound gp120. However, this reduced level of virion-bound gp120 was more resistant to shedding. Loss of a major proportion of gp120 from IIIB and RF virions resulted in reduced infectivities, and in addition, the resulting accumulation of soluble gp120 in the cultures could competitively inhibit viral infection, especially with SF-2. Increased shedding of virion gp120 also affected the neutralization of IIIB and RF particles. However, the high sensitivity to human serum neutralization characteristic of SF-2 was unaffected by soluble gp120 in cultures, suggesting that the epitopes responsible are not present on soluble gp120.
Japanese encephalitis virus neurotropism is dependent on the degree of neuronal maturity
Authors: Ogata A , Nagashima K , Hall W W , Ichikawa M , Kimura-Kuroda J , Yasui K
Published: January 2020

In a study on Fischer rats, all animals infected with Japanese encephalitis virus (JEV) before the age of 13 days died, but animals infected after the age of 14 days did not die, confirming the age-dependent resistance to JEV infection in the rat brain. A study of the kinetics of JEV infection in the developing rat brain disclosed that JEV antigen disappeared in a particular pattern, i.e., from the deeper layers to the upper layers of the motor cortex, which paralleled neuronal maturation in the cortex. Fifteen-day-old rats, which were resistant to JEV infection, received intracerebral transplants of neurons taken from 19-day embryos. When these animals were infected with JEV after transplantation, viral antigen was detected only in the embryonal neurons soon after transplantation. Thus, it can be concluded that the susceptibility to JEV infection in the rat brain is closely associated with neuronal immaturity.
Upstream promoter elements of the herpes simplex virus type 1 glycoprotein H gene
Authors: Steffy K R , Weir J P
Published: January 2020

To investigate the cis-acting sequence elements that are involved in the regulation of herpes simplex virus type 1 late-gene expression, recombinant viruses were constructed that express the Escherichia coli lacZ gene from the promoter of the glycoprotein H (gH) gene. Deletion experiments established an upstream boundary for the gH promoter of no more than 83 bp from the start of gH transcription and showed that the promoter sequences did not overlap with coding sequences of the upstream thymidine kinase (tk) gene. Sequences of the tk gene previously shown to be required for efficient processing of the tk transcript were essential for expression form the gH promoter and included a TATA-like element. In addition, the gH TATA element was specifically mutagenized to substitute the TATA elements of immediate-early, early, and other late viral promoters for the gH TATA element. The results indicated that the TATA element was an interchangeable component of herpes simplex virus type 1 promoters and did not regulate temporal expression.
Correct intranuclear localization of herpes simplex virus DNA polymerase requires the viral ICP8 DNA-binding protein
Authors: Bush M , Yager D R , Gao M , Weisshart K , Marcy A I , Coen D M , Knipe D M
Published: January 2020

We used indirect immunofluorescence to examine the factors determining the intranuclear location of herpes simplex virus (HSV) DNA polymerase (Pol) in infected cells. In the absence of viral DNA replication, HSV Pol colocalized with the HSV DNA-binding protein ICP8 in nuclear framework-associated structures called prereplicative sites. In the presence of viral DNA replication, HSV Pol colocalized with ICP8 in globular intranuclear structures called replication compartments. In cells infected with mutant viruses encoding defective ICP8 molecules, Pol localized within the cell nucleus but showed a general diffuse intranuclear distribution. In uninfected cells transfected with a plasmid expressing Pol, Pol similarly showed a diffuse intranuclear distribution. Therefore, Pol can localize to the cell nucleus without other viral proteins, but functional ICP8 is required for Pol to localize to prereplicative sites. In cells infected with mutant viruses encoding defective Pol molecules, ICP8 localized to prereplicative sites. Thus, Pol or the portions of Pol not expressed by the mutant viruses are not essential for the formation of prereplicative sites or the localization of ICP8 to these structures. These results demonstrate that a specific nuclear protein can influence the intranuclear location of another nuclear protein.
Identification of an immunodominant cytotoxic T-lymphocyte recognition site in glycoprotein B of herpes simplex virus by using recombinant adenovirus vectors and synthetic peptides
Authors: Hanke T , Graham F L , Rosenthal K L , Johnson D C
Published: January 2020

Cytotoxic T-lymphocyte (CTL) responses to herpes simplex virus (HSV) polypeptides play an important role in recovery from infection and in preventing latency. We have previously shown that glycoprotein B (gB) is a major target recognized by HSV-specific CTLs in C57BL/6 (H-2b) and BALB/c (H-2d) mice but not in CBA/J (H-2k) mice (L. A. Witmer, K. L. Rosenthal, F. L. Graham, H. M. Friedman, A. Yee, and D. C. Johnson, J. Gen. Virol. 71:387-396, 1990). In this report, we utilize adenovirus vectors expressing gB with various deletions to localize an immunodominant site in gB, recognized by H-2b-restricted anti-HSV CTLs, to a region between residues 462 and 594. Overlapping peptides spanning this region were synthesized and used to further localize the immunodominant site to residues 489 to 515, a region highly conserved in HSV type 1 (HSV-1) and HSV-2 strains. The 11-amino-acid peptide was apparently associated exclusively with the Kb major histocompatibility complex gene product and not the Db gene product. In contrast, H-2d-restricted CTLs recognized an immunodominant site between residues 233 and 379.
Virological events leading to spontaneous AKR thymomas
Authors: Stoye J P , Moroni C , Coffin J M
Published: January 2020

The spontaneous leukemias of AKR mice are caused by mink cell focus-forming (MCF) viruses. These viruses are generated by recombination between several endogenous murine retroviruses. The virological events leading to the generation of the leukemogenic agent were investigated by using an oligonucleotide specific for the U3 region of the leukemogenic virus and env-reactive oligonucleotide probes specific for the different classes of endogenous murine leukemia virus. It was shown that (i) the leukemogenic MCF virus is formed by recombination between at least three different endogenous sequences; (ii) the U3 donor for the leukemogenic virus is the inducible xenotropic virus Bxv-1; (iii) all spontaneous tumors contain viruses with duplicated enhancer regions in their long terminal repeats; (iv) enhancer duplication is a somatic event, since Bxv-1 contains only one copy; (v) the first recombinant virus detectable in mass populations of thymocytes by Southern hybridization analysis contains all structural features of the ultimate leukemogenic virus; and (vi) the multiple novel viruses in a given tumor represent progeny of the same unique recombination events. On the basis of these results, an analysis of the virological events leading to AKR thymomas is presented.
Defective transport of Sindbis virus glycoproteins in End4 mutant Chinese hamster ovary cells
Authors: Presley J F , Draper R K , Brown D T
Published: January 2020

Mutant V.24.1, a temperature-sensitive derivative of Chinese hamster ovary cells, defines the End4 complementation group of mutants selected for resistance to protein toxins and has defective lysosomes at the restrictive temperature (P. A. Colbaugh, M. Stookey, and R. K. Draper, J. Cell Biol. 108:2211-2219, 1989). We have investigated the biosynthesis of Sindbis virus envelope glycoproteins in V.24.1 cells. When the cells were infected at the restrictive temperature, the envelope glycoproteins E1 and E2 were undetectable on the cell surface and proteolytic processing of the precursor protein pE2 to envelope protein E2 did not occur. Protein retained intracellularly was sensitive to endoglycosidase H and, by immunofluorescence localization, appeared to accumulate in the endoplasmic reticulum. We conclude that the genetic defect in V.24.1 cells impairs the transport of Sindbis virus glycoproteins, apparently at the level of export from the endoplasmic reticulum.
Retrovirus promoter-trap vector to induce lacZ gene fusions in mammalian cells
Authors: Reddy S , DeGregori J V , von Melchner H , Ruley H E
Published: January 2020

A retrovirus promoter-trap vector (U3LacZ) has been developed in which Escherichia coli lacZ coding sequences were inserted into the 3' long terminal repeat (LTR) of an enhancerless Moloney murine leukemia virus. The U3LacZ virus contains the longest reported LTR (3.4 kbp); nevertheless, lacZ sequences did not interfere with the ability of the virus to transduce a neomycin resistance gene expressed from an internal promoter. Duplication of the LTR placed lacZ sequences in the 5' LTR just 30 nucleotides from the flanking cellular DNA. Approximately 0.4% of integrated proviruses expressed beta-galactosidase as judged by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining, and individual clones expressing lacZ were isolated by fluorescence-activated cell sorting. In all clones examined, beta-galactosidase expression resulted from the fusion of lacZ sequences to transcriptional promoters located in the flanking cellular DNA. Furthermore, by differential sorting of neomycin-resistant cell populations, clones were isolated in which lacZ expression was induced and repressed in growth-arrested and log phase cells, respectively.
Identification and characterization of two nonessential regions of the rabbitpox virus genome involved in virulence
Authors: Bloom D C , Edwards K M , Hager C , Moyer R W
Published: January 2020

We have developed a means to identify genes associated with particular aspects of virulence. By beginning with an avirulent deletion mutant of rabbitpox virus and systematically reintroducing overlapping segments of the deleted region, we have identified two regions of the viral genome associated with increased virulence in mice. Evaluation of illness has been aided by the exploitation of weight loss as an indicator of pathogenesis. One of the regions identified by this method contains several open reading frames and includes two previously described genes. A third, as yet undescribed, gene within this region potentially encodes a product related to the C5 protein of human complement. The second region of DNA associated with increased virulence is the HindIII M fragment, which contains only one complete open reading frame. Analysis of this previously unreported gene shows coding potential for a polypeptide of 254 amino acids (approximately 25 kDa) which is related to the C4 component of human complement. The elucidation of two new viral genes related to complement components, taken together with the recent report of the biological activity of a poxvirus-encoded complement-binding protein, suggests the importance of interactions of the virus with the complement system during a normal infection.
Molecular cloning of a novel isolate of feline immunodeficiency virus biologically and genetically different from the original U.S. isolate
Authors: Miyazawa T , Fukasawa M , Hasegawa A , Maki N , Ikuta K , Takahashi E , Hayami M , Mikami T
Published: January 2020

The Japanese isolate (TM1 strain) of feline immunodeficiency virus (FIV) which replicates in a feline CD4 (fCD4)-positive lymphoblastoid cell line (MYA-1 cells) was molecularly cloned from extrachromosomal closed circular DNA. The restriction map of the clone, termed pFTM 191 complete genome (CG), showed a considerable difference from that of the U.S. isolate (Petaluma strain) of FIV. The sequence homology in the long terminal repeat between the TM1 and Petaluma strain was 82%. The pFTM 191 CG was biologically active after transfection into Crandell feline kidney cells which were permissive for replication of FIV Petaluma. However, the progeny virions could not reinfect fCD4-negative Crandell feline kidney cells but could infect fCD4-positive MYA-1 cells. When a specific-pathogen-free cat was inoculated with the virus derived from the pFTM 191 CG, the cat seroconverted within 8 weeks postinoculation and FIV was reisolated at 4, 8, and 20 weeks postinoculation. These results indicate the infectivity of the pFTM 191 CG in vivo.
Mutations in a herpes simplex virus type 1 origin that inhibit interaction with origin-binding protein also inhibit DNA replication
Authors: Hernandez T R , Dutch R E , Lehman I R , Gustafsson C , Elias P
Published: January 2020

The herpes simplex virus type 1 genome contains three origins of replication: OriL and a diploid OriS. The origin-binding protein, the product of the UL9 gene, interacts with two sites within OriS, box I and box II. A third site, box III, which is homologous to boxes I and II, may also be a binding site for the origin-binding protein. Mutations in these three sites significantly reduce OriS-directed plasmid replication measured in transient replication assays. The reduction in replication efficiency of the mutants correlates well with the decrease in the ability to bind to the origin-binding protein, as determined by Elias et al. (P. Elias, C. M. Gustafsson, and O. Hammarsten, J. Biol. Chem. 265: 17167-17173, 1990). The effect of multiple mutations in boxes I, II, and III on plasmid replication suggests that there are multiple binding sites in OriS for the origin-binding protein. These studies indicate that proper interaction of the origin-binding protein with the OriS sequence is essential for OriS-directed DNA replication.
Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation
Authors: Jüttermann R , Hosokawa K , Kochanek S , Doerfler W
Published: January 2020

Sequence-specific methylation of the promoter and adjacent regions in mammalian genes transcribed by RNA polymerase II leads to the inhibition of these genes. So far, RNA polymerase III-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase III-transcribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by HpaII (5'-CCGG-3') and HhaI (5'-GCGC-3'). Three of these 5'-CG-3' sequences are located close to the internal regulatory region of the VAI segment. An unmethylated, a 5'-CCGG-3'- and 5'-GCGC-3'-methylated, and a 5'-CG-3'-methylated pUC18 construct containing the VAI and VAII regions were transfected into mammalian cells. In many experiments, an inactivating effect of 5'-CCGG-3' and 5'-GCGC-3' DNA methylation on the VAI region was not observed. In contrast, methylation of all 20 5'-CG-3' sequences in the VAI region by a CpG-specific DNA methyltransferase from Spiroplasma species did interfere with VAI transcription. Transcription of the VAI- and VAII- and of the VAI-containing constructs was also shown to be inhibited in an in vitro cell-free transcription system after the constructs had been methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences or at all 5'-CG-3' sequences. When an oligodeoxyribonucleotide which carried the internal control block A of the VAI region was methylated at three 5'-CG-3' sequences, the formation of a complex with HeLa nuclear proteins was abrogated. The results presented support the notion that the VAI gene transcribed by the DNA-dependent RNA polymerase III is also inactivated by methylation of the decisive 5'-CG-3' sequences.
In vitro suppression of normal human bone marrow progenitor cells by human immunodeficiency virus
Authors: Steinberg H N , Crumpacker C S , Chatis P A
Published: January 2020

Incubation of normal human nonadherent and T-cell-depleted bone marrow cells with HIVIIIB at multiplicities of infection (MOI) ranging from 0.0001:1 to 1:1 reverse transcriptase (RT) units resulted in the dose-dependent suppression of the in vitro growth of erythroid burst-forming unit (BFU-E), granulocyte-macrophage (CFU-GM), and T-lymphocyte (CFU-TL) colonies of progenitor cells. Maximum inhibition of colony formation was observed at a 1:1 ratio of virus to bone marrow cells. At this MOI, BFU-E and CFU-GM colonies were inhibited by 60 to 80%, while CFU-TL colonies were totally suppressed. Inhibition of colony formation was also observed at an MOI of 0.1:1 but not with further log dilutions of the virus. Incubation of the virus with antibody to gp160 resulted in the complete reversal of stem cell suppression and the normalization of colony growth in vitro. For BFU-E and CFU-GM colonies, this reversal was observed with dilutions of antibody up to 1:100 and was no longer observed at titers greater than 1:500. The CFU-TL colony number normalized at titers between 1:10 and 1:50. Human immunodeficiency virus (HIV) also suppressed by 50% the growth of colonies derived from CD34+ stem cell fractions. Infection of CD34+ cells and T-cell-depleted, nonadherent cell fractions was demonstrated by detection with HIV-specific DNA probe following amplification by polymerase chain reaction. The results suggest that HIV can directly infect human bone marrow progenitor cells and affect their ability to proliferate and give rise to colonies in vitro. The results indicate a direct role for the virus in bone marrow suppression and a possible mechanism for the cytopenias observed in patients with AIDS.
In vitro infection of natural killer cells with different human immunodeficiency virus type 1 isolates
Authors: Chehimi J , Bandyopadhyay S , Prakash K , Perussia B , Hassan N F , Kawashima H , Campbell D , Kornbluth J , Starr S E
Published: January 2020

Natural killer (NK) cells are a discrete subset of leukocytes, distinct from T and B lymphocytes. NK cells mediate spontaneous non-MHC-restricted killing of a wide variety of target cells without prior sensitization and appear to be involved in initial protection against certain viral infections. Depressed NK cell-mediated cytotoxicity, one of the many immunological defects observed in AIDS patients, may contribute to secondary virus infections. Here we report that clonal and purified polyclonal populations of NK cells, which expressed neither surface CD4 nor CD4 mRNA, were susceptible to infection with various isolates of human immunodeficiency virus type 1 (HIV-1). Viral replication was demonstrated by detection of p24 antigen intracellularly and in culture supernatants, by the presence of HIV DNA within infected cells, and by the ability of supernatants derived from HIV-infected NK cells to infect peripheral blood mononuclear cells or CD4+ cell lines. Infection of NK cells was not blocked by anti-CD4 or anti-Fc gamma RIII monoclonal antibodies. NK cells from HIV-infected and uninfected cultures were similar in their ability to lyse three different target cells. Considerable numbers of cells died in HIV-infected NK cell cultures. These results suggest that loss of NK cells in AIDS patients is a direct effect of HIV infection but that reduced NK cell function involves another mechanism. The possibility that NK cells serve as a potential reservoir for HIV-1 must be considered.
Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones
Authors: Kurane I , Brinton M A , Samson A L , Ennis F A
Published: January 2020

Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone recognized dengue virus types 1, 2, and 3. Four dengue virus serotype-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4. One flavivirus-cross-reactive clone recognized dengue virus types 1, 2, 3, and 4 and West Nile virus (WNV), but did not recognize yellow fever virus (YFV), whereas three flavivirus-cross-reactive clones recognized dengue virus types 1, 2, 3, and 4, WNV, and YFV. HLA restriction in the lysis by these T-cell clones was also heterogeneous. HLA-DP, HLA-DQ, and HLA-DR were used as restriction elements by various T-cell clones. We also examined the recognition of viral nonstructural protein NS3, purified from cells infected with dengue virus type 3 or WNV, by these T-cell clones. One serotype-specific clone, two dengue virus subcomplex-specific clones, and three dengue virus serotype-cross-reactive clones recognized NS3 of dengue virus type 3. One flavivirus-cross-reactive clone recognized NS3 of dengue virus type 3 and WNV. These results indicate that heterogeneous dengue virus-specific CD4+ cytotoxic T cells are stimulated in response to infection with a dengue virus and that a nonstructural protein, NS3, contains multiple dominant T-cell epitopes.
Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication
Authors: Blum H E , Galun E , Liang T J , von Weizsäcker F , Wands J R
Published: January 2020

A hepatitis B virus (HBV) genome was cloned from human liver. Numerous mutations in all viral genes define this HBV DNA as a mutant, divergent from all known HBV DNA sequences. Functional analyses of this mutant demonstrated a defect blocking viral DNA synthesis. The genetic basis of this defect was identified as a single missense mutation in the 5' region of the viral polymerase gene, resulting in the inability to package pregenomic RNA into core particles. The replication defect could be trans-complemented by a full-length wild-type, but not by a full-length mutant or 3'-truncated wild-type, polymerase gene construct. Our findings indicate a critical role of the 5' polymerase gene region in the life cycle of the virus and suggest that introducing missense mutations in this region can be a strategy to terminate viral replication in vivo.
Selection of genetic variants of simian immunodeficiency virus in persistently infected rhesus monkeys
Authors: Burns D P , Desrosiers R C
Published: January 2020

Genetic and antigenic variation may be one means by which lentiviruses that cause AIDS avoid elimination by host immune responses. Genetic variation in the envelope gene (env) was studied by comparing the nucleotide sequences of 27 clones obtained from two rhesus monkeys infected with molecularly cloned simian immunodeficiency virus. All 27 clones differed from each other and differed from the input clone in the gp120 (SU) portion of the envelope gene. Nucleotide substitutions were shown to accumulate with time at an average rate of 8.5 per 1,000 per year in SU. Surprisingly, the majority of nucleotide substitutions (81%) resulted in amino acid changes. Variation in SU was not random but occurred predominantly in five discrete regions. Within these variable regions, a remarkable 98% of the nucleotide substitutions changed the amino acid. These results demonstrate that extensive sequence variability accumulates in vivo after infection with molecularly cloned virus and that selection occurs in vivo for changes in distinct variable regions in env.
Pathways of activation of the Epstein-Barr virus productive cycle
Authors: Sinclair A J , Brimmell M , Shanahan F , Farrell P J
Published: January 2020

The promoter for the 2.8-kb RNA of Epstein-Barr virus encoding BZLF1 and BRLF1 was identified and shown to be activated by both BZLF1 and BRLF1 but not by 12-O-tetradecanoylphorbol-13-acetate. Site-directed mutagenesis suggests that two binding sites for BZLF1 within the promoter contribute to the transactivation by BZLF1. The early kinetics of induction of the 2.8- and 1.0-kb RNAs encoding BZLF1 and BRLF1 in Akata cells treated with anti-immunoglobulin indicate that both RNAs appear within 60 min. The results indicate some likely pathways of activation of Epstein-Barr virus productive cycle gene expression.
Topoisomerase I-mediated integration of hepadnavirus DNA in vitro
Authors: Wang H P , Rogler C E
Published: January 2020

Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule.
Delay time for influenza virus hemagglutinin-induced membrane fusion depends on hemagglutinin surface density
Authors: Clague M J , Schoch C , Blumenthal R
Published: January 2020

We have studied the kinetics of low-pH-induced fusion between erythrocyte membranes and membranes containing influenza virus hemagglutinin by using assays based on the fluorescence dequenching of the lipophilic dye octadecylrhodamine. Stopped-flow mixing and fast data acquisition have been used to monitor the early stages of influenza virus fusion. We have compared this with the kinetics observed for fusion of an NIH 3T3 cell line, transformed with bovine papillomavirus, which constitutively expresses influenza virus hemagglutinin (GP4f cells). Virus and GP4f cells both display a pH-dependent time lag before the onset of fluorescence dequenching, but of an order of magnitude difference, ca. 2 s versus ca. 20 s. We have adopted two strategies to investigate whether the difference in lag time reflects the surface density of acid-activated hemagglutinin, able to undergo productive conformational change. (i) Hemagglutinin expressed on the cell surface requires proteolytic cleavage with trypsin from an inactive HAO form; we have limited the extent of proteolysis. (ii) We have used infection of CV-1 cells with a recombinant simian virus 40 bearing the influenza virus hemagglutinin gene. The surface expression of hemagglutinin is a function of time postinfection. For low-pH-induced fusion of both types of cell with erythrocytes, the lag time decreases with increasing hemagglutinin densities. Our results do not indicate a cooperative phenomenon at the level of the principal rate-determining step. We also show in the instance of virus fusion, that the magnitude of the delay time is a function of the target membrane transbilayer lipid distribution. We conclude that for a given amount of pH-activated hemagglutinin per unit area of membrane, the kinetics of fusion is determined by nonspecific physical properties of the membranes involved.
Linker-scanning mutational analysis of the transcriptional activity of the human immunodeficiency virus type 1 long terminal repeat
Authors: Zeichner S L , Kim J Y , Alwine J C
Published: January 2020

We have compared the relative importance of transcription regulatory regions in the U3 and R regions of the human immunodeficiency virus type 1 long terminal repeat (LTR) by using linker-scanning mutational analysis. Twenty-six mutant LTR-chloramphenicol acetyltransferase (CAT) transient expression plasmids were prepared in which consecutive 18-bp regions of wild-type LTR were replaced with an NdeI-XhoI-SalI (NXS) polylinker. The mutant LTR-CAT plasmids were transfected into unstimulated Jurkat cells, Jurkat cells stimulated with phytohemagglutinin and tetradecanoylphorbol acetate, and Jurkat cells which constitutively express the human immunodeficiency virus type 1 trans-activator protein, Tat. Transcriptional activity was measured by analysis of CAT activity. The activities of these mutants identified one major and several minor transcription control elements in addition to previously identified elements. In addition, this fine-structure analysis identified differences in utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells. A significant regulatory region was indicated by linker-scanning mutations between nucleotides -183 and -130 (relative to the transcription start site, +1). These mutations caused marked decreases in activity of the LTR in unstimulated and especially in stimulated Jurkat cells but had no effect in Tat-expressing Jurkat cells. DNA mobility shift studies comparing probes of wild-type and mutant sequences in the -183 to -130 region indicated that alterations in specific DNA binding correspond to the altered transcriptional activity of the mutants. The effects of mutations in several regulatory regions, in addition to the -183 to -130 region described above, differ between Tat-expressing and -nonexpressing Jurkat cells. For example, the NF-kB sites are necessary for transcription in both Tat-expressing and -nonexpressing cells. However, Tat-expressing Jurkat cells primarily require only the 3'-proximal site, while both stimulated and unstimulated Jurkat cells appear to require both sites. Mutants downstream of the TATA element cause a more significant decrease in activity in Tat-expressing Jurkat cells than in the others. Finally, several mutations in the 5' half of the LTR (-453 to -184) show modest increases in transcription (1.5-fold or less) in unstimulated Jurkat cells only, suggesting possible negative regulatory sites. In summary, our studies have identified a control region (-183 to -130) upstream of the NF-kB sites and have more precisely defined significant differences in the utilization of regulatory regions between unstimulated, stimulated, and Tat-expressing Jurkat cells.