bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased viral burden and cytopathicity correlate temporally with CD4+ T-lymphocyte decline and clinical progression in human immunodeficiency virus type 1-infected individuals
Authors: Connor R I , Mohri H , Cao Y , Ho D D
Published: January 2020

The rate of clinical progression is variable among individuals infected with human immunodeficiency virus type 1 (HIV-1). Changes in viral burden which correlate with disease status have been demonstrated in cross-sectional studies; however, a detailed longitudinal study of the temporal relationship between viral burden, CD4+ T-cell numbers, and clinical status throughout the course of infection has not been reported. Multiple longitudinal blood samples were obtained from four HIV-1-infected individuals with clinically divergent profiles. Levels of HIV-1 were measured in sequential samples of peripheral blood mononuclear cells, using both end-point dilution cultures and quantitative polymerase chain reaction methods. Serial HIV-1 isolates from each case were also evaluated to determine their biological properties in vitro. For the three patients with clinical progression, a dramatic increase in the level of HIV-1 was observed concurrent with or prior to a marked drop in CD4+ T lymphocytes. This increase in viral burden was temporally associated with the emergence of a more cytopathic viral phenotype. In contrast, consistently low levels of HIV-1 were observed in the one patient who was clinically and immunologically stable for more than a decade. Moreover, viral isolates from this patient were less cytopathic in vitro compared with HIV-1 isolates from those patients with disease progression. The temporal association between increased viral burden and CD4+ T-cell decline suggests a direct role for HIV-1 in the cytopathology of CD4+ T cells in vivo. Our results indicate that the pathogenic mechanisms responsible for CD4+ T-cell depletion may be related to both quantitative and qualitative changes in HIV-1.
The acidic amino-terminal region of herpes simplex virus type 1 alpha protein ICP27 is required for an essential lytic function
Authors: Rice S A , Lam V , Knipe D M
Published: January 2020

The herpes simplex virus type 1 (HSV-1) alpha protein ICP27 regulates the transition between the delayed-early and late phases of the viral infection. Previous genetic analyses have suggested that the important functional domains of ICP27 map to its carboxyl-terminal half. One striking feature of the primary sequence of ICP27, however, is an extremely acidic region near its amino terminus. To determine whether this region is required for ICP27 function, we deleted the sequences in the ICP27 gene which encode it (codons 12 through 63). In transient expression assays, the deletion mutant was unable to efficiently repress the expression of a cotransfected reporter gene or to efficiently complement the growth of d27-1, an HSV-1 ICP27 null mutant. These results suggested that the acidic region of ICP27 is involved in a regulatory function required for lytic growth. To test this possibility further, we introduced the mutant allele into the HSV-1 genome by marker transfer. Two independently derived isolates of the mutant virus, designated d1-2a and d1-2b, were recovered and analyzed. Both isolates were defective for growth in Vero cells, exhibiting a 100-fold reduction in virus yield compared with the wild-type infection. Vero cells infected with the d1-2 isolates showed a three- to eightfold reduction in viral DNA replication, a moderate reduction in the expression of viral gamma genes, and a delay in the repression of beta genes. The phenotype of the d1-2 isolates differs substantially from the phenotypes of previously isolated ICP27 mutants, which show much more severe defects in viral gene expression. Our results demonstrate that the amino-terminal half of ICP27 participates in its regulatory activities in both infected and transfected cells.
Functional organization of the Bel-1 trans activator of human foamy virus
Authors: He F , Sun J D , Garrett E D , Cullen B R
Published: January 2020

Human foamy virus encodes a 300-amino-acid nuclear regulatory protein termed Bel-1 that is required for human foamy virus replication in culture. Bel-1 is a potent trans-activator of gene expression directed by the homologous HFV long terminal repeat as well as the long terminal repeat of human immunodeficiency virus type 1. We have used mutational analysis to define several discrete functional domains within Bel-1. The C-terminal approximately 50 amino acids of Bel-1 are shown to be essential for Bel-1 activity but can be effectively substituted by the C-terminal activation domain of VP16. We therefore conclude that the Bel-1 C terminus forms part of an activation domain. Mutations within a central, approximately 100-amino-acid segment of Bel-1 preclude trans-activation by either Bel-1 or the Bel-1/VP16 chimera. These sequences are therefore proposed to direct the interaction of Bel-1 with its viral DNA target sequences. A short Bel-1 segment located between the activation and binding domains is shown to mediate the nuclear localization of this regulatory protein. Although the functional organization of Bel-1 therefore appears comparable to that reported for other eukaryotic transcriptional activators, Bel-1 does not contain sequences homologous to known transcriptional activation or DNA-binding motifs.
Development of type-specific and cross-reactive serological probes for the minor capsid protein of human papillomavirus type 33
Authors: Volpers C , Sapp M , Komly C A , Richalet-Secordel P , Streeck R E
Published: January 2020

Human papillomavirus type 33 (HPV33) is associated with malignant tumors of the cervix. In an attempt to develop immunological probes for HPV33 infections, antisera against various bacterial fusion proteins carrying sequences of the minor capsid protein encoded by L2 were raised in animals. Antigenic determinants on the HPV33 L2 protein were identified by using truncated fusion proteins and were classified as type specific or cross-reactive with respect to HPV1, -8, -11, -16, and -18. Cross-reactive epitopes map to amino acids 98 to 107 or to amino acids 102 to 112 and 107 to 117, respectively, depending on the fusion protein used for immunization. Antibodies directed toward these epitopes detect L2 proteins of HPV11, -16, and -18, but not of HPV1 and -8, in Western immunoblots and enzyme-linked immunosorbent assays. HPV33 L2 amino acids 82 to 94 and 117 to 130 induce type-specific antibodies, with the major response directed to amino acids 117 to 130. By using a synthetic peptide corresponding to L2 amino acids 117 to 130, high-titered, type-specific antisera were obtained. These antisera should be useful as immunological probes for HPV33 infection.
Two factors that bind to highly conserved sequences in mammalian type C retroviral enhancers
Authors: Manley N R , O'Connell M , Sun W , Speck N A , Hopkins N
Published: January 2020

The transcriptional enhancers of the Moloney and Friend murine leukemia viruses (MLV) are important determinants of viral pathogenicity. We used electrophoretic mobility shift and methylation interference assays to study nuclear factors which bind to a region of these enhancers whose sequence is identical between Moloney and Friend viruses and particularly highly conserved among 35 mammalian type C retroviruses whose enhancer sequences have been aligned (E. Golemis, N. A. Speck, and N. Hopkins, J. Virol. 64:534-542, 1990). Previous studies identified sites for the leukemia virus factor b (LVb) and core proteins in this region (N. A. Speck and D. Baltimore, Mol. Cell. Biol. 7:1101-1110, 1987) as well as a site, overlapping those for LVb and core, for a third factor (N. R. Manley, M. A. O'Connell, P. A. Sharp, and N. Hopkins, J. Virol. 63:4210-4223, 1989). Surprisingly, the latter factor appeared to also bind two sites identified in the Friend MLV enhancer, Friend virus factor a and b1 (FVa and FVb1) sites, although the sequence basis for the ability of the protein to bind these diverse sites was not apparent. Here we describe the further characterization of this binding activity, termed MCREF-1 (for mammalian type C retrovirus enhancer factor 1), and the identification of a consensus sequence for its binding, GGN8GG. We also identify a factor, abundant in mouse T-cell lines and designated LVt, which binds to two sites in the Moloney MLV enhancer, overlapping the previously identified LVb and LVc binding sites. These sites contain the consensus binding site for the Ets family of proteins. We speculate on how distinct arrays of these factors may influence the disease-inducing phenotype.
Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells
Authors: Kao C , Hauser P , Reznikoff W S , Reznikoff C A
Published: January 2020

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.
Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites
Authors: Macnaughton T B , Wang Y J , Lai M M
Published: January 2020

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.
A protein-binding site with dyad symmetry in the long terminal repeat of the MCF13 murine leukemia virus that contributes to transcriptional activity in T lymphocytes
Authors: Yoshimura F K , Diem K , Chen H , Tupper J
Published: January 2020

We have previously identified regions in the long terminal repeat (LTR) of the MCF13 murine leukemia virus (MLV) that contribute to transcriptional activity in different cell types. We have observed that enhancer sequences and a region that resides 3' of the enhancer make significant contributions to transcriptional activity in T lymphocytes (T. Hollon and F. K. Yoshimura, J. Virol. 63:3353-3361, 1989). In this report, we have focused on the region of the MCF13 LTR that is 3' of the enhancer to identify binding sites for proteins that may play a role in the regulation of transcription in T cells. By gel shift and DNA footprint analyses, we have identified a single protein-binding site (MLPal) that includes a nucleotide sequence with dyad symmetry. A synthetic double-stranded oligonucleotide corresponding to this protein-binding site formed a specific protein-DNA complex. Deletion of this protein-binding site from the wild-type LTR decreased transcriptional activity in T lymphocytes but not in fibroblasts as determined by a transient expression assay. The MLPal sequence by itself cannot augment transcription in T cells but is able to do so in conjunction with enhancer sequences.
Multiple positive and negative cis-acting elements that mediate transactivation by bel1 in the long terminal repeat of human foamy virus
Authors: Lee K J , Lee A H , Sung Y C
Published: January 2020

The bel1 protein of human foamy virus (HFV), a retrovirus, regulates expression of the gene linked to the HFV long terminal repeat (LTR) and is essential for viral gene expression. The mechanism of action of the bel1 protein is unknown, but its action is mediated through the U3 region of the LTR. To determine which U3 sequences are critical for transactivation by bel1, a series of hybrid vectors consisting of a mutant HFV LTR and the chloramphenicol acetyltransferase gene were constructed and tested for their responsiveness to the bel1 protein by using transient assays after transfection. The target sequences for transactivation by bel1 were mapped to five regions in the U3 domain of the LTR: nucleotides -559 to -506, -454 to -418, -360 to -342, -327 to -284, and -116 to -89 (+1 represents the transcription initiation site). No significant sequence similarity was identified among the five target sites. The observation that the multiple distinct elements in the HFV LTR are the targets for bel1 transactivation is different from observations with other human retroviral systems. The regulation mechanism of HFV bel1 protein-mediated transactivation appears to be analogous to that of some DNA virus transactivators that increase transcription from numerous different viral promoters with little sequence similarity shared among them. We demonstrated that multiple bel1-responsive elements (BRE) can act as bel1-dependent enhancer elements, while a single copy of one BRE, BREe, can serve as an upstream activating element in both orientations. In addition, the region between -466 and -498 was identified as responsible for the downregulation of gene expression directed by BREa, which requires its upstream sequence element to act as a bel1-dependent enhancer element in a heterologous promoter.
Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly
Authors: Haynes J I , Chang D , Consigli R A
Published: January 2020

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.
Sequence, transcriptional mapping, and overexpression of p47, a baculovirus gene regulating late gene expression
Authors: Carstens E B , Lu A L , Chan H L
Published: January 2020

A 2.8-kb region of the Autographa californica nuclear polyhedrosis virus genome was sequenced and found to contain an open reading frame (p47) which was capable of rescuing a previously characterized temperature-sensitive mutant, ts317 (S. Partington, H. Yu, A. Lu, and E. B. Carstens, Virology 157:91-102, 1990). Transcriptional mapping demonstrated that an early 4.2-kb RNA encoded the p47 open reading frame and probably overlapped the 39K delayed-early gene. The p47 open reading frame was cloned behind the polyhedrin promoter in a baculovirus transfer plasmid, which was then used to prepare a recombinant baculovirus overexpressing the p47 polypeptide. The overexpressed polypeptide was used to prepare p47-specific monoclonal antibodies. These antibodies detected a polypeptide of 47 kDa in A. californica nuclear polyhedrosis virus-infected cells, demonstrating that p47 is expressed as an authentic viral product. The p47 gene product was localized to the nucleus of infected cells, supporting the hypothesis that it is involved in regulating viral transcription at late times postinfection.
Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant
Authors: Threadgill D S , Steagall W K , Flaherty M T , Fuller F J , Perry S T , Rushlow K E , Le Grice S F , Payne S L
Published: January 2020

The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J. H. Elder, D. L. Lerner, C. S. Hasselkus-Light, D. J. Fontenot, E. Hunter, P. A. Luciw, R. C. Montelaro, and T. R. Phillips, J. Virol. 66:1791-1794, 1992). Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.
Murine retrovirus-induced spongiform encephalopathy: disease expression is dependent on postnatal development of the central nervous system
Authors: Lynch W P , Portis J L
Published: January 2020

In this report, we have examined the role of central nervous system (CNS) development in the pathogenesis of neurodegenerative disease induced by murine retroviruses. This was accomplished by comparing the effect of delivering viruses, with either severe or marginal neurovirulence (J. L. Portis, S. Czub, C. F. Garon, and F. J. McAtee, J. Virol. 64:1648-1656, 1990), during the midgestational development of the mouse (gestation days 9 to 10). Midgestation inoculation of the marginally neurovirulent virus, 15-1, resulted in high level CNS infection, as determined by viral DNA and protein analysis. The high-level infection resulted in rapid, severe disease with 100% incidence and an average clinical onset on postnatal day 17 (P17). The disease onset was comparable to that observed for the highly neurovirulent virus, FrCasE, when inoculated neonatally (onset ca. P16). To evaluate whether disease could be induced even earlier in CNS development, FrCasE was inoculated during midgestation. Surprisingly, neither clinical nor histological manifestations of CNS disease were accelerated but rather appeared at the same developmental time as seen for neonatally inoculated animals (onset of neuropathology at ca. P10; onset of clinical disease at ca. P15). CNS infection, on the other hand, occurred at earlier times (< P0), at higher levels, and with a broader distribution than in neonatally inoculated animals. No infection of the neurons which ultimately degenerate was observed in any regimen of virus inoculation. It was observed, however, that the gp70 viral envelope protein from the CNS showed an increase mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis compared with the envelope protein from infected spleens or purified virions. These results indicate that a postnatal developmental event must occur to allow the presence of a neurovirulent virus to precipitate spongiform degeneration and that an altered envelope protein may be participating in the process.
A Friend virus mutant that overcomes Fv-2rr host resistance encodes a small glycoprotein that dimerizes, is processed to cell surfaces, and specifically activates erythropoietin receptors
Authors: Kozak S L , Hoatlin M E , Ferro F E , Majumdar M K , Geib R W , Fox M T , Kabat D
Published: January 2020

The env gene of Friend spleen focus-forming virus (SFFV) encodes a membrane glycoprotein (gp55) that is inefficiently (3 to 5%) processed from the rough endoplasmic reticulum to form a larger dimeric plasma membrane derivative (gp55p). Moreover, the SFFV env glycoprotein associates with erythropoietin receptors (EpoR) to cause proliferation of infected erythroblasts [J.-P. Li, A. D. D'Andrea, H. F. Lodish, and D. Baltimore, Nature (London) 343:762-764, 1990]. Interestingly, the mitogenic effect of SFFV is blocked in mice homozygous for the Fv-2r resistance gene, but mutant SFFVs can overcome this resistance. Recent evidence suggested that these mutants contain partial env deletions that truncate the membrane-proximal extracellular domain of the encoded glycoproteins (M. H. Majumdar, C.-L. Cho, M. T. Fox, K. L. Eckner, S. Kozak, D. Kabat, and R. W. Geib, J. Virol. 66:3652-3660, 1992). Mutant BB6, which encodes a gp42 glycoprotein that has a large deletion in this domain, causes erythroblastosis in DBA/2 (Fv-2s) as well as in congenic D2.R (Fv-2r) mice. Analogous to gp55, gp42 is processed inefficiently as a disulfide-bonded dimer to form cell surface gp42p. Retroviral vectors with SFFV and BB6 env genes have no effect on interleukin 3-dependent BaF3 hematopoietic cells, but they cause growth factor independency of BaF3/EpoR cells, a derivative that contains recombinant EpoR. After binding 125I-Epo to surface EpoR on these factor-independent cells and adding the covalent cross-linking reagent disuccinimidyl suberate, complexes that had immunological properties and sizes demonstrating that they consisted of 125I-Epo-gp55p and 125I-Epo-gp42p were isolated from cell lysates. Contrary to a previous report, SFFV or BB6 env glycoproteins did not promiscuously activate other members of the EpoR superfamily. Although the related env glycoproteins encoded by dualtropic murine leukemia viruses formed detectable complexes with EpoR, strong mitogenic signalling did not ensue. Our results indicate that the SFFV and BB6 env glycoproteins specifically activate EpoR; they help to define the glycoprotein properties important for its functions; and they strongly suggest that the Fv-2 leukemia control gene encodes an EpoR-associated regulatory factor.
Vaccinia virus morphogenesis is blocked by a temperature-sensitive mutation in the I7 gene that encodes a virion component
Authors: Kane E M , Shuman S
Published: January 2020

The ts16 mutation of vaccinia virus WR (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) has been mapped by marker rescue to the I7L open reading frame located within the genomic HindIII I DNA fragment. The I7 gene encodes a 423-amino-acid polypeptide. Thermolabile growth was attributed to an amino acid substitution, Pro-344-->Leu, in the predicted I7 protein. A normal temporal pattern of viral protein synthesis was elicited in cells infected with ts16 at the nonpermissive temperature (40 degrees C). Electron microscopy revealed a defect in virion assembly at 40 degrees C. Morphogenesis was arrested at a stage subsequent to formation of spherical immature particles. Western immunoblot analysis with antiserum directed against the I7 polypeptide demonstrated an immunoreactive 47-kDa polypeptide accumulating during the late phase of synchronous vaccinia virus infection. Immunoblotting of extracts of wild-type virions showed that the I7 protein is encapsidated within the virus core. The I7 polypeptide displays amino acid sequence similarity to the type II DNA topoisomerase of Saccharomyces cerevisiae.
Easily unwound DNA sequences and hairpin structures in the Epstein-Barr virus origin of plasmid replication
Authors: Williams D L , Kowalski D
Published: January 2020

The Epstein-Barr virus (EBV) origin of plasmid replication (oriP) includes two known cis-acting components, the dyad symmetry region and the family of repeats. We used P1 nuclease, a single-strand-specific endonuclease, to probe EBV oriP for DNA sequences that are intrinsically easy to unwind on a negatively supercoiled plasmid. Selective nuclease hypersensitivity was detected in the family of repeats on an oriP-containing plasmid and in the dyad symmetry region on a plasmid that lacks the family of repeats, indicating that the DNA in both cis-acting components is intrinsically easy to unwind. The hierarchy of nuclease hypersensitivity indicates that the family of repeats is more easily unwound than the dyad symmetry region, consistent with the hierarchy of helical stability predicted by computer analysis of the DNA sequence. A specific subset of the family of repeats is nuclease hypersensitive, and the DNA structure deduced from nucleotide-level analysis of the P1 nuclease nicks is a cruciform near a single-stranded bubble. The dyad symmetry region unwinds to form a broad single-stranded bubble containing hairpins in the 65-bp dyad sequence. We propose that the intrinsic ease of unwinding the dyad symmetry region, the actual origin of DNA replication, is an important component in the mechanism of initiation.
Varicella-zoster virus open reading frame 10 protein, the herpes simplex virus VP16 homolog, transactivates herpesvirus immediate-early gene promoters
Authors: Moriuchi H , Moriuchi M , Straus S E , Cohen J I
Published: January 2020

The varicella-zoster virus (VZV) open reading frame 10 (ORF10) protein is the homolog of the herpes simplex virus type 1 (HSV-1) protein VP16. These are two virion tegument proteins that have extensive amino acid sequence identity in their amino-terminal and middle domains. ORF10, however, lacks the acidic carboxy terminus which is critical for transactivation by VP16. Earlier studies showed that VZV ORF10 does not form a tertiary complex with the TAATGARAT regulatory element (where R is a purine) with which HSV-1 VP16 interacts, suggesting that ORF10 may not have transactivating ability. Using transient-expression assays, we show that VZV ORF10 is able to transactivate VZV immediate-early (IE) gene (ORF62) and HSV-1 IE gene (ICP4 and ICP0) promoters. Furthermore, cell lines stably expressing ORF10 complement the HSV-1 mutant in1814, which lacks the transactivating function of VP16, and enhance the de novo synthesis of infectious virus following transfection of HSV-1 virion DNA. These results indicate that ORF10, like its HSV-1 homolog VP16, is a transactivating protein despite the absence of sequences similar to the VP16 carboxy-terminal domain. The transactivating function of the VZV ORF10 tegument protein may be critical for efficient initiation of viral infection.
Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers
Authors: Ryden T A , de Mars M , Beemon K
Published: January 2020

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive.
Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53
Authors: Shepherd S E , Howe J A , Mymryk J S , Bayley S T
Published: January 2020

From previous studies on the induction of DNA synthesis in quiescent primary baby rat kidney cells by adenovirus type 5 (Ad5) E1A deletion mutants, we concluded that induction is prevented only when cellular proteins p300 and pRb are both uncomplexed with E1A (J.A. Howe, J.S. Mymryk, C. Egan, P.E. Branton, and S.T. Bayley, Proc. Natl. Acad. Sci. USA 87:5883-5887, 1990). We have now examined induction by these same mutants in virus lacking the E1B region, so that cellular p53 was no longer complexed to the E1B 55-kDa protein. E1A mutants that fail to bind pRb induced DNA synthesis at a significantly lower level in Ad5 lacking E1B than in Ad5 containing E1B. Apparently, therefore, uncomplexed p53 can partially replace p300 in cooperating with pRb to suppress DNA synthesis in baby rat kidney cells.
Epstein-Barr virus latent membrane protein 2A blocks calcium mobilization in B lymphocytes
Authors: Miller C L , Longnecker R , Kieff E
Published: January 2020

LMP2A is expressed in latent Epstein-Barr virus (EBV) infection and interacts with LMP1 and members of the src tyrosine kinase family in the plasma membrane. Since tyrosine kinase mediate receptor-induced changes in intracellular free calcium, the effect of LMP2A on receptor-mediated intracellular calcium mobilization was evaluated by stably expressing LMP2A in an EBV-negative Burkitt tumor cell line (BJAB) or in LMP1-converted BJAB cells. LMP2A significantly blocked calcium mobilization following class II, CD19, or immunoglobulin M cross-linking. LMP2A effects were partially reversed in LMP1-converted cell lines. These results are compatible with LMP2A acting in latent B-lymphocyte infection to downmodulate LMP1 effects on cell growth or to inhibit induction of lytic EBV infection in specific human tissues following receptor ligation.
Repression of endogenous p53 transactivation function in HeLa cervical carcinoma cells by human papillomavirus type 16 E6, human mdm-2, and mutant p53
Authors: Hoppe-Seyler F , Butz K
Published: January 2020

Somatic mutations in the p53 tumor suppressor gene represent the single most common genetic alteration observed in human cancers. Interestingly, the great majority of malignant tumors of the cervix uteri contain wild-type p53 alleles together with the DNA of specific types of human papillomaviruses (HPVs), while the small portion of HPV-negative cervical carcinomas often carry alterations in the p53 tumor suppressor gene. Transcriptional activation of yet-undefined cellular regulatory genes has been implicated to play a key role for the tumor-suppressive activity of wild-type p53, as mutant p53 in general has lost the activity to stimulate p53-responsive reporter plasmids. The detection of DNA-binding-competent and transcriptionally active p53 protein in HeLa cervical carcinoma cells enabled us to investigate the in vivo effects of putative modulators on endogenous p53 function in cervical cancer cells. We show that the transcriptional stimulatory activity of HeLa cell p53 is strongly repressed by overexpression of E6 protein from oncogenic HPV type 16 (HPV16) but is not influenced by low-risk HPV6 E6. Similar to HPV16 E6, cellular oncoproteins such as mutant p53 or the product of the human mdm-2 gene also negatively interfere with p53-mediated transactivation in HeLa cells. Our findings indicate that, within a cervical cancer cell, the expression of E6 protein from high-risk HPV16, but not from low-risk HPV6, can lead to the same functional consequences as a mutation of the p53 gene. These results could provide a biochemical basis for the inverse correlation between the presence of HPV sequences and somatic mutations of the p53 gene in cervical carcinomas.
Recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas
Authors: Sheets R L , Pandey R , Jen W C , Roy-Burman P
Published: January 2020

Using a polymerase chain reaction strategy aimed at detecting recombinant feline leukemia virus (FeLV) genomes with 5' env sequences originating from an endogenous source and 3' env sequences resulting from FeLV subgroup A (FeLV-A), we detected recombinant proviruses in approximately three-fourths of naturally occurring thymic and alimentary feline lymphosarcomas (LSAs) and one-third of the multicentric LSAs from cats determined to be FeLV capsid antigen positive by immunofluorescence assay. In contrast, only 1 of 22 naturally arising FeLV-negative feline LSAs contained recombinant proviruses, and no recombinant env gene was detected in seven samples from normal tissues or tissues from FeLV-positive animals that died from other diseases. Four preferred structural motifs were identified in the recombinants; one is FeLV-B like (recognizing that FeLV-B itself is a product of recombination between FeLV-A and endogenous env genes), and three contain variable amounts of endogenous-like env gene before crossing over to FeLV-A-related sequences: (i) a combination of full-length and deleted env genes with recombination at sites in the middle of the surface glycoprotein (SU), (ii) the entire SU encoded by endogenous-like sequences, and (iii) the entire SU and approximately half of the transmembrane protein encoded by endogenous-like sequences. Additionally, three of the thymic tumors contained recombinant proviruses with mutations in the vicinity of the major neutralizing determinant for the SU protein. These molecular genetic analyses of the LSA DNAs correspond to our previous results in vitro and support the occurrence and association of viral recombinants and mutants in vivo in FeLV-induced leukemogenesis.
Analysis of the BZLF1 promoter of Epstein-Barr virus: identification of an anti-immunoglobulin response sequence
Authors: Shimizu N , Takada K
Published: January 2020

The induction of the viral lytic cycle in latently Epstein-Barr virus (EBV)-infected B cells is initiated by activation of the BZLF1 gene, whose expression is sufficient to disrupt EBV latency, suggesting that BZLF1 acts as the switch to change from a latent to a lytic replicative cycle. In the present studies, a series of deletion plasmids encompassing positions bp -552 to +12 of the BZLF1 promoter were constructed and tested for their response to anti-immunoglobulin (anti-Ig), an inducer of the viral lytic cycle, upon transfection into EBV-negative and -positive lymphoid cells. The promoter consisted of three functionally distinct regions. Region I (bp -552 to -221) had a negative influence on promoter activity; its deletion made the promoter highly responsive to anti-Ig. Region II (bp -203 to -177) was important for conferring responsiveness to anti-Ig. The response to anti-Ig did not require the presence of the EBV genome or EBV gene products. This sequence also enhanced expression of the chloramphenicol acetyltransferase (cat) gene from the simian virus 40 promoter in response to anti-Ig, even when inserted downstream of the cat gene. Region III (-134 to -116) was a positive element that was transactivated by the BZLF1 gene product.
Herpetic stromal keratitis in the reconstituted scid mouse model
Authors: Mercadal C M , Bouley D M , DeStephano D , Rouse B T
Published: January 2020

Infections of the cornea with herpes simplex virus type 1 cause inflammatory lesions which frequently lead to blindness. The disease is suspected to be immunopathological in nature. To establish this point and to study possible mechanisms involved, corneal infections in C.B-17 scid/scid and cell-reconstituted scid mice were investigated. Whereas unreconstituted scid mice failed to develop herpetic stromal keratitis (HSK) and died of encephalitis, mice reconstituted with T lymphocytes generated severe lesions. T cells of the CD4+ subset were found to be essential mediators of the HSK lesion, while T cells of the CD8+ subset protected mice from lethality. The results confirm that HSK is an immunopathological disease and that scid mice provide a convenient model that should prove valuable in establishing the biochemical mechanisms by which HSK is mediated.
Hepatitis B virus differentially suppresses myelopoiesis and displays tropism for immature hematopoietic cells
Authors: Sing G K , Prior S , Fernan A , Cooksley G
Published: January 2020

The hematopoietic cell lines HL-60 and THP-1 were challenged with hepatitis B virus (HBV) in vitro to study interactions between the virus and host cell. Exposure to HBV suppressed the ability of HL-60 cells to differentiate into granulocytes after treatment with retinoic acid (RA) or dimethyl sulfoxide (DMSO), and RA-induced activation of the monocytic cell line THP-1 was also suppressed. Terminal differentiation of both cell lines by phorbol 12-myristate 13-acetate (PMA) was not affected by HBV. The suppressive effect on RA- or DMSO-induced differentiation was unique to HBV, since cell exposure to human cytomegalovirus, another virus that inhibits hematopoiesis, failed to block cellular differentiation. At 5 days postinfection, extracellular viral DNA was detected in immature but not in differentiated cultures and higher levels of core antigen (HBcAg) and surface antigen (HBsAg) were seen in undifferentiated cells than in RA- or PMA-treated cells. In addition, release of HBsAg into the medium was 2 to 12 times greater in untreated cultures than for RA- or PMA-treated cells. Thus, HBV suppresses hematopoiesis by blocking the maturational development of progenitors and selectively infects immature myeloid cells compared with mature end-stage cells.
Selective interference with class I major histocompatibility complex presentation of the major immediate-early protein following infection with human cytomegalovirus
Authors: Gilbert M J , Riddell S R , Li C R , Greenberg P D
Published: January 2020

Responses of cytotoxic T-cells (Tc) to human cytomegalovirus (CMV) represent the predominant mechanism by which hosts resist CMV infection. The CMV major immediate-early protein (IE) is present throughout the virus replicative cycle. Studies were performed to determine whether Tc specific for IE effectively lyse CMV-infected targets and are thus capable of providing protective immunity against infection. After in vitro stimulation of peripheral blood mononuclear cells with CMV-infected autologous fibroblasts, Tc specific for IE were not readily detectable in CMV-reactive polyclonal Tc lines. However, after stimulation of peripheral blood mononuclear cells with cells selectively expressing IE, weak but detectable IE-specific Tc responses were observed. The frequency of IE-specific Tc clones derived from cultures stimulated with IE-expressing cells was 50 to 100 times lower than the frequency of Tc clones specific for other CMV proteins isolated from cultures stimulated with CMV-infected cells. All of the IE-specific Tc clones, which efficiently lysed targets selectively expressing IE, demonstrated minimal lysis of CMV-infected fibroblasts, despite abundant IE expression in these target cells. In contrast to these results with IE, other viral proteins were efficiently presented during all phases of CMV infection. These data suggest that CMV has evolved a unique mechanism for selectively limiting the presentation of the potentially immunogenic IE protein, which may preclude IE-specific Tc from providing protective immunity to CMV infection.
Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure
Authors: Pe'ery T , Mellits K H , Mathews M B
Published: January 2020

Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems. The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations. A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo. Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist. To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure. The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule. Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell. Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell.
A single amino acid substitution in the V1 loop of human immunodeficiency virus type 1 gp120 alters cellular tropism
Authors: Boyd M T , Simpson G R , Cann A J , Johnson M A , Weiss R A
Published: January 2020

Specific point mutations which affect viral tropism have been identified in both the V3 loop and in the CD4-binding region of the human immunodeficiency virus type 1 surface glycoprotein gp120. Here we report that a single point mutation in the first variable region (V1) of human immunodeficiency virus type 1 strain JRCSF is responsible for a change in viral tropism.
A monoclonal antibody to the CDR-3 region of CD4 inhibits soluble CD4 binding to virions of human immunodeficiency virus type 1
Authors: Moore J P
Published: January 2020

The CDR-3 region of CD4 has been proposed to be involved in the fusion reaction between human immunodeficiency virus type 1 (HIV-1) and CD4+ cells, either at a stage involving virus binding or subsequent to virus binding. Part of the evidence for this has been the observation that monoclonal antibodies (MAbs) to CDR-3 block HIV infection potently without strongly inhibiting the binding of monomeric gp120 to CD4. Here I show that, in a system using oligomeric, virion-bound gp120, a MAb to CDR-3 resembles those to CDR-2 in that it inhibits soluble CD4 binding to virions. Consequently, ternary complexes of MAb-soluble CD4-gp120 cannot be detected with CDR-2 MAbs and are detectable only at a very low level with a CDR-3 MAb, but they clearly form when a control MAb to CD4 domain 4 is used. Although not in direct conflict with previously published data on the role of CDR-3 MAbs in the inhibition of HIV-1 infection, these experiments do not support the hypothesis that the CDR-3 region is specifically involved in virus entry at a postbinding stage.