bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic and genotypic characteristics of human immunodeficiency virus type 1 from patients with AIDS in northern Thailand
Authors: Yu X F , Wang Z , Beyrer C , Celentano D D , Khamboonruang C , Allen E , Nelson K
Published: January 2020

Primary human immunodeficiency virus type 1 (HIV-1) isolates were obtained from 22 patients with AIDS from northern Thailand, where HIV-1 is transmitted primarily through the heterosexual route. Viral sequences were determined for the 22 patients with AIDS, and all were subtype E HIV-1 on the basis of sequence analysis of a region from the envelope protein gp120. Syncytium-inducing (SI) viruses were detected for 16 of 22 patients with AIDS by using MT-2 cells. Characteristics of amino acid sequences in V3 which have not been reported previously for subtype B SI HIV-1 were associated with the subtype E HIV-1 SI phenotype. The SI viruses from our study population contain predominantly a GPGR or GPGH motif at the tip of the V3 loop, in contrast to the previously described subtype E HIV-1 from Thailand which contained predominantly GPGQ. All the SI viruses lost a potential N-linked glycosylation site in V3 which is highly conserved among previously described subtype E HIV-1 isolates from asymptomatic patients from Thailand. HIV-1 envelope sequences including V3 from some patients with AIDS were significantly more divergent than viruses from asymptomatic patients in Thailand characterized 2 years ago or earlier. These results suggest that emergence of subtype E SI HIV-1 variants is associated with the development of AIDS, as it is for subtype B HIV-1. The divergence of subtype E HIV-1 in patients with AIDS as the disease progresses, and the divergence of subtype E HIV-1 in the infected population as the epidemic continues in Thailand, may have important implications for vaccine development.
Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains
Authors: Jones T R , Hanson L K , Sun L , Slater J S , Stenberg R M , Campbell A E
Published: January 2020

Reduction of major histocompatibility complex class I cell surface expression occurs in adenovirus-, herpes simplex virus-, human cytomegalovirus (HCMV)-, and murine cytomegalovirus-infected cell systems. Recently, it was demonstrated that the down-regulation mediated by HCMV infection is posttranslational, as a result of increased turnover of class I heavy chains in the endoplasmic reticulum (M. F. C. Beersma, M. J. E. Bijlmakers, and H. L. Ploegh, J. Immunol. 151:4455-4464, 1993; Y. Yamashita, K. Shimokata, S. Saga, S. Mizuno, T. Tsurumi, and Y. Nishiyama, J. Virol. 68:7933-7943, 1994. To identify HCMV genes involved in class I regulation, we screened our bank of HCMV deletion mutants for this phenotype. A mutant with a 9-kb deletion in the S component of the HCMV genome (including open reading frames IRS1 to US9 and US11) failed to down-regulate class I heavy chains. By examining the effects of smaller deletions within this portion of the HCMV genome, a 7-kb region containing at least nine open reading frames was shown to contain the genes required for reduction in heavy-chain expression. Furthermore, it was determined that at least two independent loci within the 7-kb region were able to cause class I heavy-chain down-regulation. One of these, US11, encodes a 32-kDa glycoprotein which causes down-regulation of class I heavy chains in the absence of other viral gene products. Hence, a specific function associated with a phenotype of the HCMV replicative cycle has been mapped to a dispensable gene region. These loci may be important for evasion of the host's immune response and viral persistence.
Picornavirus-specific CD4+ T lymphocytes possessing cytolytic activity confer protection in the absence of prophylactic antibodies
Authors: Neal Z C , Splitter G A
Published: January 2020

Picornaviruses are a family of positive-strand RNA viruses that are responsible for a variety of devastating human and animal diseases. An attenuated strain of mengovirus (vMC24) is serologically indistinguishable from the lethal murine wild-type mengovirus and encephalomyocarditis virus (EMCV). Immunogen-specific stimulation of vMC24-immune splenocytes in vitro demonstrates preferential activation of CD4+ lymphocytes. vMC24-immune splenocytes adoptively transferred to naive recipients conferred protection against lethal EMCV challenge. Immune splenocytes, expanded in vitro, were > 92% CD4+ T lymphocytes. Interestingly, adoptive transfer of these expanded cells engendered protection against lethal challenge. In vivo depletion of CD4+ T lymphocytes prior to lethal challenge abrogated survival of transfer recipients, confirming that CD4+ T lymphocytes were essential for protection. Subsequent rechallenge of vMC24-immune splenocyte recipients with a greater EMCV dose elicited serum neutralizing antibody titers paralleling the high titers observed in vMC24-immunized mice. Unexpectedly, an augmented humoral response was absent in vMC24-specific CD4+ T-cell recipients after the secondary challenge. Moreover, comparably low serum neutralizing antibody titers failed to protect passive transfer recipients when correspondingly challenged. vMC24-immune splenocytes expanded in vitro (> 94% CD4+) lysed vMC24-infected A20.J target cells. The ability to transfer protection with primed CD4+ T cells, in the absence of primed B lymphocytes or immune sera, is novel for picornaviral infections. Consequently, mechanisms such as CD4+ cytolytic T-lymphocyte activity are implicated in mediating protection.
Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine
Authors: Bouchard M J , Lam D H , Racaniello V R
Published: January 2020

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.
In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids
Authors: Lee C S , Guo P
Published: January 2020

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA bacteriophage phi 29 per ml were assembled in vitro, with 11 proteins derived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a purified recombinant procapsid with the aid of a small viral RNA (pRNA) transcript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled capsids were subsequently converted into infectious virions after the addition of four more recombinant proteins for neck and tail assembly. Electron microscopy and genome restriction mapping confirmed the identity of the infectious phi 29 virions synthesized in this system. A nonstructural protein, gp13, was indispensable for the assembly of infectious virions. The overproduced tail protein gp9 was present in solution in mostly dimeric form and was purified to homogeneity. The purified gp9 was biologically active for in vitro phi 29 assembly. Higher-order concentration dependence of in vitro phi 29 assembly on gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to earlier findings for phages T4 and lambda. The work described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 assembly and dissection of functional domains of structural components, enzymes, and pRNA (C.-S. Lee and P. Guo, Virology 202:1039-1042, 1995). Efficient packaging of foreign DNA in vitro and synthesis of viral particles from recombinant proteins facilitate the development of phi 29 as an in vivo gene delivery system. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 29 carrying a tail fused to ligands for specific receptor of human cells.
Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase
Authors: Mansky L M , Temin H M
Published: January 2020

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.
Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene
Authors: Reddy T R , Kraus G , Suhasini M , Leavitt M C , Wong-Staal F
Published: January 2020

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.
Effect of amino acid substitutions on calmodulin binding and cytolytic properties of the LLP-1 peptide segment of human immunodeficiency virus type 1 transmembrane protein
Authors: Tencza S B , Miller M A , Islam K , Mietzner T A , Montelaro R C
Published: January 2020

Previous studies have identified two highly basic amphipathic helical regions in the human immunodeficiency virus type 1 transmembrane protein that, in vitro, display both cytolytic and calmodulin-binding and -inhibitory properties that could contribute to cellular dysfunctions and cytopathogenesis during a persistent viral infection. In the current study, the structural specificity of the cytolytic and calmodulin-binding activities of the human immunodeficiency virus type 1 lentivirus lytic peptide (LLP-1) are examined with synthetic peptide homologs and analogs. The results of these studies demonstrate that even minor changes in LLP-1 amino acid content can markedly affect these properties, suggesting that sequence variation in these highly conserved LLP sequences may correlate with alterations in viral cytopathic properties.
Involvement of a spliced and defective human foamy virus in the establishment of chronic infection
Authors: Saïb A , Koken M H , van der Spek P , Périès J , de Thé H
Published: January 2020

Human foamy retrovirus (HFV) is found as two proviruses (HFV and delta HFV) which differ by a splice-induced deletion within the bel1 transactivator gene. The defective delta HFV (which lacks a functional Bel1 but harbors an intronless bet gene) is predominantly found in nonlytic infections in vitro as well as in vivo. Here, we show that infection of cell lines stably transduced by delta HFV DNA with the highly lytic HFV leads to chronic infections characterized by an absence of lysis, a balanced ratio of HFV to delta HFV, and a persistent Bet expression accompanied by a shutoff of structural genes. While this system only partially reflects the natural situation, in which target cells are infected by HFV and delta HFV simultaneously, it strongly suggests that delta HFV is a defective interfering retrovirus. Accordingly, previous or concomitant exposure to delta HFV viruses greatly enhances the formation of lysis-resistant clones in culture after HFV infection. The inability of delta HFV proviruses encoding a mutated bet gene to induce chronic infection suggests a role for Bet in this process. Through a specific, splice-induced, genomic deletion, resulting in a switch from Bel1 to Bet expression, the lytic properties of HFV are progressively lost. Such programmed inactivation of a key gene represents a new regulatory mechanism of gene expression in retroviruses.
Regulation of human cytomegalovirus US3 gene transcription by a cis-repressive sequence
Authors: Biegalke B J
Published: January 2020

Regulation of immediate-early gene expression in human cytomegalovirus is subject to complex controls. The major immediate-early (mIE) gene is regulated by both positive and negative regulatory signals, including autoregulation mediated by a cis-repressive sequence. A second immediate-early gene, the US3 gene, is transcribed with kinetic similar to those of the mIE gene. I have identified an element present in the US3 gene located from -1 to -13 (relative to the start site of transcription) that mediates a decrease in US3 transcription. The US3 element resembles the cis-repressive element of the mIE gene in sequence, position, and function. The common theme of negative regulation of immediate-early genes shortly after infection suggests that a decrease in the level of immediate-early proteins may be critical for viral replication.
Herpes simplex virus glycoprotein K promotes egress of virus particles
Authors: Hutchinson L , Johnson D C
Published: January 2020

Herpes simplex virus (HSV) glycoprotein K (gK) is thought to be intimately involved in the process by which infected cells fuse because HSV syncytial mutations frequently alter the gK (UL53) gene. Previously, we characterized gK produced in cells infected with wild-type HSV or syncytial HSV mutants and found that the glycoprotein was localized to nuclear and endoplasmic reticulum membranes and did not reach the cell surface (L. Hutchinson, C. Roop, and D. C. Johnson, J. Virol. 69:4556-4563, 1995). In this study, we have characterized a mutant HSV type 1, denoted F-gK beta, in which a lacZ gene cassette was inserted into the gK coding sequences. Since gK was found to be essential for virus replication, F-gK beta was propagated on complementing cells which can express gK. F-gK beta produced normal plaques bounded by nonfused cells when plated on complementing cells, although syncytia were observed when the cells produced smaller amounts of gK. In contrast, F-gK beta produced only microscopic plaques on Vero cells and normal human fibroblasts (which do not express gK) and these plaques were reduced by 10(2) to 10(6) in number. Further, large numbers of nonenveloped capsids accumulated in the cytoplasm of F-gK beta-infected Vero cells, virus particles did not reach the cell surface, and the few enveloped particles that were produced exhibited a reduced capacity to enter cells and initiate an infection of complementing cells. Overexpression of gK in HSV-infected cells also caused defects in virus egress, although particles accumulated in the perinuclear space and large multilamellar membranous structures juxtaposed with the nuclear envelope were observed. Together, these results demonstrate that gK regulates or facilitates egress of HSV from cells. How this property is connected to cell fusion is not clear. In this regard, gK may alter cell surface transport of viral particles or other viral components directly involved in the fusion process.
Cytotoxic T cells are elicited during acute infection of mice with lactate dehydrogenase-elevating virus but disappear during the chronic phase of infection
Authors: Even C , Rowland R R , Plagemann P G
Published: January 2020

Lactate dehydrogenase-elevating virus (LDV) invariably establishes a life-long viremic infection in mice, which is maintained by replication of LDV in a renewable subpopulation of macrophages and escape from all host immune responses. We now demonstrate that cytotoxic T lymphocytes (CTLs) that specifically lyse LDV-infected macrophages and 3T3 cells producing the nucleocapsid protein of LDV were elicited in Swiss, B10.A, and (Swiss x B10.A)F1 mice. To detect target cell lysis, splenocytes needed to be expanded by a 5-day in vitro culture in the presence of recombinant interleukin 2 and syngeneic LDV protein-expressing cells. In vitro culture resulted in the specific expansion of CD8+ cells which mediated the lysis of target cells in a major histocompatibility complex class I-restricted manner. When CTLs were added to macrophage cultures at 1 h after infection with LDV, the lysis of the infected macrophages by the CTLs started about 5 h postinfection (p.i.) and, at an effector cell/target cell ratio of 25:1, resulted in the lysis of all LDV-infected macrophages in a culture by about 7 h p.i. However, lysis of the LDV replication in a culture was not rapid enough to significantly suppress the LDV yield in the culture. LDV replication in mice was also little affected by the presence of CTLs which were induced by immunization with 3T3 cells expressing the LDV nucleocapsid protein. Furthermore, all CTL precursor cells in infected mice had disappeared by 30 days p.i. Loss of CTL precursor cells in infected mice probably reflected high-dose clonal exhaustion, since LDV infection of a mouse results in massive production of LDV in all tissues of the mouse, but especially in lymphoidal tissues, and accumulation of LDV in newly formed germinal centers. Furthermore, slow LDV replication continues in the thymus and other lymphoidal organs.
Functional interactions between herpes simplex virus immediate-early proteins during infection: gene expression as a consequence of ICP27 and different domains of ICP4
Authors: Samaniego L A , Webb A L , DeLuca N A
Published: January 2020

Two of the five immediate-early gene products, ICP4 and ICP27, expressed by herpes simplex virus type 1 have profound effects on viral gene expression and are absolutely essential for virus replication. Functional interactions between ICP4 and ICP27 may contribute to establishing the program of viral gene expression that ensues during lytic infection. To evaluate this possibility, viral mutants simultaneously deleted for ICP27 and defined functional domains of ICP4 were constructed. These mutant viruses allowed a comparison of gene expression as a function of different domains of ICP4 in the presence and absence of ICP27. Gene expression in the absence of both ICP4 and ICP27 was also examined. The results of this study demonstrate a clear involvement for ICP27 in the induction of early genes, in addition to its known role in enhancing late gene expression during viral infection. In the absence of both ICP4 and ICP27, viral early gene expression, as measured by the accumulation of thymidine kinase and ICP6 messages was dramatically reduced relative to the amounts of these messages seen in the absence of only ICP4. Therefore, elevated levels of early gene expression as a consequence of ICP27 occurred in the absence of any ICP4 activity. Evidence is also presented regarding the modulation of the ICP4 repression function by ICP27. When synthesized in the absence of ICP27, a mutant ICP4 protein was impaired in its ability to repress transcription from the L/ST promoter in the context of viral infection and in vitro. This defect correlated with the loss of the ability of this mutant protein to bind to its recognition sequence when produced in infected cells in the absence of ICP27. These observations indicate that ICP27 can regulate the activity of at least one domain of the ICP4 protein as well as contribute to elevated early gene expression independently of ICP4.
Receptor binding site-deleted foot-and-mouth disease (FMD) virus protects cattle from FMD
Authors: McKenna T S , Lubroth J , Rieder E , Baxt B , Mason P W
Published: January 2020

Binding of foot-and-mouth disease virus (FMDV) to cells requires an arginine-glycine-aspartic acid (RGD) sequence in the capsid protein VP1. We have genetically engineered an FMDV in which these three amino acids have been deleted, producing a virus particle which is unable to bind to cells. Cattle vaccinated with these receptor binding site-deleted virions were protected from disease when challenged with a virulent virus, demonstrating that these RGD-deleted viruses could serve as the basis for foot-and-mouth disease vaccines safer than those currently in use. This strategy may prove useful in the development of vaccines for other viral diseases.
Characterization of diverse primary herpes simplex virus type 1 gB-specific cytotoxic T-cell response showing a preferential V beta bias
Authors: Cose S C , Kelly J M , Carbone F R
Published: January 2020

The glycoprotein B (gB) from herpes simplex virus type I is a major target of cytotoxic T lymphocytes (CTL) in C57BL/6 mice. The majority of these T cells are directed to a single Kb-restricted determinant, gB498-505. We have analyzed the T-cell receptor (TCR) usage in gB-specific CTL lines derived shortly after virus infection. The CTL populations preferentially used two V beta regions, a dominant V beta 10 element and a subdominant V beta 8 element. Detailed sequence analysis revealed considerable TCR beta-chain heterogeneity despite a striking level of predicted amino acid conservation at the V beta-D beta junction. This junction forms part of the third hypervariable loop of the TCR thought to directly contact the major histocompatibility complex-bound antigenic peptide. The results reveal considerable diversity within the primary T cells responding to a single viral determinant while still maintaining a high degree of TCR V beta bias and sequence conservation at the V-D-J junction.
Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells
Authors: Giorno Ralph , Kates J. R.
Published: January 2020

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.
Deoxyribonucleic Acid Polymerase Activity Associated with a Plasma Particulate Fraction from Patients with Chronic Lymphocytic Leukemia
Authors: Kiessling A. A. , Weber G. H. , Deeney A. O. , Possehl E. A. , Beaudreau G. S.
Published: January 2020

Plasma from two cases of chronic lymphocytic leukemia have been examined for deoxyribonucleic acid (DNA) polymerase activity. In both cases, detectable enzyme activity was present. In the plasma from a patient known to have chronic lymphocytic leukemia for 10 years, the enzyme activity was sufficiently high to study product formation, buoyant density of the enzyme activity, deoxyribonucleoside triphosphate requirements, and kinetics of DNA synthesis. These studies are presented in this report.
Structural Proteins of Rabies Virus
Authors: Sokol Frantisek , Stanček Daniel , Koprowski Hilary
Published: January 2020

Purified rabies virions, unlabeled or labeled with radioactive amino acids or
d
-glucosamine, were dissociated into their polypeptides by treatment with sodium dodecyl sulfate in a reducing environment and fractionated by electroiphoresis in sodium dodecyl sulfate-containing polyacrylamide gel. The molecular weights of individual polypeptides were estimated by comparison of their rate of migration with that of protein markers of known molecular weight. Purified viral nucleocapsid and a mixture of envelope components, isolated from virions disrupted by sodium deoxycholate, were analyzed by the same procedure. The number of molecules per virion of each polypeptide was estimated from the proportions of the separated components, the known molecular weight of the viral ribonucleic acid, and the chemical composition of the nucleocapsid. The protein moiety of the nucleocapsid particle was estimated to consist of 1,713 molecules of a major polypeptide (molecular weight, 62,000 daltons) and 76 molecules of a minor polypeptide (molecular weight, 55,000 daltons). In addition to 1,783 molecules of a glycoprotein component (molecular weight, 80,000 daltons), the viral envelope contains 789 and 1,661 molecules, respectively, of two other polypeptides (molecular weight, 40,000 and 25,000 daltons).
Photodynamic Action of Proflavine on Coliphage T3 I. Kinetics of Inactivation
Authors: Witmer Heman , Fraser Dean
Published: January 2020

The photodynamic inactivation of coliphage T3 was studied over a wide range of concentrations of the dye proflavine. With 2 × 10
7
phage/ml, two modes of inactivation were observed. Between 0.25 and 12 to 13 μg/ml, inactivation was biphasic. There was an initial first-order inactivation (R×1) which became temporally associated with an apparently multiorder process (R×2) at higher light doses. Dye concentrations above 12 to 13 μg/ml showed only two-target inactivation curves (R×3), except at high dye concentrations where processes kinetically identical to R×1 and R×2 reappeared. R×2 showed a normal rectangular hyperbolic saturation curve but R×1 and R×3 appeared to saturate prematurely. The saturation behavior of R×1 and R×2 was independent of phage concentration, but R×3 was lost at phage titers above 2 × 10
7
/ml. No dark inactivation was seen with R×1 and R×2 subsequent to a period of illumination. With R×3, an exponential dark inactivation was seen for at least 1 hr after a period of illumination. The dye-phage system equilibrated immediately, at any temperature, at proflavine concentrations where R×1 and R×2 occurred. With R×3, prolonged equilibration times were necessary. Moreover, there was a temperature effect. The rate of inactivation at equilibrium was temperature-dependent, whereas the initial rate at which equilibrium was approached was essentially temperature-independent.
Photodynamic Action of Proflavine on Coliphage T3 III. Damages to the Deoxyribonucleic Acid Associated with R×1 and R×2
Authors: Witmer Heman , Fraser Dean
Published: January 2020

Ultracentrifugational studies with deoxyribonucleic acid (DNA) extracted from phage exposed to light in the presence of either 0.25 or 8.5 μg of proflavine per ml reveal that the lethal damage of R×1 renders DNA alkali-labile, with lethality resulting from damage that occurs singly on either strand or simultaneously on both strands. Apparently nonlethal damages temporally associated with R×1 consist of (i) heat and alkali-labile cross-links (which produce undenatured DNA that migrates at 45
S
) and (ii) heat-labile bonds. The formation of 45
S
material is a linear function of light dose, and the production of this material ceases when R×2 appears at the higher dye concentration. No tendency to plateau is seen at 0.25 μg of dye per ml. The nature of the lethal damage of R×2 could not be determined. Damages that were temporally associated with R×2 at 8.5 μg of dye per ml were heat-labile, alkali-stable cross-links (undenatured DNA, 38 to 41
S
; alkali-denatured-reneutralized DNA, 85 to 89
S
) and some double-stranded breaks. No such changes were seen at 0.25 μg of dye per ml.
Structure and Biological Activity of Deoxyribonucleic Acid from
Bacillus
Bacteriophage φ105: Effects of
Escherichia coli
Exonucleases
Authors: Armentrout Richard W. , Skoog Lambert , Rutberg Lars
Published: January 2020

The effects of
Escherichia coli
exonuclease I, exonuclease III, and deoxyribonucleic acid (DNA) polymerase on the biological activity of mature DNA from temperate
Bacillus
bacteriophage φ105 were investigated. Intact DNA loses infectivity rapidly upon exposure to exonuclease III. Although there is an overall decrease in marker rescue from exonuclease III-digested DNA, digestion preferentially affects markers at the end of the genetic map. This is taken to indicate a nonpermuted gene sequence in mature DNA. Incubation of mature DNA in the presence of exonuclease I or DNA polymerase has no effect on its biological activity. The possible structure of the ends of mature φ105 DNA is discussed. The rate of digestion of mature φ105 DNA by exonuclease III is only about 1/20 the rate of λ DNA. Results of digestion of various DNA substrates by exonuclease III indicate that the enzyme distinguishes between different DNA terminal structures.
Effect of Myxin on Deoxyribonucleic Acid Synthesis in
Escherichia coli
Infected with T4 Bacteriophage
Authors: Behki R. M. , Lesley S. M.
Published: January 2020

Exposure of
Escherichia coli
cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.
Complete Transcription by the Transcriptase of Vesicular Stomatitis Virus
Authors: Bishop D. H. L.
Published: January 2020

Vesicular stomatitis virus (VSV) transcriptase completely transcribes the VSV genome into messenger-size ribonucleic acid (RNA) species. The replicating complexes of template and product RNA, generated in vitro by VSV transcriptase, exhibit a mass parity between product and template molecules. Purified product RNA, after annealing to small amounts of viral RNA, rendered 92% of the viral RNA ribonuclease-resistant. It is calculated that, under optimal conditions, 60 min is required before the VSV genome is completely transcribed.
Variation of 6-Methylaminopurine Content in Bacteriophage P22 Deoxyribonucleic Acid as a Function of Host Specificity
Authors: Hattman Stanley
Published: January 2020

The 6-methylaminopurine (MAP) content of P22 deoxyribonucleic acid has been analyzed as a function of the host specificity it carries. A 40 to 50% reduction in MAP level occurs as a result of growth in host cells defective in the ability to confer LT specificity.
Synergistic Effect of Herpes Simplex Virus and Cytosine Arabinoside on Human Chromosomes
Authors: O'Neill F. J. , Rapp F.
Published: January 2020

The combined treatment of cultures of human embryonic lung cells with herpes simplex virus type 2 and cytosine arabinoside produced a significantly increased number of cells containing multiple chromatid and chromosome breaks. The incidence of such cells was found to be approximately two and one half times greater than the additive effects of virus and cytosine arabinoside induced separately and is therefore synergistic.
Three Size-Classes of Intracellular Adenovirus Deoxyribonucleic Acid
Authors: Burlingham B. T. , Doerfler W.
Published: January 2020

When human adenovirus type 2 or 12 infects cells, either productively or non-productively, three classes of viral deoxyribonucleic acid (DNA) are found within the cells: (i) viral DNA which cosediments with DNA extracted from infectious adenovirions at 31.3
S
for adenovirus type 2 and at 29.0
S
for adenovirus type 12, (ii) viral DNA which sediments at about 18
S
, and (iii) viral DNA which sediments at >45
S
and is apparently integrated into the cellular DNA. A precursor-product relationship is suggested as a working hypothesis; the intact viral DNA is hydrolyzed to slowly sedimenting DNA and the slowly sedimenting DNA is integrated into the cellular DNA. Both the parental and the newly synthesized viral DNA are altered by this route. The intact viral DNA within the cells apparently is cleaved into the slowly sedimenting DNA by a preformed enzyme.
Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease
Authors: Shimizu Y , Yamaji K , Masuho Y , Yokota T , Inoue H , Sudo K , Satoh S , Shimotohno K
Published: January 2020

In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.
A conserved LXXLF sequence is the major determinant in p6gag required for the incorporation of human immunodeficiency virus type 1 Vpr
Authors: Kondo E , Göttlinger H G
Published: January 2020

The vpr gene product of human immunodeficiency virus type (HIV-1) is a virion-associated regulatory protein. A transferable virion association motif for Vpr is located in the p6 domain of the HIV-1 Gag polyprotein. To map the sequences in p6 that are involved in Vpr incorporation, we analyzed the ability of mutant forms of p6 to direct the incorporation of Vpr into chimeric viral particles. Our results show that the determinants which govern Vpr incorporation are largely confined to a C-terminal region of the p6 domain. Within this region, three hydrophobic residues in a highly conserved sequence motif (L-X-S-L-F-G) are absolutely required. Remarkably, the transfer of the conserved motif and of a single flanking residue to a heterologous Gag polyprotein was sufficient to transfer the ability to incorporate Vpr at moderate levels. The transfer of residues 32 to 46 of p6 led to Vpr incorporation levels that were comparable to those obtained with full-length HIV-1 Gag protein, indicating that this region contains essentially all the information required for efficient Vpr incorporation.
Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein
Authors: Watanabe K , Handa H , Mizumoto K , Nagata K
Published: January 2020

Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.
The A2L intermediate gene product is required for in vitro transcription from a vaccinia virus late promoter
Authors: Hubbs A E , Wright C F
Published: January 2020

Previously, the in vitro late transcription system of vaccinia virus was resolved into four components: the 17- and 30-kDa products of the A1L and G8R intermediate genes, respectively, the viral DNA-dependent RNA polymerase, and an unmapped factor sedimenting at 32 to 38 kDa. Another protein, the 26-kDa product of the A2L open reading frame was predicted to be a late transcription factor on the basis of a transient-expression assay but was not recognized as being necessary for transcriptional activity in vitro. We now report that both the unmapped factor and the 26-kDa protein are required for transcription from a vaccinia virus late promoter in vitro. Since the 26-kDa protein has now been shown to be a trans-activator of late transcription and it is the product of a known gene, we suggest that it be designated VLTF-3.
Generation and role of defective proviruses in cytopathic feline leukemia virus (FeLV-FAIDS) infections
Authors: de Noronha C M , Reinhart T A , Mullins J I
Published: January 2020

Cytopathic feline leukemia virus (FeLV) infections of feline T-cell line (FeT-cell) cultures led to the accumulation and maintenance of threefold more proviruses with deletions within the polymerase gene (pol) than minimally cytopathic FeLV infections. Over 60% of the viral DNA from cytopathic infections bore deletions in pol. Characterization of DNA sequences adjoining the deletions revealed that the junctions were most often flanked by RNA splice donor and acceptor consensus motifs. A thymidine-to-cytidine mutation introduced at the +2 position of one RNA splice donor-like motif inhibited formation of the two most prevalent viral DNA species with deletions, confirming the origin of many proviruses with deletions from reverse transcription of aberrantly spliced viral RNA species. An example of deletion by misalignment was also characterized. Viral inocula obtained from cells recovered after cytopathic infections were attenuated in their ability to cause cytopathic effects (CPE) and were able to confer superinfection resistance to naïve FeT-cells, despite maintaining envelope gene (env) sequences with full cytopathic potential. This suggested that viral genomes with deletions, rather than being required for cytopathicity, play a role in protecting cells from CPE. Indeed, expression of a molecularly cloned provirus bearing one of the characterized deletions attenuated CPE in FeT-cells caused by superinfecting cytopathic virus.
Differing T-cell requirements for recombinant retrovirus vaccines
Authors: Hasenkrug K J , Brooks D M , Nishio J , Chesebro B
Published: January 2020

Friend murine leukemia virus is a retrovirus complex that induces rapid erythroleukemia and immunosuppression in susceptible strains of adult mice. Using this model, we directly examined the T-cell subsets required for a protective retrovirus vaccine. Paradoxically, recovery in mice immunized with a chimeric envelope containing only T-helper (TH) and B-cell epitopes was dependent on CD8+ T cells as well as CD4+ T cells despite the fact that the vaccine contained no CD8+ cytolytic T-lymphocyte (CTL) epitopes. However, the requirement for CD8+ T cells was overcome by inclusion of additional TH and B-cell epitopes in the immunizing protein. These additional epitopes primed for more rapid production of virus-neutralizing antibody which appeared to limit virus spread sufficiently to protect even in the absence of CD8+ T cells. Inclusion of an immunodominant CTL epitope in the vaccine was not sufficient to overcome dependence on CD4+ T cells. These data suggest that TH priming is more critical for retrovirus immunity than CTL priming.
Primary characterization of a herpesvirus agent associated with Kaposi's sarcomae
Authors: Moore P S , Gao S J , Dominguez G , Cesarman E , Lungu O , Knowles D M , Garber R , Pellett P E , McGeoch D J , Chang Y
Published: January 2020

Detection of novel DNA sequences in Kaposi's sarcoma (KS) and AIDS-related body cavity-based, non-Hodgkin's lymphomas suggests that these neoplasms are caused by a previously unidentified human herpesvirus. We have characterized this agent using a continuously infected B-lymphocyte cell line derived from an AIDS-related lymphoma and a genomic library made from a KS lesion. In this cell line, the agent has a large episomal genome with an electrophoretic mobility similar to that of 270-kb linear DNA markers during clamped homogeneous electric field gel electrophoresis. A 20.7-kb region of the genome has been completely sequenced, and within this region, 17 partial and complete open reading frames are present; all except one have sequence and positional homology to known gammaherpesvirus genes, including the major capsid protein and thymidine kinase genes. Phylogenetic analyses using both single genes and combined gene sets demonstrated that the agent is a gamma-2 herpesvirus (genus Rhadinovirus) and is the first member of this genus known to infect humans. Evidence for transient viral transmission from infected to uninfected cells is presented, but replication-competent virions have not been identified in infected cell lines. Sera from patients with KS have specific antibodies directed against antigens of infected cell lines, and these antibodies are generally absent in sera from patients with AIDS without KS. These studies define the agent as a new human herpesvirus provisionally assigned the descriptive name KS-associated herpesvirus; its formal designation is likely to be human herpesvirus 8.
Induction of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr virus glycoproteins gp350 and gp220
Authors: Tanner J E , Alfieri C , Chatila T A , Diaz-Mitoma F
Published: January 2020

The cellular receptor for Epstein-Barr virus (EBV) is the type 2 complement receptor, CD21. At initial infection, EBV virion glycoproteins gp350 and gp220 bind to CD21. We report here that the cross-linking of CD21 by gp350/220 results in increased amounts of interleukin 6 (IL-6) RNA and IL-6 protein. This effect could be blocked with anti-gp350/220 and anti-CD21 monoclonal antibodies. Induction of IL-6 in B cells by EBV could be mimicked by treatment with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate but not with the calcium ionophore ionomycin. IL-6 induction by EBV was inhibited with the PKC-specific inhibitor bisindolylmaleimide or the protein tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and herbimycin A, indicating that the induction of IL-6 following CD21 cross-linking is mediated through PKC- and protein tyrosine kinase-dependent pathways.