bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene
Authors: Ward R L , Mason B B , Bernstein D I , Sander D S , Smith V E , Zandle G A , Rappaport R S
Published: January 2020

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.
Translation elongation factor-1 alpha interacts with the 3' stem-loop region of West Nile virus genomic RNA
Authors: Blackwell J L , Brinton M A
Published: January 2020

The conserved 3'-terminal stem-loop (3' SL) of the West Nile virus (WNV) genomic RNA was previously used to probe for cellular proteins that may be involved in flavivirus replication and three cellular proteins were detected that specifically interact with the WNV 3' SL RNA (J. L. Blackwell and M. A. Brinton, J. Virol. 69:5650-5658, 1995). In this study, one of these cellular proteins was purified to apparent homogeneity by ammonium sulfate precipitation and liquid chromatography. Amino acid sequence Western blotting, and supershift analyses identified the cellular protein as translation elongation factor-1 alpha (EF-1 alpha). Competition gel mobility shift assays demonstrated that the interaction between EF-1 alpha and WNV 3' SL RNA was specific. Dephosphorylation of EF-1 alpha by calf intestinal alkaline phosphatase inhibited its binding to WNV 3' SL RNA. The apparent equilibrium dissociation constant for the interaction between EF-1 alpha and WNV 3' SL RNA was calculated to be 1.1 x 10(-9) M. Calculation of the stoichiometry of the interaction indicated that one molecule of EF-1 alpha binds to each molecule of WNV 3' SL RNA. Using RNase footprinting and nitrocellulose filter binding assays, we detected a high-activity binding site on the main stem of the WNV 3' SL RNA. Interaction with EF-1 alpha at the high-activity binding site was sequence specific, since nucleotide substitution in this region reduced the binding activity of the WNV 3' SL RNA for EF-1 alpha by approximately 60%. Two low-activity binding sites were also detected, and each accounted for approximately 15 to 20% of the binding activity. Intracellular association between the host protein and the viral RNA was suggested by coimmunoprecipitation of WNV genomic RNA and EF-1 alpha, using an anti-EF-1 alpha antibody.
Primary structure of the alcelaphine herpesvirus 1 genome
Authors: Ensser A , Pflanz R , Fleckenstein B
Published: January 2020

Alcelaphine herpesvirus 1 (AHV-1) causes wildebeest-associated malignant catarrhal fever, a lymphoproliferative syndrome in ungulate species other than the natural host. Based on biological properties and limited structural data, it has been classified as a member of the genus Rhadinovirus of the subfamily Gammaherpes-virinae. Here, we report on cloning and structural analysis of the complete genome of AHV-1 C500. The low GC content DNA (L-DNA) region of the genome consists of 130,608 bp with low (46.17%) GC content and marked suppression of CpG dinucleotide frequency. Like in herpesvirus saimiri, the prototype of the rhadinoviruses, the L-DNA is flanked by approximately 20 to 25 GC-rich (71.83%) high GC content DNA (H-DNA) repeats of 1,113 to 1,118 nucleotides. The analysis of the L-DNA sequence revealed 70 open reading frames (ORFs), 61 of which showed homology to other herpesviruses. The conserved ORFs are arranged in four blocks collinear to other Rhadinovirus genomes. These gene blocks are flanked by nonconserved regions containing ORFs without similarities to known herpesvirus genes. Notably, a spliced reading frame with a coding capacity for a 199-amino-acid protein is located in a position homologous to the transforming genes of herpesvirus saimiri at the left end of the L-DNA. A gene with homology to the semaphorin family is located adjacent to this. Despite common biological and epidemiological properties, AHV-1 differs significantly from herpesvirus saimiri with regard to cell homologous genes, probably using a different set of effector proteins to achieve a similar T-lymphocyte-transforming phenotype.
Activation of STAT transcription factors by herpesvirus Saimiri Tip-484 requires p56lck
Authors: Lund T C , Garcia R , Medveczky M M , Jove R , Medveczky P G
Published: January 2020

Signal transducers and activators of transcription (STATs) relay signals from activated cell surface receptors directly to the nucleus. Previously, a protein required for T-cell transformation by the DNA tumor virus herpesvirus saimiri (HVS) and designated tyrosine kinase interacting protein (Tip-484) was shown to interact with and dramatically upregulate the activity of p56lck. p56lck is a nonreceptor tyrosine kinase that is essential for signaling by the T-cell receptor and also interacts with the CD4, CD8, and interleukin-2 receptors. The present data show activation of STAT1 and -3 by Tip-484. STAT1 and -3 were also found to complex with glutathione S-transferase-Tip-484 only in the presence of p56lck, and STAT3 was shown to be phosphorylated by the Tip-484-p56lck multiprotein complex in vitro. Infection of T cells with HVS or expression of recombinant Tip-484 significantly increased the DNA-binding activity of the STAT1 and STAT3 transcription factors in nuclear extracts and also increased the phosphorylation of STAT3 in vivo. This is the first report of STAT activation by a DNA tumor virus protein. Moreover, these studies demonstrate that p56lck is required for STAT activation by Tip-484.
Binding of SP1 to the immediate-early protein-responsive element of the human cytomegalovirus DNA polymerase promoter
Authors: Luu P , Flores O
Published: January 2020

Human cytomegalovirus (HCMV), a member of the herpesvirus family of DNA viruses, encodes two major immediate-early (IE) transcription factors, IE72 and IE86, that are important for regulated expression of the viral genome. The purpose of this study was to identify the host cellular components required for regulation of the HCMV DNA polymerase promoter (UL54) by HCMV IE proteins. Extensive mutagenesis defined a DNA element located between -54 and -43 relative to the transcription start site that was required for both basal transcriptional activity and transactivation by viral IE proteins. A single copy of the UL54 -54/-43 sequence enhanced the responsiveness of a heterologous minimal promoter to HCMV IE proteins. Fractionation of extracts prepared from uninfected cells led to the isolation of two cellular proteins with apparent molecular masses of 95 and 105 kDa that bound specifically to the UL54 -54/-43 element. Biochemical and immunochemical analyses identified this protein as the transcription factor SP1. Although initial inspection of the UL54 -54/-43 sequence did not predict an SP1 binding site, subsequent analyses indicated that it is indeed a nonconsensus GC box. We propose that SP1 is required to direct basal levels of promoter activity and that SP1-regulated transcription complexes allow the entry of HCMV IE proteins into the transcription cycle.
A novel type of defective viral genome suggests a unique strategy to establish and maintain persistent lymphocytic choriomeningitis virus infections
Authors: Meyer B J , Southern P J
Published: January 2020

Defective interfering RNAs have long been thought to be a causal factor of persistent RNA virus infections. Here we describe a novel type of defective genome of lymphocytic choriomeningitis virus and the unique mechanism by which these RNAs appear to contribute to the establishment and maintenance of persistent infection. The defective genomes have short deletions in the untranslated regions at their termini and additional nontemplated terminal nucleotides. This and previous work from our laboratory suggested that the RNAs were competent for replication but not for transcription. From experiments using a technique to unambiguously determine the sequences of individual RNA termini, it appears that some truncated RNAs can be repaired. The data suggest that the loss or gain of nucleotides from the RNA termini during the course of infection is the mechanism for establishing and maintaining persistence.
Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles
Authors: Chiorini J A , Yang L , Liu Y , Safer B , Kotin R M
Published: January 2020

We have cloned and characterized the full-length genome of adeno-associated virus type 4 (AAV4). The genome of AAV4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to AAV2 and AAV3. Within the inverted terminal repeat (ITR), several base changes were identified with respect to AAV2. However, these changes did not affect the ability of this region to fold into a hairpin structure. Within the ITR, the terminal resolution site and Rep binding sites were conserved; however, the Rep binding site was expanded from three GAGC repeats to four. The Rep gene product of AAV4 shows greater than 90% homology to the Rep products of serotypes 2 and 3, with none of the changes occurring in regions which had previously been shown to affect the known functions of Rep68 or Rep78. Most of the differences in the capsid proteins lie in regions which are thought to be on the exterior surface of the viral capsid. It is these unique regions which are most likely to be responsible for the lack of cross-reacting antibodies and the altered tissue tropism compared to AAV2. The results of our studies, performed with a recombinant version of AAV4 carrying a lacZ reporter gene, suggest that AAV4 can transduce human, monkey, and rat cells. Furthermore, comparison of transduction efficiencies in a number of cell lines, competition cotransduction experiments, and the effect of trypsin on transduction efficiency all suggest that the cellular receptor for AAV4 is distinct from that of AAV2.
Human immunodeficiency virus matrix tyrosine phosphorylation: characterization of the kinase and its substrate requirements
Authors: Camaur D , Gallay P , Swingler S , Trono D
Published: January 2020

During virus assembly, a subset of human immunodeficiency virus (HIV) matrix (MA) molecules is phosphorylated on C-terminal tyrosine. This modification facilitates infection of nondividing cells by allowing for the recruitment of the karyophilic MA into the viral core and preintegration complex. MA tyrosine phosphorylation is accomplished by a cellular protein kinase which is incorporated into virions. In this study, we have investigated the nature of this enzyme as well as the determinants of MA necessary for its phosphorylation. Employing an in vitro kinase assay, we found that the MA tyrosine kinase activity is present in various cultured cell lines including CEM and SupT1 T-lymphoid cells, Namalwa B cells, 293 and CV-1 kidney fibroblasts, and P4 HeLa cells. In addition, it could be detected in platelets, macrophages, and activated peripheral blood lymphocytes (PBLs) but not in erythrocytes and resting PBLs isolated from human blood. Subcellular localization of the kinase activity by cell fractionation demonstrated that it is enriched in cellular membranes. In HIV type 2 (HIV-2) particles, the MA tyrosine kinase is associated with the inner leaflet of the viral membrane, while the tyrosine-phosphorylated MA is localized to the core. Individual mutations of each of the last eight residues immediately upstream of the C-terminal tyrosine (Y132) of HIV-1 MA did not prevent Y132 phosphorylation, suggesting that the kinase does not require a highly specific sequence adjacent to the C-terminal tyrosine. Confirming this, we found that the MA of murine leukemia virus, the sequence of which is only moderately homologous to that of HIV-1 and HIV-2 MA, is also C-terminally tyrosine phosphorylated.
Activation of gene expression by herpes simplex virus type 1 ICP0 occurs at the level of mRNA synthesis
Authors: Jordan R , Schaffer P A
Published: January 2020

ICP0 is a nuclear phosphoprotein involved in the activation of herpes simplex virus type 1 (HSV-1) gene expression during lytic infection and reactivation from viral latency. Although available evidence suggests that ICP0 acts at the level of transcription, definitive studies specifically addressing this issue have not been reported. In the present study we measured the ability of ICP0 to activate gene expression (i) from promoters representing the major kinetic classes of viral genes in transient expression assays and (ii) from the same promoters during viral infection at multiplicities of infection ranging from 0.1 to 5.0 PFU/cell. The levels of synthesis and steady-state accumulation of mRNA, mRNA stability, and levels of protein synthesis were compared in cells transfected with a reporter plasmid in the presence and absence of ICP0 and in cells infected with wild-type HSV-1 or an ICP0 null mutant, n212. In transient expression assays and during viral infection at all multiplicities tested, the levels of steady-state mRNA and protein were significantly lower in the absence of ICP0, indicating that ICP0 activates gene expression at the level of mRNA accumulation. In transient expression assays and during infection at low multiplicities (< 1 PFU/cell) in the presence or absence of ICP0, marked increases in the levels of viral mRNAs accompanied by proportional increases in the levels of protein synthesis were observed with increasing multiplicity. At a high multiplicity (5 PFU/cell) in the presence or absence of ICP0, mRNA levels did not increase as a function of multiplicity and changes in the levels of protein were no longer related to changes in the levels of mRNA. Collectively, these tests indicate that transcription of viral genes is rate limiting at low multiplicities and that translation is rate limiting at high multiplicities, independent of ICP0. Consistent with the lower levels of mRNA detected in the absence of ICP0, the rates of transcription initiation measured by nuclear run-on assays were uniformly lower in cells infected with the ICP0 null mutant at all multiplicities tested, implying that ICP0 enhances transcription at or before initiation or both. No evidence was found of posttranscriptional effects of ICP0 (i.e., effects on the stability of mRNA, nuclear-cytoplasmic distribution, polyribosomal mRNA distribution, or rates of protein synthesis). Taken together, these results suggest that ICP0 activates gene expression prior to or at the level of initiation of mRNA synthesis in transient expression assays and during viral infection. Based on these findings; we hypothesize that the exaggerated multiplicity-dependent growth phenotype characteristic of ICP0 null mutants reflects the requirement for ICP0 under conditions where the steady-state level of mRNA is rate limiting, such as during low-multiplicity infection and reactivation from latency.
Interferon-independent and -induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma
Authors: Nonkwelo C , Ruf I K , Sample J
Published: January 2020

Replication of the Epstein-Barr virus (EBV) genome within latently infected cells is dependent on the EBV EBNA-1 protein. The objective of this study was to identify transcriptional regulatory proteins that mediate EBNA-1 expression via the viral promoter Qp, which is active in EBV-associated tumors such as Burkitt lymphoma and nasopharyngeal carcinoma. Results of a yeast one-hybrid screen suggested that a subset of the interferon regulatory factor (IRF) family may regulate EBNA-1 transcription by targeting an essential cis-regulatory element of Qp, QRE-2. Further investigation indicated that the transcriptional activator IRF-1 and the closely related IRF-2, a repressor of interferon-induced gene expression, are both capable of activating Qp. However, the major QRE-2-specific binding activity detected within extracts of Burkitt lymphoma cells was attributed to IRF-2, suggesting that interferon-independent activation of Qp is largely mediated by IRF-2 in these cells. We observed no effect of gamma interferon on Qp activity in transfection assays, whereas we observed a moderate but significant repression of Qp activity in response to alpha interferon, possibly mediated by either the interferon consensus sequence binding protein or IRF-7, a novel alpha interferon-inducible factor identified in this study. Since expression of IRF-1 and IRF-2 is increased in response to interferons, the Qp activity observed in the presence of interferon likely represented an equilibrium between IRF factors that activate and those that repress gene expression in response to interferon. Thus, by usurping both IRF-1 and its transcriptional antagonist IRF-2 to activate Qp, EBV has evolved not only a mechanism to constitutively express EBNA-1 but also one which may sustain EBNA-1 expression in the face of the antiviral effects of interferon.
CXCR4 expression during lymphopoiesis: implications for human immunodeficiency virus type 1 infection of the thymus
Authors: Kitchen S G , Zack J A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) infection of the human thymus results in depletion of CD4-bearing thymocytes. This depletion is initially manifested in the immature CD4+/CD8+ thymocyte subset. To determine cellular factors involved in HIV infection in the thymus, we examined the expression of the recently identified viral coreceptor, CXCR4, on fresh human thymocytes and on human cells from SCID-hu (Thy/Liv) mice. CXCR4 is a member of the chemokine receptor family which is required along with CD4 for entry into the cell of syncytium-inducing (SI) HIV-1 strains. Our analyses show that CXCR4 expression is modulated during T-lymphoid differentiation such that immature thymocytes display an increased frequency and higher surface density of the coreceptor than do more mature cells. In addition, using an SI strain of HIV-1 which directs expression of a reporter protein on the surface of infected cells, we have found that the immature CD4+/CD8+ thymocytes that express the highest levels of both CD4 and CXCR4 are the cells that are preferentially infected and depleted by the virus in vitro. Thus, high levels of both primary receptor and coreceptor may allow efficient infection of the thymus by certain HIV-1 strains. This in part may explain the rapid disease progression seen in some HIV-infected children, where the thymus is actively involved in the production of new T lymphocytes.
Transcription of the Influenza Ribonucleic Acid Genome by a Virion Polymerase I. Optimal Conditions for In Vitro Activity of the Ribonucleic Acid-Dependent Ribonucleic Acid Polymerase
Authors: Bishop David H. L. , Obijeski John F. , Simpson Robert W.
Published: January 2020

In vitro reaction conditions have been determined for the maximal synthesis of product ribonucleic acid by the influenza (WSN) virion ribonucleic acid polymerase. The reaction requires the presence of all four triphosphates, Mg
2+
and Mn
2+
ions, monovalent cations, nonionic detergent, and ribonucleoside triphosphates at concentrations above certain threshold values. The optimum
p
H for the reaction is around 8.0 to 8.2 and the kinetics of product synthesis are linear through at least 6 hr when incubated at 31 to 33 C.
Effect of Pactamycin on Synthesis of Poliovirus Proteins: a Method for Genetic Mapping
Authors: Taber Robert , Rekosh David , Baltimore David
Published: January 2020

We have studied the effect of the drug pactamycin on protein synthesis in poliovirus-infected HeLa cells. At a concentration which primarily inhibits initiation of protein synthesis, the spectrum of poliovirus proteins synthesized is markedly changed. The amount of NCVP 1, the capsid precursor, is greatly reduced relative to NCVP 2 and the amount of NCVP X is slightly reduced. Since it is believed that there is only one major site for the initiation of protein synthesis on the poliovirus genome, we interpret this differential effect on the poliovirus proteins to be an indication of their relative distance from the initiation site. On this basis, we propose a gene order for the poliovirus genome (5′ → 3′) of NCVP 1, NCVP X, NCVP 2.
Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems
Authors: Dobos P. , Kerr Ian M. , Martin E. M.
Published: January 2020

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of
35
S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.
Replication of Bacteriophage φX174 in a Temperature-Sensitive Deoxyribonucleic Acid Host Cell
Authors: Primrose S. B. , Landers W. M. , Dowell C. E.
Published: January 2020

Bacteriophage φX174 is unable to replicate in
Escherichia coli
t3 at the restrictive temperature. However, if progeny phage synthesis is initiated at the permissive temperature, it will continue after a shift to the restrictive temperature.
Proteins of a Polyhedral Cytoplasmic Deoxyvirus
Authors: Aubertin A. , Palese P. , Tan K. B. , Vilagines R. , McAuslan B. R.
Published: January 2020

The adenosine triphosphatase associated with frog virus 3 shows high specificity for ATP or deoxyadenosine triphosphate and appears to be distinct from the corresponding activity in host cells, poxvirus, or reovirus. The enzyme activity is probably integrated into virus particles since it is firmly associated with subviral particles produced when approximately 50 to 60% of the outer viral protein is removed by detergent treatment. The occurrence of adenosine triphosphatase activity within three unrelated viruses suggests that adenosine tryphosphatase might be a necessary function for most viruses that replicate in the cell cytoplasm.
Integration of Simian Virus 40 Deoxyribonucleic Acid into the Deoxyribonucleic Acid of Primary Infected Chinese Hamster Cells
Authors: Hirai K. , Lehman J. , Defendi V.
Published: January 2020

Simian virus 40 deoxyribonucleic acid (DNA) became associated in an alkaline-stable form with the DNA of Chinese hamster embryo cells at 15 to 20 hr post-infection, at the time when cell DNA synthesis and T antigen were induced. The integration process was not inhibited by
d
-arabinosyl cytosine and was only partially inhibited by cycloheximide.
Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions
Authors: McSharry James J. , Compans Richard W. , Choppin Purnell W.
Published: January 2020

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from
Streptomyces griseus
, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.
Breakdown and Exclusion of Superinfecting T-Even Bacteriophage in
Escherichia coli
Authors: Anderson Carl W. , Eigner Joseph
Published: January 2020

In bacterial strains containing the deoxyribonuclease endonuclease I (endonuclease I
+
strains), 70 to 80% of the injected superinfecting T-even phage deoxyribonucleic acid (DNA) is rapidly degraded to oligonucleotides having an average chain length of 8, the same value as that obtained by endonuclease I digestion of purified T-even phage DNA in vitro. In endonuclease I
−
strains, less than 5% of the injected superinfecting T-even phage DNA is degraded to acid-soluble components. The superinfecting phage DNA is, however, fragmented into a large segment having a molecular weight of about 90 × 10
6
and 30 or more small acid-insoluble segments having molecular weights of less than 10
6
. In both endonuclease I
+
and endonuclease I
−
strains, over 80% of the DNA from adsorbed primary T2 or T4 phage, but only 50% of the DNA from adsorbed superinfecting T2 or T4 phage, is injected. Superinfecting T4 are genetically excluded as efficiently from endonuclease I
−
strains as they are from endonuclease I
+
strains. The excluded phage cannot complement defects in either early or late gene functions carried by the primary phage. The induction of both superinfection breakdown and superinfection exclusion requires a period of protein synthesis between primary infection and addition of the superinfecting phage. These observations seem best explained by failure of superinfecting DNA to enter the host cell cytoplasm, presumably as a result of changes in the cell envelope induced by the primary phage.
Nucleotide Composition of the Ribonucleic Acid of Rabies Virus
Authors: Aaslestad H. G. , Urbano Charlotte
Published: January 2020

The nucleotide composition of the ribonucleic acid of three strains of rabies virus was determined and found to be similar to that of vesicular stomatitis virus.
Synthesis and Assembly of Simian Virus 40 I. Differential Synthesis of Intact Virions and Empty Shells
Authors: Ozer Harvey L.
Published: January 2020

Intact virions and empty shells of simian virus 40 may be rapidly separated from each other and from cell contaminants by a procedure employing a CsCl cushion. This approach permits quantitation of their respective syntheses in infected cells labeled with radioactive amino acids. As much as 5 to 10% of the total acid-precipitable radioactive lysine in infected cell extracts was incorporated into viral particles in a two-hour pulse late in infection. Evidence for multiple origins of empty shells is presented. Some of the empty shells result from breakdown of intact virions. However, empty shells can also form independently of intact virions. First, labeling for periods of 15 min to 2 hr late in the course of infection results in preferential incorporation of
3
H-lysine into empty shells. Secondly, treatment with the deoxyribonucleic acid inhibitor cytosine-β-
d
-arabinofuranoside late in infection results in a 50% inhibition in the rate of formation of intact virions with minimal reduction in the rate of appearance of empty shells.
Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected
Bacillus subtilis
I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains
Authors: Yehle C. O. , Ganesan A. T.
Published: January 2020

The effect of bacteriophage SPO1 infection of
Bacillus subtilis
and a deoxyribonucleic acid (DNA) polymerase-deficient (pol
−
) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg
2+
ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol
−
mutation had no effect on the synthesis of phage DNA or production of mature phage particles.
Viruses of
Entamoeba histolytica
I. Identification of Transmissible Virus-Like Agents
Authors: Diamond Louis S. , Mattern Carl F. T. , Bartgis I. Louise
Published: January 2020

This and a companion report deal with the identification and morphogenesis of viruses in axenized cultures of
Entamoeba histolytica.
There are probably two different types of virus each producing a different pathological picture in different amoebal strains, or, less likely, there is one type of agent having widely different morphological and morphogenetical pictures in different strains of
E. histolytica.
Both types of agent produce a lytic response in axenized amoebae and have been serially passaged to an extent assuring their replicating nature. One appears to replicate in the nucleus as multiple clusters of fine filaments which ultimately lyse the nucleus, causing cell death. The second type of agent appears to be a typical polyhedral virus, seen only in the cytoplasm and also resulting in lysis of the cell. A particle morphologically indistinguishable from this second agent is also found in late passages of the agent producing the nuclear pathology.
Bacteriophage T4 Head Morphogenesis II. Studies on the Maturation of Gene 49-Defective Head Intermediates
Authors: Luftig Ronald B. , Ganz Carol
Published: January 2020

An investigation into the metabolic requirements for maturation of gene 49-defective heads indicated that adenosine triphosphate energy and continued deoxyribonucleic acid (DNA) but not ribonucleic acid synthesis were needed. The fate of DNA present at restrictive temperatures (41.5 C) in
ts
C9 (gene 49)-infected cells was also examined. After lysis of infected cells, the 12 to 32% deoxyribonuclease-resistant DNA associated with isolated gene 49-defective heads was found to be attached to a deoxyribonuclease-sensitive complex associated with the debris. Pulsechase experiments where
3
H-thymidine was used to label the DNA at 41.5 C suggested that more DNA from this pool was present in phage recovered after rescue of the gene 49 function than could be accounted for by the deoxyribonuclease-resistant portion. Further, when these experiments were repeated with an additional density shift (
15
N
13
C-glucose to
14
N
12
C-glucose), the DNA extracted from phage rescued at 10 min after the temperature shift-down was found to be 90% conserved. These results suggest a model whereby DNA packaging into capsid precursors is separated from DNA replication and the energy from DNA synthesis provides the driving force for packaging. Pulse-chase, temperature-shift experiments with E920g (gene 66) or E920g;
ts
C9 mutant-infected cells showed that gene (49, 66)-defective heads, which were isolated as small, isometric-shaped unfilled heads, were a precursor to “petite” phage. This suggests that the maturation process is independent of the size and shape of the head membrane. Similar experiments with the double mutant
ts
C9;
am
N120 indicate that gene 49-defective heads can also be filled in the absence of tails.
Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid II. Requirement for Polyamines in T4 Infection of a Polyamine Auxotroph
Authors: Dion Arnold S. , Cohen Seymour S.
Published: January 2020

Polyamine depletion produced by exogenous arginine in
Escherichia coli
K-12
cultures defective in agmatine ureohydrolase activity resulted in a marked inhibition of the rates of growth and nucleic acid synthesis. Addition of putrescine or spermidine to such depleted cultures restored the control rate of growth and nucleic acid accumulation. The omission of lysine resulted in a further decrease in the rates of growth and nucleic acid synthesis in polyamine-depleted cells. The addition of exogenous cadaverine increased the rates of growth and ribonucleic acid synthesis to those observed in lysine-supplemented cultures, suggesting that lysine or a derivative of lysine serves a function similar to cadaverine. Addition of lysine to polyamine-depleted cultures at neutral
p
H results in the synthesis of cadaverine and a new spermidine analogue, both containing lysine carbon. This new metabolite has been isolated and identified as
N
-3-aminopropyl-1, 5-diaminopentane. T4D infection of the polyamine-depleted mutant resulted in a very low rate of DNA synthesis and phage maturation. The addition of putrescine or spermidine 15 min before infection restored phage DNA synthesis and phage maturation to control rates, i.e., rates observed in infected cells grown in the absence of arginine.
Gene Order of the Poliovirus Capsid Proteins
Authors: Rekosh David
Published: January 2020

Two methods were used to determine the genetic map of the poliovirus capsid proteins. The first method uses pactamycin, a drug which selectively inhibits the initiation of protein synthesis and causes a change in the relative amounts of capsid proteins synthesized. This differential effect on each of the capsid proteins is interpreted as indicating the relative distance of each protein from the initiation site of protein synthesis. The second method involves an analysis of coat precursor molecules released from polyribosomes after a series of short pulses of different length terminated by addition of emetine, a drug which stops all protein synthesis almost immediately after its addition. As the pulse length is increased, each of the capsid proteins within the precursor gains radioactivity with different kinetics. From these kinetics, it is possible to determine the gene order of the capsid proteins within the precursor as well as a rate of protein synthesis. Both methods indicate a gene order for the region of the ribonucleic acid coding for the capsid proteins as (5′ → 3′) VP 4 - VP 2 - VP 3 - VP 1.
Bacteriophage Lysozyme Synthesis in
Escherichia coli
K-12(λ) Infected with rII Mutant of Bacteriophage T4
Authors: Mark Kai-Keung
Published: January 2020

When
Escherichia coli
K-12(λ) was infected with a T4rII bacteriophage, synthesis of lysozyme appeared at the normal time but stopped 15 min after infection. The lysozyme produced was 1% of the normal level.
Transcription of the Adenovirus Genome by an α-Amanitine-Sensitive Ribonucleic Acid Polymerase in HeLa Cells
Authors: Price Richard , Penman Sheldon
Published: January 2020

The properties of the ribonucleic acid (RNA) polymerase activity which transcribes the major portion of the adenovirus genome were studied. Nuclei were prepared from infected cells and incubated in vitro. Virus-specific RNA was determined by hybridization to adenovirus deoxyribonucleic acid (DNA). Adenovirus DNA is transcribed principally by an activity which resembles closely polymerase II of the host cell. This activity is inhibited by α-amanitine and stimulated by (NH
4
)
2
SO
4
. Its product is high-molecular-weight heterogeneous RNA. The polymerase activity measured early in infection (3 to 5 hr) resembles that found late in infection (16 to 18 hr).
Morphology and Physical Properties of
Staphylococcus
Bacteriophage P11-M15
Authors: Brown Dennis T. , Brown Neal C. , Burlingham Byron T.
Published: January 2020

The Group B
Staphylococcus
phage P11-M15 is shown to be 51% protein and 49% deoxyribonucleic acid (DNA). The intact virion has a molecular weight of 66.7 × 10
6
daltons. The purified viral DNA has a molecular weight of 32.7 × 10
6
daltons. The intact virion is shown to be composed of a polyhedral head which is attached at one of its vertices to a flexible tail having helical symmetry. The tail structure is terminated by a complex baseplate which has sixfold symmetry. The virion contains a single molecule of double-stranded DNA which has no apparent single-strand nicks or single-stranded terminal redundancies.
Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus
Authors: Wagner Robert R. , Kiley Michael P. , Snyder Ruth M. , Schnaitman Carl A.
Published: January 2020

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS
1
, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.
Isolation of a Polyvalent Bacteriophage for
Escherichia coli, Klebsiella pneumoniae
, and
Aerobacter aerogenes
Authors: Souza K. A. , Ginoza H. S. , Haight R. D.
Published: January 2020

A lytic bacteriophage isolated from sewage was found to attack strains of
Aerobacter aerogenes, Escherichia coli
, and
Klebsiella pneumoniae
, but not members of the genera
Salmonella, Proteus
, and
Serratia.
The phage, designated φmp, contained deoxyribonucleic acid with a 50% guanine plus cytosine ratio and a molecular weight of 23.1 × 10
6
daltons. Single-step growth experiments of φmp plated at 37 C on
A. aerogenes
A2 gave a mean latent period of 20 min, an average burst size of 103 plaque-forming units/infected cell, and an average adsorption rate constant of 3 × 10
−10
ml/min. Electron microscopy of φmp revealed a phage with a flexible tail (165 nm long and 6 nm wide). The phage head had a hexagonal outline (62 nm in diameter).
Effects of Ionizing Radiation on the Capacity of
Escherichia coli
to Support Bacteriophage T4 Growth
Authors: Marsden Howard , Ginoza William , Pollard Ernest C.
Published: January 2020

The loss in capacity of irradiated bacteria to support the growth of T4 phage has been studied for two strains,
Escherichia coli
B and
E. coli
B
s—1
. Following a dose of 25 krads, the capacity is quite rapidly lost during postirradiation incubation so that after two hr of such incubation at 37 C only 12% remains in strain B and 3% in strain B
s—1
. Evidence that capacity is lost in an all-or-none fashion was provided by two types of experiments: (i) a single-burst analysis of those cells which survived to give a burst, and (ii) an analysis of the regulation of an early phage enzyme. Several processes were examined to try to determine the cause of the loss in capacity. These were the ability of the irradiated and postirradiation incubated cells to respire, to allow phage adsorption and injection of phage deoxyribonucleic acid, to support the transcription and translation of an early gene, and to support replication of phage deoxyribonucleic acid. Of these processes, transcription and translation appeared to be the most closely associated with the capacity loss, although respiration was reduced to 50% after 2 hr.
Absence of Ribonucleic Acid Methylase in the Avian Myeloblastosis Virus Core
Authors: Gantt Raymond , Stromberg Kurt , Julian Barbara
Published: January 2020

N
2
-guanine-ribonucleic acid-methyltransferase, which is associated with avian myeloblastosis virus, is not a component of the viral core.
Metabolism of T4 Bacteriophage Ghost-Infected Cells II. Do Ghosts Cause a Generalized Permeability Change?
Authors: Duckworth Donna Hardy , Winkler Herbert H.
Published: January 2020

Ghosts appear to alter the barrier function of the cell membrane, allowing the release of phosphorylated sugars which normally cannot pass through the cell membrane, whereas phage do not. No increased influx of normally impermeable compounds is observed in the presence of ghosts, indicating that the loss of membrane function after ghost infection cannot be attributed to a generalized breakdown of the permeability barrier.
Comparative Analyses of Vertebrate Gut Microbiomes Reveal Convergence between Birds and Bats
Authors: Song Se Jin , Sanders Jon G. , Delsuc Frédéric , Metcalf Jessica , Amato Katherine , Taylor Michael W. , Mazel Florent , Lutz Holly L. , Winker Kevin , Graves Gary R. , Humphrey Gregory , Gilbert Jack A. , Hackett Shannon J. , White Kevin P. , Skeen Heather R. , Kurtis Sarah M. , Withrow Jack , Braile Thomas , Miller Matthew , McCracken Kevin G. , Maley James M. , Ezenwa Vanessa O. , Williams Allison , Blanton Jessica M. , McKenzie Valerie J. , Knight Rob
Published: January 2020

In this comprehensive survey of microbiomes of >900 species, including 315 mammals and 491 birds, we find a striking convergence of the microbiomes of birds and animals that fly. In nonflying mammals, diet and short-term evolutionary relatedness drive the microbiome, and many microbial species are specific to a particular kind of mammal, but flying mammals and birds break this pattern with many microbes shared across different species, with little correlation either with diet or with relatedness of the hosts. This finding suggests that adaptation to flight breaks long-held relationships between hosts and their microbes.
Pangenomics Analysis Reveals Diversification of Enzyme Families and Niche Specialization in Globally Abundant SAR202 Bacteria
Authors: Saw Jimmy H. W. , Nunoura Takuro , Hirai Miho , Takaki Yoshihiro , Parsons Rachel , Michelsen Michelle , Longnecker Krista , Kujawinski Elizabeth B. , Stepanauskas Ramunas , Landry Zachary , Carlson Craig A. , Giovannoni Stephen J.
Published: January 2020

The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Information about microbial interactions with this vast resource is limited, despite broad recognition that DOM turnover has a major impact on the global carbon cycle. To explain patterns in the genomes of marine bacteria, we propose hypothetical metabolic pathways for the oxidation of organic molecules that are resistant to oxidation via common pathways. The hypothetical schemes we propose suggest new metabolic pathways and classes of compounds that could be important for understanding the distribution of organic carbon throughout the biosphere. These genome-based schemes will remain hypothetical until evidence from experimental cell biology can be gathered to test them. Our findings also fundamentally change our understanding of the ecology of SAR202 bacteria, showing that metabolically diverse variants of these cells occupy niches spanning all depths and are not relegated to the dark ocean.
Medication-related osteonecrosis (MRONJ) of the mandible and maxilla
Authors: Dunphy Louise , Salzano Giovanni , Gerber Barbara , Graystone Jennifer
Published: January 2020

In 2003, Marx reported the first case of osteonecrosis of the jaw in 36 cases related to zoledronic acid or pamidronate. Painful bone exposure in the mandible or maxilla unresponsive to medical or surgical management was observed. In 2014, the American Association of Oral and Maxillofacial Surgeons proposed the term ‘medication-related osteonecrosis of the jaw’ (MRONJ). However, a non-exposed variant may also occur. MRONJ can lead to debilitating clinical sequelae with limited treatment options. We present the case of a 73-year-old woman with metastatic breast cancer and MRONJ of her mandible and maxilla following treatment with intravenous zoledronic acid and denosumab. Six months following dental extractions, she was referred to the Department of Oral and Maxillofacial Surgery for assessment of extensive necrosis of her maxilla and mandible. Extraoral draining sinuses were observed. A CT mandible showed cortical destruction with an ill-defined mixed sclerotic–lucent pattern in keeping with osteonecrosis. Due to her metastatic breast cancer, the extent of her necrosis and poor performance status, free flap reconstruction of her mandible was ruled out. She was treated conservatively.
Cautionary study on the effects of pay for performance on quality of care: a pilot randomised controlled trial using standardised patients
Authors: Green Ellen , Peterson Katherine S , Markiewicz Kathy , O'Brien Janet , Arring Noel M
Published: January 2020

Background

Due to the difficulty of studying incentives in practice, there is limited empirical evidence of the full-impact pay-for-performance (P4P) incentive systems.

Objective

To evaluate the impact of P4P in a controlled, simulated environment.

Design

We employed a simulation-based randomised controlled trial with three standardised patients to assess advanced practice providers’ performance. Each patient reflected one of the following: (A) indicated for P4P screenings, (B) too young for P4P screenings, or (C) indicated for P4P screenings, but screenings are unrelated to the reason for the visit. Indication was determined by the 2016 Centers for Medicare and Medicaid Services quality measures.

Intervention

The P4P group was paid $150 and received a bonus of $10 for meeting each of five outcome measures (breast cancer, colorectal cancer, pneumococcal, tobacco use and depression screenings) for each of the three cases (max $300). The control group received $200.

Setting

Learning resource centre.

Participants

35 advanced practice primary care providers (physician assistants and nurse practitioners) and 105 standardised patient encounters.

Measurements

Adherence to incentivised outcome measures, interpersonal communication skills, standards of care, and misuse.

Results

TheType apatient was more likely to receive indicated P4P screenings in the P4P group (3.82 out of 5 P4P vs 2.94 control, p=0.02), however, received lower overall standards of care under P4P (31.88 P4P vs 37.06 control, p=0.027). TheType bpatient was more likely to be prescribed screenings not indicated, but highlighted by P4P: breast cancer screening (47% P4P vs 0% control, p<0.01) and colorectal cancer screening (24% P4P vs 0% control, p=0.03). The P4P group over-reported completion of incentivised measures resulting in overpayment (average of $9.02 per patient).

Limitations

A small sample size and limited variability in patient panel limit the generalisability of findings.

Conclusions

Our findings caution the adoption of P4P by highlighting the unintended consequences of the incentive system.