bluuebunny/crossref_metadata_2025 huggingface dataset:
The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity
Authors: Buss J E , Kamps M P , Gould K , Sefton B M
Published: January 2020

We have constructed two point mutants of Rous sarcoma virus in which the amino-terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur.
Frequent partial deletion of human adult T-cell leukemia virus type I proviruses in experimental transmission: pattern and possible implication
Authors: Hiramatsu K , Yoshikura H
Published: January 2020

Human T-cell leukemia virus type I (HTLV I) propagated in human diploid fibroblast IMR90 was transmitted to human promyelocytic leukemia HL60 cells by coculture. Of 14 provirus-positive HL60 clones, five harbored only defective proviruses, five had defective proviruses in addition to full-sized HTLV I, and four had full-sized proviruses integrated in their chromosomes. The frequency of defective proviruses was unexpectedly high (41% of total proviruses). Analysis of the genomic structure of these defective proviruses revealed polarity of deletion, that is, preferred conservation of the 3' end of the proviral genome (pX and the 3' long terminal repeat). The implication of these findings are discussed with reference to the replication and pathogenesis of HTLV I.
Biological activity and electron microscopy of poliovirus 14S particles obtained from alkali-dissociated procapsids
Authors: Onodera S , Cardamone J J , Phillips B A
Published: January 2020

Highly purified 14S subunit particles were obtained from alkali-dissociated poliovirus type 1 procapsids (naturally occurring empty capsids in poliovirus-infected cells) to compare their morphological and biophysical properties with those of naturally occurring 14S particles. Procapsid-derived 14S particles (PC-14S), like naturally occurring 14S particles, were capable of self-assembly into an empty shell in buffer or extracts from uninfected cells. These empty capsids always exhibited pIs more acidic than those of procapsids but were themselves distinguishable by their respective pIs. Nevertheless, if PC-14S or naturally occurring 14S particles were incubated with extracts made from poliovirus-infected cells, procapsidlike empty shells were formed. This clearly showed that the 14S particle, however obtained, possesses the information to form an empty shell of correct dimensions but of improper conformation, unless a factor present in poliovirus-infected cells is present. With the electron microscope, the PC-14S subunit frequently was seen as a pentagonal structure with a diameter of 20.4 +/- 1.4 nm, a size somewhat larger than expected for a subunit composing 1/12th of the poliovirus surface. Upon self-assembly in vitro, the empty shell formed exhibited a diameter of 29 +/- 1 nm and a wall thickness of ca. 6 to 7 nm. It was necessary to avoid CsCl banding of procapsids in their preparation as this treatment altered both their pI and their sensitivity to alkali dissociation into 14S subunits. The relevance of these findings to the nature and role of procapsids and the requirement for a morphopoietic factor in poliovirus morphogenesis is discussed.
Identification of an amino acid essential to the normal assembly of Autographa californica nuclear polyhedrosis virus polyhedra
Authors: Carstens E B , Krebs A , Gallerneault C E
Published: January 2020

We compared the DNA sequence of the Autographa californica nuclear polyhedrosis virus polyhedrin gene with that of the polyhedrin gene from a morphology mutant called M5. A single point mutation was found at the BamHI restriction site within the polyhedrin coding sequence. This point mutation caused a substitution of leucine for proline at amino acid 58 in the M5 polyhedrin. This point mutation was shown to be responsible for both the appearance of cubic polyhedra and the altered mobility of the polypeptide on sodium dodecyl sulfate-polyacrylamide gels by transferring the M5 polyhedrin gene to the wild-type virus by cotransfection. Recombinants were found which assembled cubic polyhedra in infected cells, had the BamHI restriction site missing, and had an altered mobility of their polyhedrin polypeptide. Computed-predicted secondary-structure analysis indicated that the amino acid at position 58 could be critical to the proper folding of polyhedrin.
Organization of nonstructural genes of the autonomous parvovirus minute virus of mice
Authors: Cotmore S F , Tattersall P
Published: January 2020

Regions of open reading frame (ORF) from the genome of the autonomous parvovirus minute virus of mice (MVM) were cloned into a procaryotic expression vector, and bacterial fusion proteins containing MVM-specific amino acid sequences were isolated. Antibodies raised against these proteins were used to immunoprecipitate viral proteins synthesized in vitro in a rabbit reticulocyte lysate translation system programmed with mRNA isolated from cells infected with MVM and a number of different parvoviruses. These studies demonstrated that: the 83-kilodalton nonstructural protein NS-1 and the 25-kilodalton nonstructural protein NS-2 have a common amino-terminal sequence which is encoded by the single ORF located between nucleotides 225 and 534 in the viral genome; the ORF located between nucleotides 1110 and 1638 is only expressed in the NS-1 protein; and the sequence encoded in a small alternative ORF between nucleotides 2075 and 2291 is expressed exclusively in NS-2. These data confirm that NS-1 is the product of the 4.8-kilobase R1 viral transcript and demonstrate that NS-2 is synthesized from the 3.3-kilobase R2 transcript which arises from the left-hand promoter at map unit 4 on the viral genome. Antibodies against the MVM fusion proteins also cross-reacted with similar proteins encoded by the viruses H-1 and LuIII, but although antibodies against the carboxy-terminal half of NS-1 cross-reacted with a similar protein in CPV, we were unable to demonstrate an NS-2 protein encoded by this virus.
Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis
Authors: Shade R O , Blundell M C , Cotmore S F , Tattersall P , Astell C R
Published: January 2020

The nucleotide sequence of an almost-full-length clone of human parvovirus B19 was determined. Whereas the extreme left and right ends of this genomic clone are incomplete, the sequence clearly indicates that the two ends of viral DNA are related by inverted terminal repeats similar to those of the Dependovirus genus. The coding regions are complete in the cloned DNA, and the two large open reading frames which span almost the entire genome are restricted to one strand, as has been found for all other parvoviruses characterized to date. From the DNA sequence we conclude that the organization of the B19 transcription units is similar although not identical to those of other parvoviruses. In particular, we predict that the B19 genome may utilize a fourth promoter to transcribe mRNA encoding the major structural polypeptide, VP2. Analysis of the putative polypeptides confirms that B19 is only distantly related to the other parvoviruses but reveals that there is a small region in the gene probably encoding the major nonstructural protein of B19, which is closely conserved between all of the parvovirus genomes for which sequence information is currently available.
Nucleotide sequence analysis of squirrel monkey retrovirus reveals a novel primer-binding site for tRNALys1,2
Authors: Chiu I M , Skuntz S F
Published: January 2020

Nucleotide sequences of a DNA fragment containing the long terminal repeat (LTR) of squirrel monkey retrovirus (SMRV) were determined. Sequence analysis showed that the SMRV LTR is 456 base pairs (bp) long and is bounded by 2-bp inverted repeats. Within the U3 region, there are two 43-bp repeats and two 42-bp repeats which are homologous to each other. These repeats are likely to provide enhancer activities commonly observed in other enhancer sequences. Following the repeats are transcriptional regulatory sequences including a CAT box, a Goldberg-Hogness box, and a polyadenylation signal, all positioned within the U3 region of SMRV LTR. A 22-nucleotide sequence immediately downstream from the LTR was found to be complementary to tRNALys1,2, suggesting that tRNALys1,2 serves as the primer for the reverse transcription of SMRV viral RNA.
Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer
Authors: Cole S T , Streeck R E
Published: January 2020

The 7,909-nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer, has been determined and used to deduce the corresponding genome arrangement. Extensive sequence homologies and other genetic features are shared with the related oncogenic virus, human papillomavirus type 16, especially in the major reading frames. A surprising difference was found in the noncoding region of human papillomavirus type 33 as, unlike all other sequenced papillomaviruses, it contains a perfect 78-base pair tandem repeat.
Transcription unit mapping in adenovirus: regions of termination
Authors: Iwamoto S , Eggerding F , Falck-Pederson E , Darnell J E
Published: January 2020

Using a series of single-stranded clones of adenovirus DNA, we determined the extent of RNA polymerase II transit early in infection for two rightward-reading transcription units. RNA synthesis beginning at the major late promoter (16.5 on the genomic map) continued until approximately 65 to 70 map units so that differential choices of mRNAs within that region were not based primarily on transcriptional decisions but rather on posttranscriptional decisions. Transcription from the major late promoter beginning at 16.5 map units, however, did greatly decrease before approximately 75 map units, ensuring that no mRNAs were formed with sequences beyond approximately 75 map units. Early transcription from E3 then began just past 75 map units (at a higher rate than transcription from the major late promoter); E3 transcripts terminated at least 2 kilobases downstream from a second and final poly(A) site in this transcription unit. The effectiveness of termination in E3 was greater than 95 to 99%.
Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells
Authors: Simek S L , Stephens R M , Rice N R
Published: January 2020

The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it.
AKR ecotropic murine leukemia virus SL3-3 forms envelope gene recombinants in vivo
Authors: Thomas C Y
Published: January 2020

The ecotropic AKR virus SL3-3 was injected into neonatal mice of the high-leukemia strains HRS/J and CWD/J and the low-leukemia strains CBA/J, SEA/J, and NIH Swiss. SL3-3 was highly leukemogenic in each strain, and 90% of the inoculated animals died by 6 months of age. T1 oligonucleotide fingerprint analysis of the genomic RNAs of viruses recovered from 9 of 13 leukemic animals revealed the presence of the SL3-3 virus and recombinant viruses with polytropic virus-related envelope gene sequences. Recombinant proviruses were detected by the Southern blot technique in the DNAs of 17 of 18 tumors. The pattern of substitutions within the envelope genes of the SL3-3 recombinant viruses appeared to be dependent on the strain of the animal. These observations indicate that the SL3-3 virus formed envelope gene recombinants in vivo in each of the strains that were studied. However, the role of these recombinants during leukemogenesis remains to be defined.
In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein
Authors: Ruyechan W T , Chytil A , Fisher C M
Published: January 2020

The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.
Interruption-deficient mutants of bacteriophage T5: analysis of single-site mutants
Authors: Rhoades M
Published: January 2020

The properties of viable mutants of bacteriophage T5 that lack, singly, each of the four major sites at which single-chain interruptions normally occur in T5 DNA are described. The mutations responsible for loss of each interruption were mapped by analysis with HhaI, a restriction endonuclease with a cleavage site (pGCGC) that occurs at the 5' termini of the major interruptions (B. P. Nichols and J. E. Donelson, J. Virol. 22:520-526, 1977). For each mutant tested, loss of a specific interruption resulted in loss of a specific HhaI cleavage site. Multiple single-site mutants were constructed to determine the effect of loss of more than one interruption on phage viability. These recombinants, including a phage that lacks the four major interruptible sites, were fully viable and did not exhibit a compensating increase in the frequency of minor interruptions. The effect of loss of a specific interruption on genetic recombination was tested in two-factor crosses with markers that occur close to, but on opposite sites of, the interruption. Loss of the interruptible site did not affect recombination frequency.
Eradication of herpes simplex virus persistence in rat trigeminal ganglia by retrograde axoplasmic transport
Authors: Iwasaki Y , Yamamoto T , Konno H , Iizuka H , Kudo H
Published: January 2020

Potential use of retrograde axoplasmic flow to eradicate virus persistence in ganglionic cells was studied in a new herpes simplex virus (HSV) persistence model in rat trigeminal ganglia. After injection of the F strain of HSV type 1 into the mental nerve, viral antigens were detectable in the ganglia by the immunofluorescence and peroxidase methods between postinoculation (p.i.) days 3 and 6 but not thereafter. None of 82 inoculated rats showed signs of acute illness, and some survived for more than 502 days without symptoms. By cocultivation of ganglion tissues with Vero cells, the virus was isolated from 42 of 49 ganglia (85.7%) between 15 and 386 days (p.i.). HSV DNA was solely localized in the nucleus of neurons by immunoperoxidase staining of paraffin sections with a biotinylated HSV DNA probe, and the presence of HSV DNA-positive cells was confirmed in four of four ganglia on p.i. day 6 and in five of six on p.i. day 502. The efficacy of axoplasmic flow in drug delivery to ganglionic cells was investigated by injection of doxorubicin (ADM) into the nerve once used for virus inoculation. As early as 19 h after injection, strong ADM-specific autofluorescence was seen in the nuclei of neurons parental to the mental nerve and in those of adjacent Schwann cells, and the death of ADM-positive cells subsequently ensued. A single injection of ADM reduced the virus isolation rate from 31/37 (84%) to 3/37 (8%).
Expression of molecular clones of v-myb in avian and mammalian cells independently of transformation
Authors: Lipsick J S , Ibanez C E , Baluda M A
Published: January 2020

We demonstrated that molecular clones of the v-myb oncogene of avian myeloblastosis virus (AMV) can direct the synthesis of p48v-myb both in avian and mammalian cells which are not targets for transformation by AMV. To accomplish this, we constructed dominantly selectable avian leukosis virus derivatives which efficiently coexpress the protein products of the Tn5 neo gene and the v-myb oncogene. The use of chemically transformed QT6 quail cells for proviral DNA transfection or retroviral infection, followed by G418 selection, allowed the generation of cell lines which continuously produce both undeleted infectious neo-myb viral stocks and p48v-myb. The presence of a simian virus 40 origin of replication in the proviral plasmids also permitted high-level transient expression of p48v-myb in simian COS cells without intervening cycles of potentially mutagenic retroviral replication. These experiments establish that the previously reported DNA sequence of v-myb does in fact encode p48v-myb, the transforming protein of AMV.
Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone
Authors: Adachi A , Gendelman H E , Koenig S , Folks T , Willey R , Rabson A , Martin M A
Published: January 2020

We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.
Characterization of Rous sarcoma virus-related sequences in the Japanese quail
Authors: Chambers J A , Cywinski A , Chen P J , Taylor J M
Published: January 2020

We detected sequences related to the avian retrovirus Rous sarcoma virus within the genome of the Japanese quail, a species previously considered to be free of endogenous avian leukosis virus elements. Using low-stringency conditions of hybridization, we screened a quail genomic library for clones containing retrovirus-related information. Of five clones so selected, one, lambda Q48, contained sequence information related to the gag, pol, and env genes of Rous sarcoma virus arranged in a contiguous fashion and spanning a distance of approximately 5.8 kilobases. This organization is consistent with the presence of an endogenous retroviral element within the Japanese quail genome. Use of this element as a high-stringency probe on Southern blots of genomic digests of several quail DNA demonstrated hybridization to a series of high-molecular-weight bands. By slot hybridization to quail DNA with a cloned probe, it was deduced that there were approximately 300 copies per diploid cell. In addition, the quail element also hybridized at low stringency to the DNA of the White Leghorn chicken and at high stringency to the DNAs of several species of jungle fowl and both true and ruffed pheasants. Limited nucleotide sequencing analysis of lambda Q48 revealed homologies of 65, 52, and 46% compared with the sequence of Rous sarcoma virus strain Prague C for the endonuclease domain of pol, the pol-env junction, and the 3'-terminal region of env, respectively. Comparisons at the amino acid level were also significant, thus confirming the retrovirus relatedness of the cloned quail element.
Adenovirus type 12 E1A protein expressed in Escherichia coli is functional upon transfer by microinjection or protoplast fusion into mammalian cells
Authors: Krippl B , Andrisani O , Jones N , Westphal H , Rosenberg M , Ferguson B
Published: January 2020

We efficiently expressed, in Escherichia coli, and purified the protein product encoded by the human adenovirus type 12 (Ad12) 13S mRNA. The functional properties of the E1A protein were analyzed by introducing the protein by microinjection or protoplast fusion into living mammalian cells. We showed that the E. coli-expressed E1A protein induces gene expression of the adenovirus type 5 (Ad5) E1A deletion mutant Ad5dl312. The purified E1A protein rapidly and quantitatively localized to the cell nucleus after microinjection into the cytoplasm. In addition, we raised high-titered monospecific antibodies to the purified Ad12 E1A protein. Using deleted forms of an adenovirus type 2 and Ad5 hybrid (Ad2/5) E1A protein, we showed that all of the epitopes conserved between Ad2/5 E1A and Ad12 E1A protein that are recognized by the Ad12 E1A-specific antiserum map to within the first exon-encoded amino-terminal half of the protein.
Herpes simplex virus (HSV)-specific human T-cell clones recognize HSV glycoprotein D expressed by a recombinant vaccinia virus
Authors: Zarling J M , Moran P A , Lasky L A , Moss B
Published: January 2020

Human cytotoxic T-cell (CTL) clones that lyse autologous cells infected with herpes simplex virus (HSV) type 1 or 2 were generated by stimulating lymphocytes with a recombinant vaccinia virus (recombinant vaccinia-gD-1 virus) that expresses HSV type 1 glycoprotein D (gD-1). Furthermore, CTL clones generated with HSV type 1 or with cloned gD-1 lysed autologous cells infected with the recombinant vaccinia-gD-1 virus. Our findings thus showed that gD serves as a target antigen for human CTLs and that a recombinant vaccinia-gD virus activates HSV-specific human CTL.
Serological characterization of human reassortant rotaviruses
Authors: Garbarg-Chenon A , Bricout F , Nicolas J C
Published: January 2020

We analyzed the serological properties of two human wild-type cell culture-adapted rotaviruses (strains 308 and 46) and of 308 X 46 reassortants which were previously obtained and genetically characterized. Strain 308, exhibiting a so-called long RNA pattern, was found to belong to human rotavirus subgroup II, serotype 1, whereas strain 46, exhibiting a so-called short RNA pattern, represented subgroup I, serotype 2. Among the 308 X 46 reassortants we analyzed, two belonged to subgroup II, serotype 1, and exhibited short RNA patterns. This showed that the correlation observed between human subgroups I and II rotaviruses and the short and long electrophoretic patterns is not supported by any molecular basis (i.e., gene segment 10 or 11 was not involved in the subgroup specificity).
Putative glycoprotein gene of varicella-zoster virus with variable copy numbers of a 42-base-pair repeat sequence has homology to herpes simplex virus glycoprotein C
Authors: Kinchington P R , Remenick J , Ostrove J M , Straus S E , Ruyechan W T , Hay J
Published: January 2020

A strain variation of varicella-zoster virus that maps to the UL region of the genome was found to be due to different copy numbers of a high GC 42-base-pair repeat. DNA sequence analysis of this variable region showed the sequence to be 5-GCGGGATCGGGCTTTCGGG(A/T)AGCGGCCGAGGTGGGCGCGACG-3. Strains Scott and Webster both contain 7 and 32/42 copies of the repeat, whereas strain Oka has exactly 4 copies less. Microheterogeneity exists within the repeated sequences, depending on the strain and the repeat number. Sequencing of the entire EcoRI P fragment (which contains the repeated sequences) and part of the adjacent EcoRI M and EcoRI Q fragments from strain Scott showed that the repeats are part of a large open reading frame that could code for a polypeptide core with a molecular weight of 66,000. Several potential TATA boxes exist upstream and two polyadenylation signals are found downstream of the open reading frame. The predicted protein bears several characteristics of a glycoprotein. The region is transcriptionally active in varicella-zoster virus-infected cells, specifying at least three RNA species of 1.7, 1.95, and 2.5 kilobases, which are transcribed from the same DNA strand. Part of the predicted protein has a high degree of homology to the herpes simplex virus type 1 glycoprotein gC.
Primate cytomegalovirus glycoproteins: lectin-binding properties and sensitivities to glycosidases
Authors: Benko D M , Gibson W
Published: January 2020

The lectin-binding properties and glycosidase sensitivities of the virion glycoproteins of primate cytomegaloviruses (CMVs) were examined. Three simian CMV (SCMV) strains, including Colburn, and four human CMV (HCMV) strains were compared. Their proteins were separated in denaturing polyacrylamide gels and electrotransferred onto nitrocellulose, and the glycosylated species were visualized with iodinated concanavalin A or wheat germ agglutinin (WGA). Virions of both HCMV and SCMV strains contained six principal and several minor lectin-reactive bands. Neuraminidase treatment abolished WGA binding and reduced the charge and charge heterogeneity of the SCMV (i.e., Colburn) virion glycoproteins and had a similar, although less dramatic, effect on those of HCMV. The specificities of concanavalin A and WGA in these assays were evaluated with endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F, and a combination of lectins and glycosidases was used to demonstrate that many of the primate CMV glycoproteins contain both high-mannose and complex, N-linked oligosaccharides. Results suggest that the HCMV virion glycoproteins are more extensively glycosylated or have more completely processed carbohydrate side chains, or both, than their SCMV counterparts.
Enhanced susceptibility to cytotoxic T lymphocytes of target cells isolated from virus-infected or interferon-treated mice
Authors: Bukowski J F , Welsh R M
Published: January 2020

Bone marrow cells and thymocytes isolated from virus-infected or interferon (IFN)-treated mice had marked increases in sensitivity to lysis by allospecific cytotoxic T lymphocytes (CTL) and in expression of class I histocompatibility antigens. Cultured fibroblasts treated with IFN in vitro yielded similar findings in addition to having increased sensitivity to lysis by virus-specific CTL. This indicates that virus-induced IFN may condition target cells in vivo for surveillance by CTL.
Ribonucleic Acid Transcriptase Activity in Purified Wound Tumor Virus
Authors: Black D. R. , Knight C. A.
Published: January 2020

Purified wound tumor virus was found to possess an associated ribonucleic acid (RNA) transcriptase. The product of transcriptase synthesis was shown to be single-stranded RNA which annealed specifically to wound tumor viral RNA.
Increased Fragility of
Escherichia coli
After Infection with Bacteriophage M13
Authors: Roy A. , Mitra S.
Published: January 2020

Male strains of
Escherichia coli
infected with filamentous phage M13 released the progeny phage particles from intact cells. At the same time, the cells continued to grow and multiply at a slightly lower rate than the uninfected cells. Concomitant with the phage release, lipopolysaccharide from the cell wall of the infected cells was also released. The buoyant density of
E. coli
HfrC in diaginol, 1.25 g/cc, did not change as a result of infection. Detergents like sodium dodecyl sulfate and Sarkosyl specifically lysed the infected cells. The infected cells showed enhanced fragility as indicated by inactivation by various stresses, namely heat, osmotic shock, and freezing and thawing. It is concluded that the infection with M13 causes certain alterations in the surface structure of
E. coli
, thus making the cells more fragile.
Inhibition of Poxvirus Replication by Streptovaricin
Authors: Quintrell Nancy A. , McAuslan B. R.
Published: January 2020

A component of streptovaricin complex inhibits the replication of poxvirus. To be effective, the inhibitor must be introduced early in the replication cycle; it appears to inhibit early messenger ribonucleic acid synthesis by viral cores, thus interfering with all subsequent events. Neither of the two major components of the complex, streptovaricin A or C, was the active component.
Arenoviruses in Vero Cells: Ultrastructural Studies
Authors: Murphy Frederick A. , Webb Patricia A. , Johnson Karl M. , Whitfield Sylvia G. , Chappell W. Adrian
Published: January 2020

Thin-section electron microscopy was carried out on Vero green monkey kidney cell cultures infected with some viruses of the newly constituted arenovirus group. Junin, Machupo, Amapari, Pichinde, Parana, Tamiami, and Latino viruses were morphologically identical and indistinguishable from lymphocytic choriomeningitis virus, the prototype virus of the group. Virus particles were round, oval, or pleomorphic, 60 to 280 nm in diameter, and matured via budding from plasma membranes. Most characteristically, particles contained various amounts of homogeneous, 20- to 25-nm, dense granules; these granules in large masses also formed distinctive intracytoplasmic inclusions. In negative-contrast preparations from infected Vero cell culture supernatant fluids, several of the viruses appeared as pleomorphic membrane-bound forms with rather pronounced surface projections. Most particles were between 90 and 220 nm in diameter, although some reached 350 nm in their longest dimension. Internal structure was not resolved by negative-contrast electron microscopy. All observations supported the current delineation of a distinct arenovirus group.
Properties of
Caulobacter
Ribonucleic Acid Bacteriophage φCb5
Authors: Bendis Inakaren , Shapiro Lucille
Published: January 2020

The ribonucleic acid (RNA) bacteriophage φCb5, which specifically infects only one form of the dimorphic stalked bacterium
Caulobacter crescentus
, has been obtained in high yield. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. Phage φCb5 was studied with respect to the physical and chemical properties of both the phage and its RNA. In an electron microscope, the phage particles appear as small polyhedra, 23 nm in diameter. The phage is similar to the
Escherichia coli
RNA phages in that it (i) sediments at an
S
20, w
of 70.6
S
, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. Phage φCb5 differs from the
E. coli
RNA phages in (i) host specificity, (ii) salt sensitivity, and (iii) the presence of histidine, but not methionine, in the coat protein.
Capsid Size and Deoxyribonucleic Acid Length: the Petite Variant of Bacteriophage T4
Authors: Eiserling Frederick A. , Geiduschek E. Peter , Epstein Richard H. , Metter E. Jeffrey
Published: January 2020

A mutant which produces a small-headed (“petite”) variant of bacteriophage T4 is described. The mutation (E920g) maps in a new gene (66) between genes 23 and 24. Petite phage particles composed up to 70% of the phage yield. The petite phage was nonviable upon single infection but produced progeny when two or more infected a cell. Its genome was shortened by a random deletion of about 30%, and deoxyribonucleic acid (DNA) extracted from the particles was 0.68 the length of normal T4 DNA. The reduction in DNA length was accompanied by a proportional reduction in head volume. Double mutants between E920g and head-defective mutants in gene 21 produced unusually high frequencies of spherical capsidlike structures (τ-particles).
Localization of discontinuous epitopes of herpes simplex virus glycoprotein D: use of a nondenaturing ("native" gel) system of polyacrylamide gel electrophoresis coupled with Western blotting
Authors: Cohen G H , Isola V J , Kuhns J , Berman P W , Eisenberg R J
Published: January 2020

Previously, a panel of monoclonal antibodies (MCAb) was used to define specific epitopes of herpes simplex virus glycoprotein D (gD) (R. J. Eisenberg et al., J. Virol. 53:634-644, 1985). Three groups of antibodies recognized continuous epitopes; group VII reacted with residues 11 to 19 of the mature protein (residues 36 to 44 of the predicted sequence), group II reacted with residues 272 to 279, and group V reacted with residues 340 to 356. Four additional antibody groups recognized discontinuous epitopes of gD, since their reactivity was lost when the glycoprotein was denatured by reduction and alkylation. Our goal in this study was to localize more precisely the discontinuous epitopes of gD. Using a nondenaturing system of polyacrylamide gel electrophoresis ("native" gel electrophoresis) coupled to Western blotting, we analyzed the antigenic activity of truncated forms of gD. These fragments were generated either by recombinant DNA methods or by cleavage of purified native gD-1 (gD obtained from herpes simplex virus type 1) and gD-2 (gD obtained from herpes simplex virus type 2) with Staphylococcus aureus protease V8. Antibodies in groups III, IV, and VI recognized three truncated forms of gD-1 produced by recombinant DNA methods, residues 1 to 287, 1 to 275, and 1 to 233. Antibodies in group I recognized the two larger forms but did not react with the gD-1 fragment of residues 1 to 233. On the basis of these and previous results, we concluded that a protion of epitope I was located within residues 233 to 259 and that epitopes III, IV, and VI were upstream of residue 233. Antibodies to continuous epitopes identified protease V8 fragments of gD-1 and gD-2 that contained portions of either the amino or carboxy regions of the proteins. None of the V8 fragments, including a 34K polypeptide containing residues 227 to 369, reacted with group I antibodies. This result indicated that a second portion of epitope I was located upstream of residue 227. Two amino-terminal fragments of gD-1, 33K and 30K, reacted with group III, IV, and VI antibodies. A 33K fragment of gD-2 reacted with group III antibodies. Based on their size and reactivity with endo-beta-N-acetylglycosaminidase F, we hypothesized that the 33K and 30K molecules represented residues 1 to 226 and 1 to 182 of gD-1, respectively. These results suggest that epitopes III, IV, and VI are located within the first 182 residues of gD.(ABSTRACT TRUNCATED AT 400 WORDS)
Antibodies to pre-S and X determinants arise during natural infection with ground squirrel hepatitis virus
Authors: Persing D H , Varmus H E , Ganem D
Published: January 2020

The DNA sequence of the ground squirrel hepatitis virus (GSHV) genome predicts the existence of several proteins in addition to the major surface (S) and core antigens. These include the pre-S1 and pre-S2 proteins, initiated at sites within the open reading frame preceding and continuous with the coding region for the S gene product, and the X protein, the putative product of an independent reading frame. Using an antibody directed against a peptide predicted by codons 130 to 143 of the pre-S1 reading frame, we identified a 43-kilodalton product of the pre-S1 coding region in preparations of GSHV surface antigen purified from the sera of infected animals. In addition, by immunoprecipitation of S- and pre-S-specific in vitro translation products with ground squirrel sera obtained after GSHV infection, we determined that antibodies arise to both S and pre-S determinants. The antibody response to pre-S includes, in some cases, reactivity to pre-S1-specific domains and is not always associated with an anti-S response. Similarly, by production of the viral X gene product in vitro followed by immunoprecipitation with ground squirrel sera, we showed that antibodies to this viral gene product also arise during infection, indicating that X antigenic determinants are synthesized during viral infection and are recognized by the host immune system.
Tumor progression in murine leukemia virus-induced T-cell lymphomas: monitoring clonal selections with viral and cellular probes
Authors: Cuypers H T , Selten G C , Zijlstra M , de Goede R E , Melief C J , Berns A J
Published: January 2020

Clonal selections occurring during the progression of Moloney murine leukemia virus (MuLV)-induced T-cell lymphomas in mice were examined in primary and transplanted tumors by monitoring various molecular markers: proviral integration patterns, MuLV insertions near c-myc and pim-1, and rearrangements of the immunoglobulin heavy chain and beta-chain T-cell receptor genes. The results were as follows. Moloney MuLV frequently induced oligoclonal tumors with proviral insertions near c-myc or pim-1 in the independent clones. Moloney MuLV acted as a highly efficient insertional mutagen, able to activate different (putative) oncogenes in one cell lineage. Clonal selections during tumor progression were frequently marked by the acquisition of new proviral integrations. Independent tumor cell clones exhibited a homing preference upon transplantation in syngeneic hosts and were differently affected by the route of transplantation.
Reovirus guanylyltransferase is L2 gene product lambda 2
Authors: Cleveland D R , Zarbl H , Millward S
Published: January 2020

Reovirus guanylyltransferase, studied as a covalent enzyme-GMP intermediate, was used to guanylate appropriate acceptor molecules in vitro to produce authentic cap structures. Guanylyltransferase activity was associated with lambda 2, the 140-kilodalton product of the L2 gene segment of reovirus serotypes 1 and 3.
Identification of attenuating mutations on the reovirus type 3 S1 double-stranded RNA segment with a rapid sequencing technique
Authors: Bassel-Duby R , Spriggs D R , Tyler K L , Fields B N
Published: January 2020

Reovirus type 3 variants with mutations in the major neutralization domain of the sigma 1 protein have attenuated neurovirulence and restricted neurotropism. We devised a variation of the rapid RNA sequencing technique to facilitate the analysis of double-stranded RNA. We sequenced the S1 double-stranded RNA segment, which encodes the sigma 1 protein, of five attenuated reovirus type 3 variants. Four of the variants have changes in codon 419, and a fifth variant has a change at codon 340, all of which resulted in amino acid substitutions in the sigma 1 protein. We identified two sites on the reovirus type 3 sigma 1 protein that play a critical role in neurovirulence.
Identification of two nuclear subclasses of the adenovirus type 5-encoded DNA-binding protein
Authors: Voelkerding K , Klessig D F
Published: January 2020

The synthesis, accumulation, and subcellular distribution of the adenovirus serotype 5 DNA-binding protein (DBP) has been examined during the infectious cycle in HeLa cells. With the onset of viral DNA replication and entry into the late phase, two nuclear subclasses of DBP are distinguishable by immunofluorescence microscopy and can be separately isolated by in situ cell fractionation. The first subclass, represented by diffuse-staining DBP, is released by the addition of 1% Nonidet P-40-150 mM NaCl. The second subclass of DBP, which is sequestered into intranuclear globular structures, requires a high ionic strength (2 M NaCl) for extraction and appears to be associated with centers of active viral DNA replication. This association is based on the observations that: DBP within the globules and viral DNA, as detected by in situ hybridization, form identical structures that colocalize within the nuclei of infected cells, the formation of DBP globular structures requires the onset and continuation of viral DNA replication, and once formed, the globular structures can be perturbed by modulating viral DNA synthesis.
Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates
Authors: Friedman H M , Glorioso J C , Cohen G H , Hastings J C , Harris S L , Eisenberg R J
Published: January 2020

The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.
Persistence of vesicular stomatitis virus in cloned interleukin-2-dependent natural killer cell lines
Authors: Rosenthal K L , Zinkernagel R M , Hengartner H , Groscurth P , Dennert G , Takayesu D , Prevec L
Published: January 2020

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.
Transduction of host cellular sequences by a retroviral shuttle vector
Authors: Jhappan C , Vande Woude G F , Robins T S
Published: January 2020

We studied the frequencies and types of excision events which can occur with a retroviral shuttle vector containing the simian virus 40 origin of DNA replication. Analysis of the recloned vector plasmids by size and restriction enzyme mapping indicated that most contain one long terminal repeat. By hybridizing the plasmids to a mouse genomic repetitive DNA probe, we also determined that approximately 1 to 3% contain transduced cellular DNA sequences.
Complex formation of simian virus 40 large T antigen with cellular protein p53
Authors: Montenarh M , Kohler M , Henning R
Published: January 2020

We investigated the formation of native complexes between simian virus 40 large T antigen and the cellular protein p53 (T-p53) by using simian virus 40 tsA58-transformed mouse fibroblasts (tsA58 F2b). We observed that newly synthesized p53 bound to all structural subclasses of large T antigen detectable on sucrose density gradients. This led to various intermediates of T-p53 complexes which converted within 2 h into typical mature aggregates. The final levels of stable T-p53 complexes seemed to be determined by p53 rather than by large T antigen.
Role of adenovirus type 2 early region 4 in the early-to-late switch during productive infection
Authors: Yoder S S , Berget S M
Published: January 2020

Deletion of 98% of the adenovirus type 2 E4 transcription unit resulted in a delay of several events characteristic of the intermediate stage of infection, including viral DNA synthesis, induction of the cellular gene coding for dihydrofolate reductase, and the onset of the switch from viral early to late gene expression. Although delayed, both viral DNA replication and dihydrofolate reductase induction eventually approached wild-type levels. Events characteristic of the late stage of infection were both delayed and diminished in mutant-infected cells. Therefore, the viral E4 gene family is involved in the transition from the early to late stages of infection, and events whose timing is coincident or dependent upon the early-to-late switch are delayed.