bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct measurement of the poliovirus RNA polymerase error frequency in vitro
Authors: Ward C D , Stokes M A , Flanegan J B
Published: January 2020

The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 x 10(-4) to 5.4 x 10(-3), depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two different templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.
Inducible and constitutive enhancer domains in the noncoding region of human papillomavirus type 18
Authors: Gius D , Grossman S , Bedell M A , Laimins L A
Published: January 2020

The noncoding region of human papillomavirus type 18 (HPV-18) is shown to contain at least three enhancer elements. Two of these elements are responsive to papillomavirus-encoded trans-acting factors, and the third element functions as a constitutive enhancer, requiring only cellular factors for activity. The first enhancer (IE2) is located proximal to the E6 cap site and is responsive to papillomavirus E2 trans-activator. The second enhancer (IE6) is located approximately 500 base pairs upstream of the E6 cap site and is dependent upon the viral E6 gene product for function. A third enhancer (C) is located between 200 and 400 base pairs upstream of the E6 cap site and possesses a constitutive activity, requiring no HPV-18-encoded factors for function. The constitutive enhancer element exhibits some cell type preference for epithelial cell lines, but also functions in rodent fibroblast lines. Each of these enhancers manifests activity independent of the other elements and may reflect separate transcriptional control elements for different stages of the HPV-18 virus life cycle.
Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus
Authors: Whitton J L , Gebhard J R , Lewicki H , Tishon A , Oldstone M B
Published: January 2020

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.
Strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses
Authors: Donahue P R , Hoover E A , Beltz G A , Riedel N , Hirsch V M , Overbaugh J , Mullins J I
Published: January 2020

We report the first complete nucleotide sequence (8,440 base pairs) of a biologically active feline leukemia virus (FeLV), designated FeLV-61E (or F6A), and the molecular cloning, biological activity, and env-long terminal repeat (LTR) sequence of another FeLV isolate, FeLV-3281 (or F3A). F6A corresponds to the non-disease-specific common-form component of the immunodeficiency disease-inducing strain of FeLV, FeLV-FAIDS, and was isolated from tissue DNA of a cat following experimental transmission of naturally occurring feline acquired immunodeficiency syndrome. F3A clones were derived from a subgroup-A-virus-producing feline tumor cell line. Both are unusual relative to other molecularly cloned FeLVs studied to date in their ability to induce viremia in weanling (8-week-old) cats and in their failure to induce acute disease. The F6A provirus is organized into 5'-LTR-gag-pol-env-LTR-3' regions; the gag and pol open reading frames are separated by an amber codon, and env is in a different reading frame. The deduced extracellular glycoproteins of F6A, F3A, and the Glasgow-1 subgroup A isolate of FeLV (M. Stewart, M. Warnock, A. Wheeler, N. Wilkie, J. Mullins, D. Onions, and J. Neil, J. Virol. 58:825-834, 1986) are 98% homologous, despite having been isolated from naturally infected cats 6 to 13 years apart and from widely different geographic locations. As a group, their envelope gene sequences differ markedly from those of the disease-associated subgroup B and acutely pathogenic subgroup C viruses. Thus, F6A and F3A correspond to members of a highly conserved family and represent prototypes of the horizontally transmitted, minimally pathogenic FeLV present in all naturally occurring infections.
Failure of simian virus 40 small t antigen to disorganize actin cables in nonpermissive cell lines
Authors: Phillips B , Rundell K
Published: January 2020

Mouse C3H 10T1/2 cell lines expressing the simian virus 40 (SV40) small t antigen were obtained by cotransfection of pSV2neo and plasmids which encode small t. Cell lines derived from two plasmids which encode small t in the absence of stable deletion fragments of the large T antigen were morphologically normal and grew to slightly higher saturation densities in low serum than control cell lines. Unexpectedly, the clones had highly organized actin cables, as did parental 10T1/2 cells infected with wild-type SV40. These observations and comparisons of rat F111 cells infected with either polyomavirus or SV40 suggest that the SV40 small t antigen does not directly affect cytoskeletal organization.
Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies
Authors: Hardy W R , Strauss J H
Published: January 2020

Plasmids were constructed which contained a large portion of each of the four nonstructural genes of Sindbis virus fused to the N-terminal two-thirds of the trpE gene of Escherichia coli. The large quantity of fusion protein induced from cells containing these plasmids was subsequently used as an antigen to generate polyclonal antisera in rabbits. Each antiserum was specific for the corresponding nonstructural protein and allowed ready identification of each nonstructural protein and of precursors containing the sequences of two or more nonstructural proteins. These antisera were used to determine the stability of the mature nonstructural proteins and to examine the kinetics of processing of the nonstructural proteins from their respective precursors in vivo. Pulse-chase experiments showed that the precursor P123 is cleaved with a half-life of approximately 19 min to produce P12 and nsP3; P12 is then cleaved with a half-life of approximately 9 min to produce nsP1 and nsP2. Thus, although the rate of cleavage between nsP1 and nsP2 is faster than that between nsP2 and nsP3, the latter cleavage must occur first and is therefore the rate-limiting step. The rate at which P34 is chased suggests that the cleavage between nsP3 and nsP4 is the last to occur; however the regulation of nsP4 function in Sindbis virus-infected cells may be even more complex than was previously thought. The products nsP1 and nsP2 (and nsP4) are relatively stable; nsP3, however, is unstable, with a half-life of about 1 h, and appears to be modified to produce heterodisperse, higher-molecular-mass forms. In general, the processing schemes used by Sindbis virus and Semliki Forest virus appear very similar, the major difference being that most nsP3 in Sindbis virus results from termination at an opal condon, whereas in Semliki Forest virus cleavage of the P34 precursor is required.
Induction of antibodies to the Epstein-Barr virus glycoprotein gp85 with a synthetic peptide corresponding to a sequence in the BXLF2 open reading frame
Authors: Oba D E , Hutt-Fletcher L M
Published: January 2020

Epstein-Barr virus codes for at least three envelope glycoproteins, one of which, gp85, has not yet been mapped to the viral genome. The publication and analysis of the entire Epstein-Barr virus DNA sequence has allowed identification of open reading frames with potential for encoding membrane glycoproteins. To determine whether one of these candidate open reading frames, BXLF2, codes for gp85, an antibody was made to a 17-residue peptide derived from positions 518 to 533 of the predicted BXLF2 protein. The reactivity of the antipeptide antibody was then compared with that of the monoclonal antibody F-2-1, which was originally used to define and characterize gp85. Antipeptide antibody and F-2-1 immunoprecipitated glycosylated molecules with identical electrophoretic mobilities; digestion of the two immunoprecipitated proteins with V8 protease generated comparable peptides; and the antipeptide antibody reacted in Western immunoblots with the gp85 glycoprotein that had been immunoprecipitated by F-2-1 before transfer to nitrocellulose. In addition, a monospecific rabbit antibody, made against native gp85, reacted with the peptide used for immunization. These results are compatible with the hypothesis that the BXLF2 open reading frame codes for gp85.
A fast and sensitive method for detecting specific viral RNA in mammalian cells
Authors: Paeratakul U , De Stasio P R , Taylor M W
Published: January 2020

A quick and sensitive method to quantitate viral RNA synthesis has been developed. Utilizing glutaraldehyde to fix infected cells onto nitrocellulose paper, viral RNA can be probed directly in situ. Viral message can be detected from as few as 10(4) infected cells. This technique can be used to study viral gene expression and can be adapted to screen the activity of antiviral agents such as interferon.
Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product
Authors: Arisaka F , Nakako T , Takahashi H , Ishii S
Published: January 2020

The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method. To confirm the deduced amino acid sequence of the tail sheath protein (gp18) that is encoded by gene 18, gp18 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography. Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified. Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N- and C-terminal sequences. Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined.
Psoralen-cross-linking study of the organization of intracellular adenovirus nucleoprotein complexes
Authors: Wong M L , Hsu M T
Published: January 2020

We used the psoralen-cross-linking technique to investigate the structures of adenovirus nucleoprotein complexes during infection. At late times after infection, three types of psoralen cross-linking patterns were observed. A high cross-linking pattern (type I), with about one cross-link in every 10 to 17 base pairs, was found for the newly synthesized and the bulk of the adenovirus late chromatin. Viral templates involved in replication, transcription, and recombination were all found to exhibit this cross-linking pattern. These results suggest that there is no nucleosome-like organization in the unpackaged late adenovirus nucleoprotein complexes. The second type of cross-linking pattern (type II) had a low cross-linking density of about one cross-link in every 700 to 1,000 base pairs. This cross-linking pattern was found to be associated with the viral DNA in the mature virus particles. The sequences at the termini of the virion DNAs, however, were found to have higher cross-linking densities, as shown by electron microscopy. The third type of cross-linking pattern (type III) was composed of a mixture of various proportions of type I and type II patterns in a single molecule. This mixed cross-linking pattern suggests that these molecules are virion assembly intermediates, with viral DNA being partially packaged in the virus particles. The organization of adenovirus nucleoprotein complexes at early times after infection was analyzed by the gel electrophoresis technique following digestion of the DNA with a restriction enzyme that was inhibited by cross-links. Our data suggest that the viral nucleoprotein complexes at early times after infection have accessibility to psoralen cross-linking between the virion DNA and the late viral nucleoprotein complexes. The observed cross-linking density of the early nucleoprotein complex DNA, however, was inconsistent with the nucleosomelike organization suggested by previous investigators.
Molecular analysis of viral RNAs in mice persistently infected with lymphocytic choriomeningitis virus
Authors: Francis S J , Southern P J
Published: January 2020

Infection of newborn mice with lymphocytic choriomeningitis virus (LCMV) results in a lifelong persistent infection. Persistently infected animals continuously produce low levels of infectious virus and accumulate large amounts of intracellular viral nucleic acid (P. J. Southern, P. Blount, and M. B. A. Oldstone, Nature [London] 312:555-558, 1984). We have used gel electrophoresis and hybridization techniques to analyze viral RNAs that appear during the establishment and maintenance of a persistent LCMV infection in vivo to identify any role for defective and/or defective interfering RNAs. We have found a complex, heterogeneously sized population of viral RNAs in multiple independent tissues that is uniquely associated with persistent infections in vivo, but we have not yet established whether these RNAs have a causal or a consequential association with persistent infection by LCMV. Within the complex virus RNA population, full-length genomic L and S RNAs were readily detectable and represented the most abundant individual viral RNA species. RNAs apparently corresponding in size to the viral nucleoprotein and glycoprotein mRNAs could also be detected in these tissue RNA samples. The presence of glycoprotein mRNA indicates a potential mechanism of posttranscriptional regulation to account for the previously documented restriction in viral glycoprotein expression in persistently infected mice (M. B. A. Oldstone and M. J. Buchmeier, Nature (London) 300:360-362, 1982).
Inhibition of adenovirus DNA replication by vesicular stomatitis virus leader RNA
Authors: Remenick J , Kenny M K , McGowan J J
Published: January 2020

Vesicular stomatitis virus (VSV) leader RNA and a synthetic oligodeoxynucleotide of the same sequence were found to inhibit the replication of adenovirus DNA in vitro. In contrast, the small RNA transcribed by the VSV defective interfering particle DI-011 did not prevent adenovirus DNA replication. The inhibition produced by leader RNA was at the level of preterminal protein (pTP)-dCMP complex formation, the initiation step of adenovirus DNA replication. Initiation requires the adenovirus pTP-adenovirus DNA polymerase complex (pTP-Adpol), the adenovirus DNA-binding protein, and nuclear factor I. Specific replication in the presence of leader RNA was restored when the concentration of adenovirus-infected or uninfected nuclear extract was increased or by the addition of purified pTP-Adpol or HeLa cell DNA polymerase alpha-primase to inhibited replication reactions. Furthermore, the activities of both purified DNA polymerases could be inhibited by the leader sequence. These results suggest that VSV leader RNA is the viral agent responsible for inhibition of adenovirus and possibly cellular DNA replication during VSV infection.
A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA
Authors: Feitelson M A , DeTolla L J , Zhou X D
Published: January 2020

BALB/c nude mice were injected intrahepatically with hepatitis B virus (HBV) DNA prepared from recombinant plasmids. Hepatitis B surface antigen appeared in the circulation in 19 of 23 mice (82%) 3 to 20 weeks postinjection and persisted for more than 6 months in most animals. Hepatitis B e antigen appeared transiently in the circulation in 12 of the 23 mice (52%) within a few weeks after the appearance of hepatitis B surface antigen. Antibodies to the core, X, and/or polymerase gene products of HBV have also been observed in 14 (61%) of the mice. Histopathological examination of the livers at 7 months postinjection demonstrated that nearly half had characteristics consistent with chronic hepatitis. HBV DNA appeared to be integrated into host liver DNA. No evidence of viral replication was observed in sera or livers from these mice at 7 months postinjection. These results demonstrate that an HBV chronic carrierlike state can be established in mice and that such a model could be used to study host and virus factors important in the establishment and maintenance of HBV-associated chronic liver disease.
The hepatitis B virus enhancer modulates transcription of the hepatitis B virus surface antigen gene from an internal location
Authors: Bulla G A , Siddiqui A
Published: January 2020

We have used a transient expression system to examine the influence of the hepatitis B virus enhancer on the transcription of the hepatitis B virus surface antigen (HBsAg) gene. This enhancer is located 3' to HBsAg coding sequences but is contained within the mature HBsAg transcripts. Removal of the enhancer resulted in a significant reduction in HBsAg gene expression in liver cells. By using an in vitro nuclear run-on assay, this effect was determined to be at the level of transcription. We have further noted that HBsAg expression can be restored by the reinsertion of the enhancer sequences in the enhancerless vector. However, this reconstitution of HBsAg expression appears to depend on location of the enhancer with regard to the HBsAg promoters.
Expression of herpes simplex virus type 1 latency-associated transcripts in the trigeminal ganglia of mice during acute infection and reactivation of latent infection
Authors: Spivack J G , Fraser N W
Published: January 2020

Herpes simplex virus type 1 (HSV-1) establishes a latent infection in the trigeminal ganglia of mice infected via the eye. In these ganglia three viral transcripts, of 2.0, 1.5, and 1.45 kilobases (kb), which are at least partially colinear, have been identified by Northern (RNA) blot analysis. These RNAs partially overlap ICPO, but are transcribed in the opposite direction (J. G. Spivack and N. W. Fraser, J. Virol. 61:3841-3847, 1987). The accumulation of these latency-associated transcripts, as well as other viral RNAs, was studied during an acute infection and the reactivation of a latent HSV-1 infection in mice. The 2.0-kb latency-associated transcript was detected in trigeminal ganglia of mice as early as 4 days postinfection, and the 1.45- and 1.5-kb RNA doublet was detected at 14 days postinfection. The levels of these latency-associated transcripts increased steadily over a 60-day period. In contrast, other HSV-1 transcripts were detected at 2 to 3 days postinfection, reached a peak on day 4, and rapidly declined below detectable levels by day 7. The data indicate that the temporal expression of the latency-associated genes during acute infection in the trigeminal ganglia of mice is different from the temporal expression of genes involved in HSV-1 replication. During the reactivation of latent HSV-1 from explanted trigeminal ganglia, the latency-associated RNAs decreased about twofold, but were present at significant levels even after HSV-1 DNA increased and infectious virus was recovered. The decrease of the latency-associated transcripts occurred when reactivation was blocked by phosphonoacetic acid or novobiocin, which suggests that this decrease may be an early event in the entry of latent HSV-1 into the viral replication cycle.
Production of antibodies of predetermined specificity against herpes simplex virus DNA polymerase and their use in characterization of the enzyme
Authors: Thomas M S , Banks L M , Purifoy D J , Powell K L
Published: January 2020

Peptides from preselected regions of the herpes simplex virus DNA polymerase were used to generate monospecific antisera to defined regions of the enzyme. The antisera were used to localize the polymerase within the infected cell and to determine the time of synthesis during productive infection. Comparison with a neutralizing polyclonal antiserum was used to show the specificity of the peptide antisera. By using the antisera the stabilities of the DNA polymerase, the alkaline nuclease, and the major DNA-binding protein were determined, and the state of phosphorylation of the DNA polymerase was compared with each of these proteins.
Rabbit model of rotavirus infection
Authors: Conner M E , Estes M K , Graham D Y
Published: January 2020

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.
The polyomavirus enhancer comprises multiple functional elements
Authors: Mueller C R , Muller W J , Hassell J A
Published: January 2020

The polyomavirus enhancer occupies 244 base pairs within noncoding sequences between the early and late transcription units. To define more precisely the DNA sequences that make up the enhancer, we cloned it together with the viral early promoter upstream of a reporter gene, isolated mutants bearing deletions introduced in vitro in the enhancer, and measured the capacity of the various mutant genomes to express the cat gene after transient transfection into mouse 3T3 cells. Analysis of a large number of deletion mutants revealed that the enhancer is between 102 and 172 base pairs long and can be divided into at least three functional elements. Relative to the entire enhancer, individual elements possessed little or no enhancer activity. However, pairs of elements enhanced transcription to levels much higher than the sum of individual elements approximating the activity of the complete enhancer. These findings support the view that the polyomavirus enhancer is composed of multiple sequence elements that function combinatorily and imply that a measure of cooperation exists in the interaction between cellular protein factors bound to their cognate sites in the enhancer and the transcriptional machinery of the cell.
Alternative promoters in the development of bacteriophage plasmid P4
Authors: Dehó G , Zangrossi S , Ghisotti D , Sironi G
Published: January 2020

Infection of Escherichia coli with the satellite virus P4 without its helper bacteriophage P2 leads either to the immune integrated state or to the nonimmune multicopy plasmid condition. We analyzed the transcription pattern of the phage plasmid P4 early and late after infection and during the stable plasmid or lysogenic condition. The early postinfection phase is characterized by the leftward transcription of an operon including the genes cI (P4 immunity) and alpha (replication). This early transcript starts from the promoter PLE, which shows a good homology with the E. coli sigma 70 promoter. At later times, the transcription of this operon starts from a different promoter, PLL, located 400 base pairs upstream of PLE, and sharing little homology with the canonical E. coli promoter sequence; a longer transcript encoding an additional open reading frame is thus produced. PLL shares two boxes of homology with the P4 late promoter PSID, positively regulated by the P4 delta gene product, and depends on delta function for its full activation. In the multicopy plasmid state, the transcription pattern is similar to that observed at late times after infection. Since in the plasmid state not only is P4 immunity not expressed but its establishment is prevented, even though the P4 cI gene is transcribed, the P4 cI function may be regulated at the posttranscriptional level. In the immune state, transcription starts from PLE but does not continue to cover the P4 alpha gene. This suggests that P4 immunity acts by prematurely terminating transcription initiated at PLE.
Simian retrovirus D serogroup 1 has a broad cellular tropism for lymphoid and nonlymphoid cells
Authors: Maul D H , Zaiss C P , MacKenzie M R , Shiigi S M , Marx P A , Gardner M B
Published: January 2020

Simian acquired immunodeficiency syndrome is a fatal immunosuppressive disease caused by type D retroviruses such as simian acquired immunodeficiency syndrome retrovirus type 1 (SRV-1). The disease is characterized by generalized lymphadenopathy, opportunistic infections, and lymphoid depletion with defects in both humoral and cell-mediated immunity. To understand how SRV-1 infection relates to the immune defect, we studied in vivo-infected lymphocytes from SRV-1-positive macaques with and without clinical signs of immunosuppressive disease. B and T helper/inducer and T suppressor/cytotoxic lymphocytes were purified by panning or by flow cytometry. Neutrophils were purified by dextran sedimentation, and platelets were purified by low-speed centrifugation. In vitro infection studies were also done with HUT78, H9, K562, rhesus lung fibroblast, rhesus monkey kidney, and bat lung cells. SRV-1 in lymphocytes or culture supernatants was detected by the induction of syncytia in cocultivated Raji cells and was confirmed by immunofluorescence, electron microscopy, or reverse transcriptase assay. We found that B and T helper/inducer lymphocytes were infected in all animals tested. The number of infected T suppressor/cytotoxic cells was generally lower than that of the other cell subsets, and not all animals in this subset had SRV-1 infections. All other cells exposed in vitro to SRV-1, except bat lung cells, were able to be infected. These findings show that SRV-1 has a broad cell tropism for lymphoid and nonlymphoid cell types.
In vitro synthesis of infectious retroviral RNA
Authors: Flamant F , Sorge J A
Published: January 2020

A plasmid was constructed in which a T7 RNA polymerase promoter was placed upstream of a recombinant amphotropic retrovirus genome containing a selectable neomycin resistance gene. To test the infectivity of the RNA produced by T7 RNA polymerase in vitro, the RNA was microinjected into the nuclei of psi 2 packaging cells. Infectious particles conferring G418 resistance were released.
Presentation and immunogenicity of the hepatitis B surface antigen and a poliovirus neutralization antigen on mixed empty envelope particles
Authors: Delpeyroux F , Peillon N , Blondel B , Crainic R , Streeck R E
Published: January 2020

Insertion of a synthetic DNA fragment encoding a poliovirus neutralization epitope into the S gene encoding the major envelope protein of hepatitis B virus has yielded hybrid (HBsPolioAg) particles closely resembling authentic 22-nm antigen (HBsAg) particles by expression of the modified gene in mammalian cells. In mice, these hybrid particles induce neutralizing antibodies against poliovirus but only weak immune response to HBsAg (F. Delpeyroux, N. Chenciner, A. Lim, Y. Malpièce, B. Blondel, R. Crainic, S. Van der Werf, and R. E. Streeck, Science 233:472-474, 1986). By cotransfection with different plasmids carrying either modified or unmodified S genes, we have now obtained mixed particles presenting both HBsAg and HBsPolioAg. When such particles were inoculated into rabbits, antibodies to both poliovirus and to HBsAg were induced. Moreover, the titers of neutralizing antibodies to poliovirus induced by HBsPolioAg were much higher than those previously obtained in mice. The design of multivalent particles carrying various peptide sequences or presenting several heterologous epitopes may therefore be possible.
Proviral-activated c-erbB is leukemogenic but not sarcomagenic: characterization of a replication-competent retrovirus containing the activated c-erbB
Authors: Pelley R J , Moscovici C , Hughes S , Kung H J
Published: January 2020

Avian leukosis virus (ALV) induces erythroblastosis in chickens by integrating its DNA into the host c-erbB locus and by activating expression of truncated c-erbB transcripts. Although there is a 100% correlation of c-erbB activation with ALV-induced erythroblastosis, direct evidence that the activated c-erbB is oncogenic has not been established. We have constructed a replication-competent retrovirus containing the activated c-erbB to investigate its transforming potential. The Rous c-erbB virus (REB-c) was constructed by inserting the activated c-erbB cDNA into a Rous sarcoma virus vector in place of src. When transfected into transformed quail fibroblasts (QT6), the REB-c construct stably integrates and expresses c-erbB-specific transcripts and produces infectious virus. The REB-c retrovirus produces short-latency polyclonal erythroblastosis in chickens. However, in contrast to avian erythroblastosis virus which contains v-erbB, the REB-c construct does not transform chicken embryo fibroblasts in vitro, nor does the REB-c virus produce sarcomas when injected into the wing web of chickens. Our results provide the first direct evidence that the activated c-erbB which lacks the amino-terminal extracellular domain but which retains the entire carboxy-terminal sequences is leukemogenic but not sarcomagenic.
Altered expression of two Epstein-Barr virus early genes localized in BamHI-A in nonproducer Raji cells
Authors: Zhang C X , Decaussin G , Daillie J , Ooka T
Published: January 2020

The Epstein-Barr virus-carrying lymphoblastoid cell line Raji has two major genomic deletions and is incapable of virus production. Two cDNA clones, c70 and c55, were constructed from early mRNA of P3HR-1 cells and localized, respectively, in BALF-2 and BARF-1 open reading frames where one of the major genomic deletion in Raji cells is situated. These were used to search the different early viral transcripts in producer P3HR-1 and nonproducer Raji lines. c70 and c55 hybridized with their corresponding mRNAs only in producer lines. Analysis with in vitro-synthesized RNA probes showed quite a different transcriptional profile in Raji cells than in P3HR-1 cells. In the P3HR-1 line, BALF-2 encodes a 3.4-kilobase (kb) mRNA during the early phase and a 3.3-kb mRNA during the late phase, and in the Raji line, the probe corresponding to BALF-2 hybridized with three mRNAs of 5.0, 3.1, and 2.4 kb; in P3HR-1 cells, BARF-1 encodes a group of 3'-conterminal transcripts (0.8, 1.2, 1.7, 2.7, 3.2, and 5.0 kb) during both the early and late stages; in Raji cells, however, 0.8-, 1.2-, and 1.7-kb mRNAs are absent, the only mRNAs transcribed being upstream of the deletion and of 5.0, 2.6, and 2.0 kb in size. In vivo and in vitro experiments demonstrated that the BALF-2 open reading frame encodes an early 135-kilodalton (kDa) protein which possesses DNA-binding ability and can be recognized by a herpes simplex virus ICP-8 antiserum. The BARF-1 open reading frame encodes in vitro a 26- to 33-kDa early protein recognized by anti-EA serum. The proteins of both two genes expressed in psi AM 22b cells were localized in nuclei. According to their properties, both proteins, particularly the BALF-2-encoded 135-kDa DNA-binding protein, could play a role in virus replication.
Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells
Authors: Cohen G H , Wilcox W C , Sodora D L , Long D , Levin J Z , Eisenberg R J
Published: January 2020

Glycoprotein D (gD) is a viron envelope component of herpes simplex virus types 1 and 2. We have previously defined seven monoclonal antibody (MAb) groups which recognize distinct epitopes on the mature gD-1 protein of 369 amino acids. MAb groups VII, II, and V recognize continuous epitopes at residues 11-19, 272-279, and 340-356, respectively. MAb groups I, III, IV, and VI recognize discontinuous epitopes. Recent studies have focused on epitopes I, III, and VI. Using truncated forms of gD generated by recombinant DNA methods and proteolysis, epitopes III, IV, and VI were located within amino acids 1-233. A portion of discontinuous epitope I was located in a region within residues 233-275. For this study, we used recombinant DNA methods to create mutations in the gD-1 gene and studied the effects of those mutations on gD as expressed in mammalian cells. Plasmid pRE4, containing the coding sequence of gD-1 and the Rous sarcoma virus long terminal repeat promoter, was transfected into mammalian cells. The expressed protein, gD-1-(pRE4), was identical in size and antigenic properties to gD-1 from infected cells. Six in-frame deletion mutations were subsequently constructed by using restriction enzymes to excise portions of the gD-1 gene. Plasmids carrying these mutated forms were transfected into cells, and the corresponding proteins were examined at 48 h posttransfection for antigenicity and glycosylation patterns. Three deletions of varying size were located downstream of residue 233. Analysis of these mutants showed that amino acids within the region 234-244 were critical for binding of DL11 (group I), but not for other MAb groups. Three other deletion mutants lost all ability to bind MAbs which recognize discontinuous epitopes. In addition, much of the gD expressed by these mutants was observed to migrate as high-molecular-weight aggregated forms in nondenaturing gels. Each of these mutations involved the loss of a cysteine residue, suggesting that disulfide linkages play an essential role in the formation of discontinuous epitopes. The extent of glycosylation of the mutant gD molecules accumulated at 48 h posttransfection suggested altered carbohydrate processing. In one case, there was evidence for increased O-linked glycosylation. Those proteins which had lost a cysteine residue as part of the deletion did not accumulate molecules processed beyond the high-mannose stage. The results suggest that carbohydrate processing during synthesis of gD is very sensitive to alterations in structure, particularly changes involving cysteine residues.
Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex
Authors: Tersmette M , de Goede R E , Al B J , Winkel I N , Gruters R A , Cuypers H T , Huisman H G , Miedema F
Published: January 2020

Human immunodeficiency virus isolates were studied with respect to syncytium-inducing capacity, replicative properties, and host range. Five of 10 isolates from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex were able to induce syncytia in cultures of peripheral blood mononuclear cells (MNC). In contrast, only 2 of 12 isolates from asymptomatic individuals had syncytium-inducing capacity. Syncytium-inducing isolates were reproducibly obtained from the same MNC sample in over 90% of the cases, independent of the donor MNC used for propagation. Syncytium-inducing capacity was shown to be a stable property of an isolate, independent of viral replication rates. Evidence was obtained that the high replication rate of syncytium-inducing isolates observed during primary isolation may be due to higher infectivity of these isolates. The finding that only syncytium-inducing isolates could be transmitted to the H9 cell line is compatible with this higher infectivity. The frequent isolation of syncytium-inducing isolates from individuals with AIDS-related complex or AIDS and the apparent higher in vitro infectivity of these isolates suggest that syncytium-inducing isolates may unfavorably influence the course of human immunodeficiency virus infection.
Identification of immunoreactive antigens of human papillomavirus type 6b by using Escherichia coli-expressed fusion proteins
Authors: Jenison S A , Firzlaff J M , Langenberg A , Galloway D A
Published: January 2020

Human papillomavirus (HPVs) infect the genital epithelium and are found in proliferative lesions ranging from benign condylomata to invasive carcinomas. The immunological response to these infections is poorly understood because of the lack of purified viral antigens. In this study, bacterially derived fusion proteins expressing segments of all the major open reading frames (ORFs) of HPV type 6b (HPV-6b) have been used in Western blot (immunoblot) assays to detect antibodies directed against HPV-encoded proteins. The most striking reactivities present in sera from patients with genital warts were to the HPV-6b L1 ORF protein and, to a lesser extent, to the HPV-6b L2 ORF protein. Two cases of reactivity to HPV-6b E2 ORF were observed, but no reactivities were seen with other HPV-6b constructs. Two sera reacted with the HPV-16 L2 fusion protein, and two sera reacted with the HPV-16 E4 protein. The antibodies directed against the HPV-6b fusion proteins showed no cross-reactivity with comparable regions of the HPV-16 ORFs. This assay provides a useful approach for further studies of HPV serology.
Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro
Authors: Polo J M , Davis N L , Rice C M , Huang H V , Johnston R E
Published: January 2020

Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.
Identification and procaryotic expression of the gene coding for the highly immunogenic 28-kilodalton structural phosphoprotein (pp28) of human cytomegalovirus
Authors: Meyer H , Bankier A T , Landini M P , Brown C M , Barrell B G , Rüger B , Mach M
Published: January 2020

Human cytomegalovirus contains a structural polypeptide that is 28 kilodaltons in apparent molecular size and is reactive in Western blot (immunoblot) analysis with the majority of human sera. The gene coding for this polypeptide was mapped on the genome of human cytomegalovirus strain AD169. A monoclonal antibody specific for the 28-kilodalton polypeptide was used to screen a cDNA library constructed from poly(A)+ RNA of human cytomegalovirus-infected cells in the procaryotic expression vector lambda gt11. Hybridization of cDNA with cosmid and plasmid clones mapped the gene to the HindIII R fragment. The gene was transcribed into a late 1.3-kilobase RNA. The nucleotide sequence of the coding region was determined. Parts of the 28-kilodalton polypeptide were expressed in Escherichia coli as hybrid proteins fused to beta-galactosidase. In Western blots these proteins were recognized by human sera. Antibodies raised against the hybrid proteins reacted specifically with the viral antigen in immunoprecipitations and Western blots. In vitro phosphorylation of HCMV virions and immunoprecipitation showed that the 28-kilodalton polypeptide was phosphorylated.
An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis
Authors: Trono D , Andino R , Baltimore D
Published: January 2020

Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides.
The truncated form of the Epstein-Barr virus latent-infection membrane protein expressed in virus replication does not transform rodent fibroblasts
Authors: Wang D , Liebowitz D , Kieff E
Published: January 2020

The gene encoding the Epstein-Barr virus membrane protein LMP, expressed in latent infection, is known to induce morphologic changes and some loss of contact inhibition in NIH 3T3 cells as well as profound loss of contact inhibition and of anchorage dependence in Rat-1 cells. Another form of LMP (D1LMP), deleted for the amino terminus and first four putative transmembrane domains of LMP, was recently shown to be expressed late in Epstein-Barr virus replication. We now demonstrate that D1LMP has no transformation-associated phenotypic effect in Rat-1 cells and does not significantly affect LMP-induced Rat-1 cell transformation. LMP activity and D1LMP inactivity in inducing anchorage-independent growth are not restricted to Rat-1 cells, but are also evident in BALB/c 3T3 cells. In both cell types, loss of contact inhibition and anchorage independence are acutely evident after LMP expression. Although newly transfected polyclonal Rat-1 or BALB/c cells have a lower agar cloning efficiency than established LMP-expressing clones, this is attributable, at least in part, to their lower average LMP expression, since among clones of transfected cells, higher cloning efficiencies correlated with higher levels of LMP. LMP is bound to the vimentin cytoskeletal network in rodent fibroblasts as it is in transformed lymphocytes, whereas D1LMP showed no detectable cytoskeletal binding, suggesting that cytoskeletal association may be integral to LMP-mediated cell transformation. LMP association with the cytoskeleton in latently infected, growth-transformed lymphocytes and LMP-transformed rodent fibroblasts, correlated with the lack of both rodent cell-transforming activity and cytoskeletal association of D1LMP supports working hypothesis that cytoskeletal association is important in LMP transforming activity.