bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of 5'-terminal modifications on the biological activity of defective interfering RNAs of Sindbis virus
Authors: Tsiang M , Weiss B G , Schlesinger S
Published: January 2020

We have been studying defective interfering (DI) genomes of the RNA enveloped virus Sindbis virus. Deletion mapping of a DI cDNA demonstrated that only sequences at the 3' and 5' termini of the genome are required for the DI RNA to be biologically active. We constructed a series of cDNAs that transcribe DI RNAs differing only in 5'-terminal sequences. Two of the 5' termini identical to ones found in naturally occurring DI RNAs are the 5' terminus of the virion RNA (DI-549) and the first 142 nucleotides from the 5' terminus of the subgenomic 26S mRNA attached to the 5' terminus of the virion RNA (DI-15). The latter has a 42-nucleotide deletion from nucleotides 25 to 66 in the 26S RNA sequence. These DI RNA transcripts were biologically active, but one (DI-526) which did not have the 42-nucleotide deletion of DI-15 was not replicated. The DI RNA isolated after the presumed amplification of the DI-526 transcript had deleted the first 54 nucleotides of the 26S RNA sequences. The 5' terminus of Sindbis virion RNA contains a stem and loop region that is conserved among alphaviruses. An 11-nucleotide deletion in DI-549 that disrupted this stem and loop rendered this DI RNA inactive. In contrast, this same deletion in DI-15 and one that removed an additional 100 nucleotides of the virion 5' terminus did not prevent its amplification. We did not detect by computer analysis any common secondary structures among the biologically active DI RNAs that distinguished them from those RNAs that were not amplified. Our results support the conclusion that tertiary structure or the ability of the RNA to adapt its structure upon interaction with protein is important in the recognition process.
The avian retrovirus env gene family: molecular analysis of host range and antigenic variants
Authors: Bova C A , Olsen J C , Swanstrom R
Published: January 2020

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.
Induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein E1
Authors: Charley B , Laude H
Published: January 2020

Epithelial cells infected with the coronavirus transmissible gastroenteritis virus (TGEV) and fixed by glutaraldehyde induced a high alpha interferon (IFN-alpha) production in nonimmune porcine as well as human or bovine peripheral blood mononuclear cells (PBMC). IFN-alpha was detected as early as 3 h after exposure of PBMC to infected cells and at producer/inducer cell ratios as low as 1/1. Two of four monoclonal antibodies directed against the viral transmembrane glycoprotein E1 could block the IFN-inducing capacity of both TGEV-infected cells and viral particles. On the other hand, IFN-alpha induction was not markedly affected by monoclonal antibodies directed against other E1 epitopes, against peplomer glycoprotein E2, or against nucleocapsid protein. Thus, these findings strongly imply that IFN induction by TGEV results from interactions between an outer membrane domain of E1 and the PBMC membrane.
Theiler's virus infection in nude mice: viral RNA in vascular endothelial cells
Authors: Zurbriggen A , Fujinami R S
Published: January 2020

Infection of athymic (nu/nu) mice with Theiler's murine encephalomyelitis virus results in an acute encephalitis which resembles poliomyelitis. Immunohistochemistry and in situ hybridization were used to delineate the presence of viral proteins and RNA in the nervous systems of nude mice infected with the Daniels strain of Theiler's virus. This system permits the analysis of a viral infection in the absence of an effective immune response. By immunohistochemistry, viral antigen was found in the processes and cell bodies of neurons and glial cells. Besides the presence of viral antigen in these cell types, by in situ hybridization, Theiler's virus RNA was also found in cells associated with vascular endothelium in the brains and spinal cords of these infected mice. Theiler's virus RNA-positive endothelial cells were observed not only near the primary lesions but also away from demonstrable lesions in normal-appearing regions in the central nervous system. Earlier work had suggested an intra-axonal dissemination for this virus (M. C. Dal Canto and H. L. Lipton, Am. J. Pathol. 106:20-29, 1982). Our findings are consistent with this model but also suggest an additional mechanism for virus spread within the central nervous system, i.e., by infecting vascular cells and crossing the blood-brain barrier. Lastly, after Theiler's murine encephalomyelitis virus infection, not only glial cells but also endothelial cells express major histocompatibility complex class II (la) antigen on their surface (M. Rodriguez, M. L. Pierce, and E. A. Howie, J. Immunol. 138:3438-3442, 1987). Our demonstration of Theiler's virus-infected endotheliumlike cells may explain interactions of virus products in stimulating antigen presentation.
Demonstration in vitro of temperature-sensitive elongation of RNA in Sindbis virus mutant ts6
Authors: Barton D J , Sawicki S G , Sawicki D L
Published: January 2020

Characterization of conditionally lethal mutants of alphaviruses, Sindbis virus and Semliki Forest virus, has indicated that in almost all the RNA-negative mutants the temperature-sensitive (ts) defect prevents the formation of active transcription complexes at nonpermissive temperature (40 degrees C), but such complexes retain activity at 40 degrees C if formed first at permissive temperature (30 degrees C). Our recent results have extended the characterization of one exception to this finding: Sindbis ts6 transcription complexes, once formed at 30 degrees C, do not function at 40 degrees C. We used an in vitro assay for viral RNA synthesis to determine whether the ts defect was the result of dissociation of the complex or of a failure to elongate RNA chains in a stable complex. Our results indicated that the phenotype of ts6 observed in vivo was retained in vitro. In vivo incorporation into single-stranded 49S and 26S RNA was inhibited simultaneously with its incorporation into replicative intermediates upon shifting ts6-infected cells to 40 degrees C, which was compatible with a defect in elongation. Complexes formed at 30 degrees C and inactivated in vivo by shifting to 40 degrees C were reactivated by incubation in vitro at 30 degrees C but not at 40 degrees C. Thus, the transcription complexes were stable. Nascent RNA chains initiated in vivo and pulse-labeled in vitro were chased into single-stranded 49S and 26S RNA only when incubation was at 30 degrees C, indicating that the ts6 transcription complex was temperature sensitive in elongation. It should be possible to study in vitro other alphavirus RNA-negative mutants that demonstrate a change in viral RNA synthesis after shift to 40 degrees C. These would include ts mutants in the synthesis of subgenomic 26S mRNA and of minus-strand RNA.
Altered replicase specificity is responsible for resistance to defective interfering particle interference of an Sdi- mutant of vesicular stomatitis virus
Authors: Giachetti C , Holland J J
Published: January 2020

The in vitro resistance of an Sdi- mutant of vesicular stomatitis virus to interference by wild-type defective interfering (DI) particles was expressed quantitatively in a cell-free replication system derived from mutant-infected cells. Added wild-type DI particle templates were replicated very poorly by extracts of Sdi- mutant-infected cells. However, the addition of purified viral polymerase (a complex of L and NS proteins) from wild-type vesicular stomatitis virus allowed efficient replication of wild-type DI particle genomes in these cell extracts. Added wild-type NS protein alone did not complement DI particle genome replication in these cell extracts, but it did complement a defect in the in vitro transcriptional activity of Sdi- mutant virus. These results clearly implicate the vesicular stomatitis virus polymerase complex in the inability of Sdi- mutants to replicate DI particles and in the quantitative escape from DI particle interference in evolving virus populations.
Epstein-Barr virus-specific cytotoxic T-cell recognition of transfectants expressing the virus-coded latent membrane protein LMP
Authors: Murray R J , Wang D , Young L S , Wang F , Rowe M , Kieff E , Rickinson A B
Published: January 2020

Cytotoxic T cells from Epstein-Barr virus (EBV)-immune individuals specifically kill EBV-transformed B cells from HLA class I antigen-matched donors even though the latently infected cells express only a restricted set of virus genes. The virus-induced target antigens recognized by these immune T cells have not been identified. In our experiments, EBV DNA sequences encoding the virus latent gene products Epstein-Barr nuclear antigen (EBNA)1, EBNA 2, and EBNA-LP and the latent membrane protein (LMP) were individually expressed in a virus-negative human B-lymphoma cell line, Louckes. Transfected clones expressing LMP were killed by EBV-specific cytotoxic T-cell preparations from each of three virus-immune donors HLA matched with Louckes through HLA-A2, B44 antigens; control transfectants or clones expressing one of the EBNA proteins were not recognized. Expression of LMP in a second virus-negative B-cell line, BL41, sensitized these cells to EBV-specific cytolysis restricted through the HLA-A11 antigen. To distinguish between the viral protein and an induced human B-cell activation antigen as the target for T-cell recognition, LMP was then expressed in a murine mastocytoma cell line, P815-A11-restricted human T cells. The LMP-expressing P815-A11 transfectants were susceptible to lysis by EBV-specific cytotoxic T cells from three HLA-A11-positive individuals. Both Louckes and P815-A11 cells were also transfected with constructs capable of encoding a truncated form of LMP (Tr-LMP) which lacks the N-terminal 128 amino acids of the full-length protein. Tr-LMP-expressing transfectants were not recognized by the above T-cell preparations. The results suggest that LMP, and, in particular, epitopes derived from the N-terminal region of the protein, provides one of the target antigens for the EBV-induced human cytotoxic T-cell response.
Redundancy of signal and anchor functions in the NH2-terminal uncharged region of influenza virus neuraminidase, a class II membrane glycoprotein
Authors: Brown D J , Hogue B G , Nayak D P
Published: January 2020

Class II membrane glycoproteins share a common topology of the NH2 terminus inside and the COOH terminus outside the cell. Their transport to the cell surface is initiated by the function of a single hydrophobic domain near the NH2 terminus. This functional domain serves both as an uncleaved signal sequence and as a transmembrane anchor. We examined the signal and anchor functions of influenza virus neuraminidase, a prototype class II membrane glycoprotein, by deletion analysis of its long, uncharged amino-terminal region. The results presented here show that the entire stretch of 29 uncharged amino acids (7 to 35) is not required for either a signal sequence or an anchor sequence function. On the basis of translocation and membrane stability data for different mutants, we suggest that the first 20 amino acid residues (7 to 27) are likely to provide the hydrophobic core for these functions and that within this putative subdomain some sequences are more efficient than the other sequences in providing a translocation function. Finally, it appears that neuraminidase and its mutant proteins are translocated with the proper orientation, regardless of the characteristics of the flanking sequences.
Isolation and characterization of a hepatitis B virus endemic in herons
Authors: Sprengel R , Kaleta E F , Will H
Published: January 2020

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.
Vaccinia virus recombinants expressing an 11-kilodalton beta-galactosidase fusion protein incorporate active beta-galactosidase in virus particles
Authors: Huang C , Samsonoff W A , Grzelecki A
Published: January 2020

Recombinant plasmids in which vaccinia virus transcriptional regulatory sequences were fused to the Escherichia coli lacZ gene were constructed for insertion of the lacZ gene into the vaccinia virus genome. beta-Galactosidase (beta-gal) was found in some purified recombinant vaccinia virions. By enzyme activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and microscopic techniques, the evidence suggested that beta-gal accounted for 5% of the total protein in the virion. These recombinant viruses were constructed so that a portion of the coding sequences of a late vaccinia virus structural polypeptide was fused to the amino terminus of beta-gal to produce the fusion protein. Removal of the coding sequences resulted in the complete loss of beta-gal activity. This demonstrated that a vaccinia virus DNA segment from a late structural gene is responsible for the incorporation of beta-gal into the virion.
Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs
Authors: Makino S , Soe L H , Shieh C K , Lai M M
Published: January 2020

Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free leader RNA species. As a result, each virus-specific mRNA contains an identical leader RNA derived from the 5', end of the genomic RNA. In this study, we demonstrate by primer extension studies that the leader-fusion sites on a given species of coronavirus subgenomic mRNA are heterogeneous. The heterogeneity was due to variation in the number of pentanucleotide (UCUAA) repeats present at the leader fusion site. This pentanucleotide repeat region was complementary between the free leader RNA and the transcription start sites on the template RNA. This result suggests that the discontinuous transcription of coronavirus mRNAs occurs within the complementary sequences localized in two different RNA segments and that RNA joining occurs at variable sites.
Synthesis of functional human immunodeficiency virus tat protein in baculovirus as determined by a cell-cell fusion assay
Authors: Jeang K T , Shank P R , Rabson A B , Kumar A
Published: January 2020

The human immunodeficiency virus tat protein is a strong trans-activator of the expression of mRNAs originating from the viral long terminal repeat. We have expressed the first 72 amino acids (coding exon 1) of this protein in eucaryotic Spodoptera frugiperda SF9 cells by using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. We show that the baculovirus vector stably produced the 72-amino-acid form of the tat protein but was unable to stably synthesize a larger 101-amino-acid full-length version of the same polypeptide. The 72-amino-acid tat protein, when introduced into mammalian fibroblasts by using a cell-cell fusion technique, functionally trans-activated the expression of the human immunodeficiency virus long terminal repeat.
Passive transfer of respiratory syncytial virus (RSV) antiserum suppresses the immune response to the RSV fusion (F) and large (G) glycoproteins expressed by recombinant vaccinia viruses
Authors: Murphy B R , Olmsted R A , Collins P L , Chanock R M , Prince G A
Published: January 2020

In young infants who possess maternally derived respiratory syncytial virus (RSV) antibodies, the antibody response to RSV glycoproteins is relatively poor, despite extensive replication of RSV. In the present study, it was found that cotton rat RSV hyperimmune antiserum suppressed the antibody response to the RSV glycoproteins but not the response to vaccinia virus antigens when the antiserum was passively transferred to cotton rats prior to infection with vaccinia recombinant viruses expressing the RSV envelope glycoproteins. The cotton rats which had their immune responses suppressed by passively transferred antibodies were more susceptible to infection with RSV than were animals inoculated with control serum lacking RSV antibodies. Furthermore, many of the immunosuppressed animals infected with the vaccinia recombinant viruses developed RSV glycoprotein antibodies which had abnormally low neutralizing activities. Thus, preexisting serum RSV antibodies had dramatic quantitative and qualitative effects on the immune response to RSV glycoproteins, which may explain, in part, the poor RSV antibody response of young human infants to infection with RSV. Our observations also suggest that immunosuppression by preexisting, passively acquired RSV antibodies may constitute a major obstacle to RSV immunoprophylaxis during early infancy, when immunization is most needed.
Inhibition of human immunodeficiency virus by using an oligonucleoside methylphosphonate targeted to the tat-3 gene
Authors: Zaia J A , Rossi J J , Murakawa G J , Spallone P A , Stephens D A , Kaplan B E , Eritja R , Wallace R B , Cantin E M
Published: January 2020

Antiviral effects were characterized for two oligodeoxyribonucleoside methylphosphonates synthesized in an antisense (3'-TCTTAACC-5') or a sense (5'-AGAATTGG-3') orientation, based on the RNA sequence of the first splice acceptor site of the tat-3 gene of human immunodeficiency virus (HIV) (5'...AGAAUUGG...3'). The development of syncytial cells and supernatant reverse transcriptase was inhibited by a single exposure to the antisense HIV, and HIV RNA synthesis was inhibited by both antisense and sense methylphosphonates but not by a control herpes simplex virus antisense sequence.
Identification of mouse adenovirus type 1 early region 1: DNA sequence and a conserved transactivating function
Authors: Ball A O , Williams M E , Spindler K R
Published: January 2020

The left end of the genome of mouse adenovirus type 1 (also known as strain FL) was characterized by determination of the DNA sequence, amino acid similarities with early region proteins of primate adenoviruses, and a functional assay. Several specific DNA sequence features were similar to those found in human adenoviruses, and open reading frames from this region could encode proteins similar to human adenovirus early region 1A and early region 1B proteins. DNAs from this region were tested in transient-expression assays in human and mouse cells were found to transactivate the human adenovirus type 5 early region 3 promoter fused to the chloramphenicol acetyltransferase gene. The data indicate structural and functional homologies between mouse adenovirus type 1 early region 1 and early region 1 of primate adenoviruses.
X-region-specific transcript in mammalian hepatitis B virus-infected liver
Authors: Kaneko S , Miller R H
Published: January 2020

In vitro gene expression systems for hepatitis B virus have demonstrated that the virus genome is capable of producing an X-region-specific transcript of approximately 0.7 kilobases (kb). However, this transcript has not been detected in virus-infected cells. We now report the presence of a heterogeneous X-region-specific transcript of approximately 0.65 kb that is found primarily in the nucleus of liver cells infected with the woodchuck hepatitis virus. Interestingly, the majority of the transcripts are not polyadenylated. The transcript, which represents less than 1% of total virus-specific RNA, is found in animals with both acute and chronic woodchuck hepatitis virus infections. While it is probable that the 0.65-kb transcript is involved in the expression of the X gene protein, it may also direct the translation of a protein encoded by a newly identified open reading frame, ORF5, that is present in all hepadnavirus genomes analyzed.
Bovine papillomavirus type 1 E1 replication-defective mutants are altered in their transcriptional regulation
Authors: Lambert P F , Howley P M
Published: January 2020

Bovine papillomavirus type 1 (BPV-1) is capable of replicating as a stable, high-copy-number plasmid in transformed rodent cells. The BPV-1 E1 open reading frame (ORF) encodes multiple functions involved in viral DNA replication. Mutations which disrupt the translational integrity of the E1 ORF disable the viral genome from replicating as a stable plasmid and result in the integration of the viral genome into the host chromosome generally at a low copy number. Despite the low copy number of the integrated genomes, BPV-1 E1 mutants transform rodent cells to anchorage independence very efficiently, at levels equal to or greater than that of wild-type (wt) BPV-1. Studies were performed to provide insight into why these low-copy-number, replication-defective mutants are capable of expressing an equal or greater transformation potential than wt BPV-1. Analysis of viral RNA revealed higher rates of transcription per viral genome in cells harboring E1 mutated BPV-1 DNA than in cells containing wt BPV-1 DNA. Furthermore, the levels of viral RNA mapping the P89 promoter were found to be 15- to 35-fold higher in cells transformed by E1 mutated DNAs compared with wt BPV-1 transformants. This promoter controls expression of the BPV-1 E6 transforming gene and is regulated by the viral E2 gene products. The studies presented in this report determined that the E1 mutants were perturbed in their E2 transcriptional regulation, suggesting a possible explanation for the observed P89 induction. Mutations throughout the E1 ORF, in either of the two regions previously identified as encoding distinct replication functions, were altered in viral transcription.
Fine mapping of the latency-related gene of herpes simplex virus type 1: alternative splicing produces distinct latency-related RNAs containing open reading frames
Authors: Wechsler S L , Nesburn A B , Watson R , Slanina S M , Ghiasi H
Published: January 2020

The latency-related (LR) gene of herpes simplex virus type 1 (HSV-1) is transcriptionally active during HSV-1 latency, producing at least two LR-RNAs. The LR gene partially overlaps the immediate-early gene ICP0 and is transcribed in the opposite direction from ICP0, producing LR-RNAs that are complementary (antisense) to ICP0 mRNA. The LR gene is thought to be involved in HSV-1 latency. We report here the fine mapping and partial sequence analysis of this HSV-1 LR gene. 32P-labeled genomic DNA restriction fragments and synthetic oligonucleotides were used as probes for in situ hybridizations and Northern (RNA) blot hybridizations of RNA from trigeminal ganglia of rabbits latently infected with HSV-1. The two most abundant LR-RNAs appeared to share their 5' and 3' ends and to be produced by alternative splicing. These LR-RNAs were approximately 2 and 1.3 to 1.5 kilobases in length and were designated LR-RNA 1 and LR-RNA 2, respectively. Their 5' ends started approximately 1,210 nucleotides downstream from the 3' end of the ICP0 mRNA. Their 3' ends overlapped ICP0 by nearly 1,000 nucleotides. LR-RNA 1 appeared to have at least one intron removed, while LR-RNA 2 appeared to have at least two introns removed. The LR-RNAs contained two potential long open reading frames, suggesting the possibility that one or more of the LR-RNAs may be a functional mRNA.
Expression of bicistronic measles virus P/C mRNA by using hybrid adenoviruses: levels of C protein synthesized in vivo are unaffected by the presence or absence of the upstream P initiator codon
Authors: Alkhatib G , Massie B , Briedis D J
Published: January 2020

The measles virus (MV) P/C mRNA is functionally bicistronic. Translation is presumed to initiate at both the first and second 5'-proximal AUG codons, leading, respectively, to synthesis of the P and C polypeptides from different overlapping reading frames. To study the function and differential expression of these polypeptides, we have constructed hybrid human adenoviruses capable of expressing high levels of P and C together or of C alone. Cloned cDNA corresponding to the MV P/C gene was coupled to the adenovirus type 2 (Ad2) major late promoter, most of the Ad2 tripartite leader sequence, and the simian virus 40 3'-end processing signal and then used to replace most of the E1a-E1b region of the Ad5 genome in two hybrid adenoviruses: one (Ad5MV/PC13) which contained both 5'-proximal AUG codons of the P/C mRNA and another (Ad5MV/C3) which retained only the second. The sequence context for the P protein initiator AUG codon in Ad5MV/PC13 was made more favorable (GAGAUGG) than the relatively unfavorable context (CCGAUGG) seen in the native MV P/C mRNA. After infection of 293 cells (which provide complementary E1a-E1b functions), both viruses directed equal amounts of P/C-specific mRNA transcription. Ad5MV/PC13 directed the synthesis of both P and C proteins, while Ad5MV/C3 directed the synthesis of C protein alone. Ad5-expressed P protein was phosphorylated, while C was not. C protein had a similar diffuse cytoplasmic localization in both MV and Ad5-infected cells. Ad5MV/C3 and Ad5MV/PC13 directed equal amounts of C protein expression in 293 cells at a level approximately 15 times greater than that seen in MV-infected cells. Thus the level of C protein expression was unaffected by the presence or absence of an out-of-frame upstream AUG codon in a favorable sequence context. This observation cannot be explained by the scanning model for ribosomal initiation and suggests that ribosomes may be binding directly at an internal mRNA site at or near the initiator AUG codon for the C protein.
Differences in cytopathogenicity and host cell range among infectious molecular clones of human immunodeficiency virus type 1 simultaneously isolated from an individual
Authors: Sakai K , Dewhurst S , Ma X Y , Volsky D J
Published: January 2020

A cytopathic human immunodeficiency virus type 1 (HIV-1) isolate containing multiple virus genotypes was molecularly cloned, and the biological activity of six randomly selected clones was assessed by transfection into human lymphoid or glial cell lines. Five infectious clones of HIV-1, termed N1T-A through -E, were isolated in this manner. Clones N1T-A, -B, -C, and -E could be distinguished by restriction endonuclease mapping whereas clones N1T-B and -D had identical maps with the enzymes used. Each clone exhibited a distinct host cell range as well as markedly different infection kinetics and cytopathogenic properties when tested in human cell lines of T-lymphocytic, monocytic, and astrocytic origin. In particular, infection with HIV-1 clone N1T-E was characterized by slow kinetics and lack of significant cytopathic effects in acutely and chronically infected cells. Clone N1T-A, similar to the parental isolate N1T, exhibited a wide host cell range, fast kinetics of infection, and high cytopathogenicity. These data indicate that HIV-infected individuals may carry multiple HIV-1 genotypes with distinct cytopathogenic potential and cell tropism. Analysis of virus isolates must take into account the contribution, or masking, of individual virus clones.
Characterization of major recognition sequences for a herpes simplex virus type 1 origin-binding protein
Authors: Koff A , Tegtmeyer P
Published: January 2020

To investigate early initiation events in the replication of herpes simplex virus type 1, we analyzed interactions of proteins from infected cell extracts with the small origin of herpes simplex virus type 1 (oris1). Using the mobility shift assay, we detected two origin-specific binding interactions. We characterized the more prominent interaction on both strands of the DNA duplex with DNase I protection and methylation interference assays. Protein binding protects 17 bases of DNA on each strand from DNase I. These sequences are located at the left end of the central palindrome and are shifted four bases relative to one another. On the basis of the DNase protection pattern, we believe this protein to be related to the origin-binding protein defined by Elias et al. (P. Elias, M.E. O'Donnell, E.S. Mocarski, and I.R. Lehman, Proc. Natl. Acad. Sci. 83:6322-6326, 1986). Our DNase I footprint shows both strong and weak areas of protection. The regions strongly protected from DNase I align with the essential contact residues identified by interference footprinting. Methylation interference defines a small binding domain of 8 base pairs: 5'-GTTCGCAC-3'/3'-CAAGCGTG-5'. This recognition sequence contains two inverted 5'-GT(T/G)CG-3' repeats which share a 2-base overlap; thus, the origin-binding protein probably binds to the inverted repeats as a dimer.
Structural analysis of a hepatitis B virus genome integrated into chromosome 17p of a human hepatocellular carcinoma
Authors: Zhou Y Z , Slagle B L , Donehower L A , vanTuinen P , Ledbetter D H , Butel J S
Published: January 2020

Hepatitis B virus (HBV) is clearly a factor in the development of hepatocellular carcinoma, but its mechanism of action remains obscure. One possibility is that the HBV integration event alters the expression of a nearby growth-regulatory cellular gene. A 9-kilobase (kb) DNA fragment containing an HBV insert plus flanking cellular sequences was cloned from a hepatoma specimen from Shanghai, People's Republic of China. Restriction mapping of the insert revealed a large inverted repeat structure consisting of both viral sequences (encompassing all of the core and pre-S regions and portions of the X and S genes) and at least 3 kb of unique cellular sequences. The virus-cell junction mapped 11 nucleotides from the DR1 region, in a position within the HBV X gene and included in the cohesive overlap region. A probe generated from 1.0 kb of the flanking cellular DNA mapped the viral insert to chromosome 17 in the region designated 17p11.2-17p12, which is near the human proto-oncogene p53. Sequence data from a portion of the flanking cellular DNA revealed a stretch of approximately 70 base pairs that showed highly significant homology with a conserved region of a number of functional mammalian DNAs, including the human autonomously replicating sequence 1 (ARS1).
Specific interaction between coronavirus leader RNA and nucleocapsid protein
Authors: Stohlman S A , Baric R S , Nelson G N , Soe L H , Welter L M , Deans R J
Published: January 2020

Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.
Receptors for Theiler's murine encephalomyelitis virus: characterization by using rabbit antiviral antiserum
Authors: Rubio N , Cuesta A
Published: January 2020

An immunological assay was developed to characterize the binding of Theiler's murine encephalomyelitis virus to BHK-21 cell receptors. After absorption of the virus and formaldehyde fixation, rabbit antibodies and Staphylococcus aureus protein A labeled with 125I formed a specific complex on the surfaces of the cells. The optimal multiplicity of infection in this system was 10 PFU per cell. The virus was internalized at 33 and 37 degrees C, but internalization did not take place at 25 or 4 degrees C. The binding was proportional to the number of cells and was significant within 30 s. Cell surface receptors were still active after fixation, and only intact viruses were bound, as demonstrated by the lack of binding of the purified, isolated virion proteins VP1, VP2, and VP3.
Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein
Authors: Roberts M S , Boundy A , O'Hare P , Pizzorno M C , Ciufo D M , Hayward G S
Published: January 2020

In transient-expression assays, the IE175 (alpha 4) promoter region of herpes simple virus is down-regulated after cotransfection with DNA encoding its own protein product (IE175 or ICP4). The inhibition by IE175 proved to be highly specific for its own promoter region and did not act on either the herpes simplex virus type 1 IE110 (alpha 0) or human cytomegalovirus major immediate-early promoters. Furthermore, the inhibition was still exhibited by IE175 effector plasmids driven by strong heterologous promoters and therefore must be a direct autoregulatory response that cannot be explained by promoter competition effects. In gel mobility retardation assays with infected-cell nuclear extracts, a prominent and specific DNA-protein complex was formed with DNA fragments containing sequences from -108 to +30 in the IE175 promoter region. This activity was not present in mock-infected samples. Even stronger binding occurred with a fragment containing sequences from -128 to +120 in the IE110 promoter, but this second locus was not associated with any detectable response phenotype in cotransfection assays. Supershift experiments with an anti-IE175 monoclonal antibody confirmed the presence of the IE175 protein in both DNA-protein complexes. In the IE175 promoter, specific binding correlated closely with the presence of an intact autoregulatory signal near the cap site as judged by the loss of both activities in a 3'-deleted promoter fragment lacking sequences from -7 to +30. Insertion of a cloned 30-mer synthetic oligonucleotide sequence from positions -8 to +18 in IE175 restored both IE175 binding activity and the down-regulation phenotype. Direct shift-up assays with a similar 30-base-pair (bp) oligonucleotide containing 21 bp from positions -75 to -55 of IE110 (which encompasses a consensus ATCGTC motif) also produced a specific DNA-protein complex containing the IE175 protein. This ATCGTC motif proved to be a necessary component of both the IE110 and IE175 binding sites, but was insufficient on its own for complex formation. Finally, deletion of 2 bp from positions -3 and -4 within the ATCGTC sequence in the IE175 cap site region abolished both binding activity and the IE175-dependent autoregulation phenotype.
Molecular and antigenic characterization of porcine rotavirus YM, a possible new rotavirus serotype
Authors: Ruiz A M , López I V , López S , Espejo R T , Arias C F
Published: January 2020

In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein.
Evaluation of the interaction of phi X174 gene products E and K in E-mediated lysis of Escherichia coli
Authors: Bläsi U , Young R , Lubitz W
Published: January 2020

Gene K of bacteriophage phi X174 was cloned, and its gene product was localized in the cell envelope of Escherichia coli. Compared with the sole expression of the phi X174 lysis gene E, the simultaneous expression of the K and E genes had no effect on scheduling of cell lysis. Therefore, a direct interaction of proteins E and K could be excluded. In contrast, phi X174 infection of a host carrying a plasmid expressing gene K resulted in a delayed lysis and an apparent increase in phage titer.
Sequence and genetic organization of adenovirus type 35 early region 3
Authors: Flomenberg P R , Chen M , Horwitz M S
Published: January 2020

The early transcription region 3 (E3) of group B adenovirus type 35 (Ad35), a serotype isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders, has been partially sequenced. We had previously identified an Ad35 29-kilodalton (kDa) early glycoprotein which, analogous to group C Ad2 E3-19K, associated with major histocompatibility complex class I antigens in the endoplasmic reticulum of infected cells. The open reading frame (ORF) of the Ad35 29-kDa protein has now been identified within a 2-kilobase-pair cloned Ad35 E3 fragment. The predicted amino acid sequence was very similar to that of group B Ad3 E3-19K. In contrast, homology between the Ad35 and Ad2 glycoproteins was limited to five cysteines in identical positions and a 20-amino-acid region proximal to the transmembrane domain. In addition, 20.3- and 20.6-kDa ORFs have been identified downstream from the ORF for the Ad35 glycoprotein. Analogous 20-kDa ORFs are present in the Ad3 E3 region but are not present in Ad2 and Ad5. In contrast, the region analogous to an Ad2 11.6-kDa ORF, which is 9 kDa in size in Ad3, was absent from the expected position within the Ad35 E3 region. Because the E3 region is likely to play an important role in the interaction between virus and host, analysis of the function of the Ad35 E3 proteins should further our understanding of adenovirus pathogenesis.
Mapping an antibody-binding site and a T-cell-stimulating site on the 1A protein of respiratory syncytial virus
Authors: Nicholas J A , Mitchell M A , Levely M E , Rubino K L , Kinner J H , Harn N K , Smith C W
Published: January 2020

A synthetic peptide modeled on residues 45 to 60 of the 1A protein of respiratory syncytial (RS) virus [1A(45-60)] was constructed and used for immunization of mice and rabbits. The immunoglobulin G fraction of the resulting rabbit antibody, purified on protein A-Sepharose, immunoprecipitated from RS-infected HEp-2 cells a protein with a molecular size of approximately 9.5 kilodaltons, which corresponds to the previously published molecular size of the 1A protein (Y. T. Huang, P. L. Collins, and G. W. Wertz, Virus Res. 2:157-173, 1985). To investigate the T-cell-inducing properties of 1A(45-60), six strains of mice were immunized and their popliteal lymph node cells were tested for proliferation upon restimulation with peptide in vitro. The lymph node cells of all six strains of mice were responsive to restimulation with 1A(45-60) and showed high- and low-responder strain variation. These peptide-primed lymph node cells also proliferated upon in vitro restimulation with RS virus-infected cells. Correlation of proliferation with interleukin 2 production suggested that the responding lymphocytes were T-helper cells. The antibody-binding and T-cell-stimulating sites of 1A were mapped by constructing a series of overlapping synthetic peptides and testing each for ability to react with antiserum prepared by immunization of BALB/C mice with free peptide 1A(45-60) or for ability to restimulate proliferation in 1A(45-60)-primed lymph node cells of BALB/C mice. Human antibody, obtained during confirmed RS virus infection, was similarly tested with the truncated peptides. Antibody-binding activity was reduced after truncation from the carboxy terminus, and a binding site was mapped to residues 51 through 60, the smallest peptide tested. T-cell-stimulating activity in mice was relatively resistant to truncation from the carboxy terminus and sensitive to truncation from the amino terminus. The smallest region which retained significant T-cell-stimulating activity mapped to residues 46 through 56. However, addition of the naturally occurring Cys at residue 45 and extension of the C terminus to residue 62 resulted in maximum T-cell-stimulating activity of the peptide. These data define both a T-cell epitope and a B-cell epitope of the 1A protein of RS virus and suggest that the carboxy terminus of 1A contains a B-cell epitope, involving residues 51 through 60, which is recognized during natural human infection.
Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates
Authors: Haffey M L , Stevens J T , Terry B J , Dorsky D I , Crumpacker C S , Wietstock S M , Ruyechan W T , Field A K
Published: January 2020

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.
Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1
Authors: Viglianti G A , Mullins J I
Published: January 2020

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.