bluuebunny/crossref_metadata_2025 huggingface dataset:
Two conditional tsA mutant simian virus 40 T antigens display marked differences in thermal inactivation
Authors: Reynisdóttir I , Prives C
Published: January 2020

We have characterized the simian virus 40 (SV40) origin-containing DNA (ori-DNA) replication functions of two SV40 conditional mutant T antigens: tsA438 A-V (tsA58) and tsA357 R-K (tsA30). Both tsA mutant T antigens, immunopurified from recombinant baculovirus-infected insect cells, mediated replication of SV40 ori-DNA in vitro to similar extents as did wild-type T antigen in reactions at 33 degrees C. However, at 41 degrees C, the restrictive temperature, while tsA438 T antigen still generated substantial levels of replication products, tsA357 T antigen did not support any detectable DNA synthesis. Furthermore, preincubation for approximately fourfold-longer time periods at 41 degrees C was required to heat inactivate tsA438 T antigen than to heat inactivate tsA357 T antigen. Unexpectedly, results of analyses of the various DNA replication activities of the two mutant T antigens did not correlate with results from ori-DNA replication reactions. In particular, although tsA357 T antigen was incapable of mediating replication at 41 degrees C at all protein concentrations examined, it displayed either wild-type levels or only partial reductions of the several T-antigen replication-associated activities. These data suggest either that tsA357 T antigen is defective in an as yet unidentified replication function of T antigen or that the combination of its partial defects result in a protein that is unable to support replication. The data also show that two conditional mutant T antigens can be markedly different with respect to thermal sensitivity.
Sequence similarity between Borna disease virus p40 and a duplicated domain within the paramyxovirus and rhabdovirus polymerase proteins
Authors: McClure M A , Thibault K J , Hatalski C G , Lipkin W I
Published: January 2020

We report the sequence of a Borna disease virus clone (pBDV-40) that encodes a 40-kDa protein (p40) found in the nuclei of infected cells. Comparative sequence analysis indicates that p40 is distantly similar to two different regions in the L-polymerase proteins encoded by paramyxoviruses and rhabdoviruses. The p40 sequence similarity indicates a previously undetected duplication in these viral polymerases. Phylogenetic reconstruction suggests that the gene that encodes p40 last shared a common ancestor with these viral polymerase genes prior to the duplication event. These findings support the hypothesis that Borna disease virus is a negative-strand RNA virus and suggest that p40 is involved in transcription and/or replication. The discovery of a duplication within the polymerase proteins of paramyxoviruses and rhabdoviruses has profound implications for the mapping of enzymatic activities within these multifunctional proteins.
Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein
Authors: Felsenstein K M , Goff S P
Published: January 2020

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo.
Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus
Authors: Corapi W V , Olsen C W , Scott F W
Published: January 2020

Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26- to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and all but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin G1 subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the N- or M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE.
Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2
Authors: Lloyd R M , Shatkin A J
Published: January 2020

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.
The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection
Authors: Yokomori K , Lai M M
Published: January 2020

The SJL mouse strain is resistant to infection by some strains of the murine coronavirus mouse hepatitis virus (MHV), such as JHM and A59. The block to virus infection has been variously attributed to defects in virus receptors or virus spread. Since the cellular receptors for MHV, mmCGM1 and mmCGM2, have recently been identified as members of the carcinoembryonic antigen family, we reexamined the possible defectiveness of the MHV receptors in SJL mouse strain. Cloning and sequencing of the cDNAs of both mmCGMs RNAs from SJL mice revealed that they were identical in size to those of the susceptible C57BL/6 (B6) mouse. There was some sequence divergence in the N terminus of the mmCGM molecules between the two mouse strains, resulting in a different number of potential glycosylation sites. This was confirmed by in vitro translation of the mmCGM RNAs, which showed that the glycosylated mmCGM2 of SJL was smaller than that of B6 mice. However, transfection of either mmCGM1 or mmCGM2 from SJL mice into MHV-resistant Cos 7 cells rendered the cells susceptible to MHV infection. The ability of the SJL mmCGM molecules to serve as MHV receptors was comparable to that of those from B6. These molecules are expressed in SJL mouse brain and liver in a similar ratio and in amounts equivalent to those in the B6 mouse. Furthermore, we demonstrated that an SJL-derived cell line was susceptible to A59 but resistant to JHM infection. We concluded that the MHV receptor molecules in the SJL mouse are functional and that the resistance of SJL mice to infection by some MHV strains most likely results from some other factor(s) required for virus entry or some other step(s) in virus replication.
Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines
Authors: Ravnan J B , Gilbert D M , Ten Hagen K G , Cohen S N
Published: January 2020

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.
Varicella-zoster virus open reading frame 61 protein is functionally homologous to herpes simplex virus type 1 ICP0
Authors: Moriuchi H , Moriuchi M , Smith H A , Straus S E , Cohen J I
Published: January 2020

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.
Repression of liver-specific hepatitis B virus enhancer 2 activity by adenovirus E1A proteins
Authors: Chen S T , Su H , Yee J K
Published: January 2020

Two regions of the hepatitis B virus (HBV) genome have been shown to display properties of a transcriptional enhancer. Enhancer 1 is active in most hepatoma lines examined as well as in some non-hepatocyte-derived cell lines. In contrast, enhancer 2 activity is strictly liver specific. In this study, we show that adenovirus E1A expression in the highly differentiated human hepatoma line Huh6 strongly inhibits HBV enhancer 2-stimulated transcription while having no effect on HBV enhancer 1 activity. A sequence motif in HBV enhancer 2 which is essential for its enhancer function is the target for E1A-mediated repression. The repression of HBV enhancer 2 activity is mediated through the N-terminal region of the E1A proteins known to bind a 300-kDa cellular protein. Our results suggest that HBV enhancer function may be modulated by a cellular mechanism similar to E1A-mediated transcriptional repression.
The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle
Authors: Lear A L , Rowe M , Kurilla M G , Lee S , Henderson S , Kieff E , Rickinson A B
Published: January 2020

In Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell lines exhibiting the latency I form of infection (i.e., EBV nuclear antigen 1 [EBNA1] positive in the absence of other latent proteins), the EBNA1 mRNA has a unique BamHI Q/U/K splice structure and is expressed from a novel promoter, Fp, located near the BamHI FQ boundary. This contrasts with the situation in EBV-transformed lymphoblastoid cell lines (LCLs) exhibiting the latency III form of infection (i.e., positive for all latent proteins), in which transcription from the upstream Cp or Wp promoters is the principal source of EBNA mRNAs. We carried out cDNA amplifications with oligonucleotide primer-probe combinations to determine whether Fp is ever active in an LCL environment. The results clearly showed that some LCLs express a Q/U/K-spliced EBNA1 mRNA in addition to the expected Cp/Wp-initiated transcripts; this seemed inconsistent with the concept of Cp/Wp and Fp as mutually exclusive promoters. Here we show that Fp is indeed silent in latency III cells but is activated at an early stage following the switch from latency III into the virus lytic cycle. Four pieces of evidence support this conclusion: (i) examples of coincident Cp/Wp and Fp usage in LCLs are restricted to those lines in which a small subpopulation of cells have spontaneously entered the lytic cycle; (ii) transcripts initiating from Fp can readily be demonstrated in spontaneously productive lines by S1 nuclease protection; (iii) the presence of Fp-initiated transcripts is not affected by acyclovir blockade of the late lytic cycle; and (iv) infection of latently infected LCLs with a recombinant vaccinia virus encoding the EBV immediate-early protein BZLF1, a transcriptional transactivator which normally initiates the lytic cycle, results in the appearance of the diagnostic Q/U/K-spliced transcripts.
Independent evolution of monkeypox and variola viruses
Authors: Douglass N , Dumbell K
Published: January 2020

Smallpox was eradicated more than 10 years ago, but infection with another Orthopoxvirus, monkeypox virus, can result in a clinical picture resembling smallpox. Human infection with monkeypox virus is extremely rare, not easily transmitted, and confined to the rain forest belt of Africa (Z. Jezek and F. Fenner, p. 81-102, in Human Monkeypox, 1988). Evidence that variola virus, the causative agent of smallpox, might be readily derived from monkeypox virus was presented [S. S. Marennikova and E. M. Shelukhina, Nature (London) 276:291-292, 1978; S. S. Marennikova, E. M. Shelukhina, N. N. Maltseva, and G. R. Matsevich Intervirology 11:333-340, 1979], but this was not confirmed [K. R. Dumbell and L. C. Archard, Nature (London) 286:29-32, 1980] and was subsequently discounted (J. J. Esposito, J. H. Nakano, and J. F. Obijeski, Bull. W.H.O. 63:695-703, 1985). Although enough difference between the genomes of monkeypox and variola viruses to rule out a simple interconversion has been demonstrated [K. R. Dumbell and L. C. Archard, Nature (London) 286:29-32, 1980; J. J. Esposito and J. C. Knight, Virology 143:230-251, 1985; J. J. Esposito, J. H. Nakano, and J. F. Obijeski, Bull. W.H.O. 63:695-703, 1985; M. Mackett and L. C. Archard, J. Gen. Virol. 45:683-701, 1979], the possibility that monkeypox virus was a more remote ancestor of variola virus remained. We have now identified a sequence in monkeypox virus DNA which is a homolog of a 1,065-bp open reading frame in the conserved region of the variola virus genome but which has multiple deletions. This is strong evidence that monkeypox virus is not ancestral to variola virus and strengthens confidence in the long-term success of smallpox eradication.
Transfection-mediated recombination of influenza A virus
Authors: Bergmann M , García-Sastre A , Palese P
Published: January 2020

Several mechanisms, including a high mutation rate and reassortment of genes, have been found to be responsible for the variability of influenza A viruses. RNA recombination would be another mechanism leading to genetic variation; however, recombination has only rarely been reported to occur in influenza viruses. During ribonucleoprotein transfection experiments designed to generate viable influenza viruses from in vitro-synthesized RNA, we discovered several viruses which must have originated from recombination events. The ribonucleoprotein transfection system may enhance the formation of viruses which result from jumping of the viral polymerase between RNAs or from ligation of different viral RNAs. Five different recombinant viruses are described. Two of these, REC1 and REC2, contain a neuraminidase (NA) gene whose defective polyadenylation signal has been repaired via intergenic recombination; 124 and 95 nucleotides have been added, respectively. Another virus, REC5, must have originated by multiple recombination events since it contains a mosaic gene with sequences derived from the NA gene of influenza A/WSN/33 virus and the matrix, polymerase protein PB1, and NA genes of influenza A/PR/8/34 virus.
Characterization of early pathogenic effects after experimental infection of calves with bovine immunodeficiency-like virus
Authors: Carpenter S , Miller L D , Alexandersen S , Whetstone C A , VanDerMaaten M J , Viuff B , Wannemuehler Y , Miller J M , Roth J A
Published: January 2020

The early pathogenic effects of bovine immunodeficiency-like virus (BIV) were studied in calves experimentally inoculated with BIV. All animals inoculated with BIV R29-infected cells seroconverted by 6 weeks postinoculation, and BIV was recoverable from each animal at 2 weeks postinoculation. However, levels of BIV replication in vivo appeared to be low. In situ hybridization studies indicated that during peak periods of viral replication in vivo, less than 0.03% of peripheral blood mononuclear cells were expressing detectable levels of viral RNA. Moreover, the levels of viral RNA in these cells in vivo were less than 1/10 the levels observed in persistently infected cells in vitro. BIV-inoculated calves had significantly higher numbers of circulating lymphocytes, and follicular hyperplasia was observed in lymph nodes, hemal nodes, and spleen. The histopathological changes observed in BIV-infected calves were similar to changes found early after infection with the immunosuppressive lentiviruses, including human immunodeficiency virus type 1.
Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts
Authors: Bean W J , Schell M , Katz J , Kawaoka Y , Naeve C , Gorman O , Webster R G
Published: January 2020

The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.
Association between the herpes simplex virus major DNA-binding protein and alkaline nuclease
Authors: Thomas M S , Gao M , Knipe D M , Powell K L
Published: January 2020

Herpes simplex virus encodes seven proteins which have been shown to be both necessary and sufficient for in vitro replication of origin-containing plasmids. We have shown previously that one of these proteins, the major DNA-binding protein mDBP, forms a complex with alkaline nuclease, which is not one of the seven essential proteins. In this study, we have employed immunological reagents and a series of deletion mutants to investigate this complex further. We have determined the regions of mDBP which are important in the formation of this complex, and we have shown that the intranuclear locations of alkaline nuclease and major DNA-binding protein overlap.
A novel particulate influenza vaccine induces long-term and broad-based immunity in mice after oral immunization
Authors: Pang G T , Clancy R L , O'Reilly S E , Cripps A W
Published: January 2020

The immunogenicity of a novel particulate oral influenza vaccine was examined in terms of antibody response and protection in mice. Oral immunization with chicken erythrocytes (CRBC) adsorbed with gamma-irradiated influenza A virus induced high levels of immunoglobulin G antibodies and protection in the lung compared with gamma-irradiated virus administered alone or CRBC. Immunoglobulin A antibodies were the predominant antibodies in nasal washings, and their presence did not correlate with protection as well as immunoglobulin G antibodies. Immunity was not specific for the immunizing virus subtype, as antibodies and enhanced lung clearance of virus were demonstrated with different virus subtypes. However, mice were not protected when challenged with live influenza B virus. The antibody response and the degree of protection were dependent on both the concentration of virus adsorbed to CRBC and number of CRBC adsorbed to virus. Virus-adsorbed CRBC given subcutaneously failed to induce antibodies or protection. Oral immunization with A/Qld/6/72 (H3N2) virus gave a high level of protection over 12 weeks, which could be demonstrated with different subtypes. Protection correlated with antibody levels in the lung determined by both enzyme-linked immunosorbent and hemagglutination inhibition assays, although the levels detected by the latter declined over time.
Finite life span of hybrids formed by fusion of different simian virus 40-immortalized human cell lines
Authors: Whitaker N J , Kidston E L , Reddel R R
Published: January 2020

Simian virus 40 (SV40) genes are able to induce immortalization of normal human cells after a culture crisis during which unknown cellular genetic changes presumably occur. To determine whether these genetic changes are always identical, we performed somatic cell hybridization analysis of an SV40-immortalized human bronchial epithelial cell line, BET-1A. Fusion of BET-1A with an SV40-immortalized fibroblast cell line resulted in hybrids that senesced, indicating that these cell lines are in different complementation groups for immortalization.
Identification of cognate genes among heterologous strains of group B rotavirus
Authors: Eiden J J , Allen J R
Published: January 2020

The genetic relatedness of group B rotavirus (GBR) strains has previously been documented by hybridization with probes derived from whole genomic sequences, but the relationship of individual genes of heterologous GBR strains has not been evaluated. Definition of cognate GBR genes would facilitate investigation of the determinants of group specificity, serotype identity, and neutralization epitopes. Therefore, we investigated the genetic relatedness of three GBR strains by means of Northern (RNA) blot hybridization with isotopically labeled probes prepared from each of the 11 genes of the IDIR strain of GBR. Under low-stringency conditions, hybridization between each of the IDIR gene probes and genomic RNA from the ADRV strain of GBR was observed. Genomic RNA obtained from a bovine strain of GBR hybridized with 9 of the 11 IDIR gene probes. In most cases, cognate genes of each of the GBR strains appeared to migrate to similar positions following polyacrylamide gel electrophoresis. However, the electropherotype positions of GBR genes 5, 6, and 7 were different for each of the three GBR strains. Identification of these genomic segments among GBR strains should prove helpful in future evaluations of GBR structure and function.
v-mos proteins encoded by myeloproliferative sarcoma virus and its ts159 mutant
Authors: Singh B , Stocking C , Walker R , Yang Y D , Ostertag W , Arlinghaus R B
Published: January 2020

The myeloproliferative sarcoma virus (MPSV) v-mos protein was predicted to be identical in size to p39c-mos because of an observed one-base deletion in the seventh codon of the env-mos open reading frame, which would allow translation to initiate at the methionine equivalent to codon 32 of the env-mos gene. On the basis of published results, p39c-mos is known to have greatly reduced in vitro protein kinase activity compared with p37env-mos encoded by Moloney murine sarcoma virus. Unexpectedly, the relative activity of the MPSV v-mos protein kinase was comparable to that of p37env-mos. Consistent with this finding, the size of MPSV v-mos protein was found to be similar to the size of p37env-mos. Moreover, the pattern and sizes of phosphorylated bands produced by autophosphorylation of the MPSV v-mos protein were similar to those of p37env-mos. These results were confirmed by in vitro transcription-translation of the MPSV v-mos gene. Resequencing portions of the MPSV mos gene failed to show the deletion within codon 7. Except for the codon 262 deletion, other mutations characteristic of MPSV and temperature-sensitive MPSV v-mos genes were confirmed. A glycine-to-arginine mutation at residue 338 of the MPSV env-mos sequence, previously shown to cause thermosensitivity of the mutant virus (termed ts159) transforming function, yielded a v-mos protein that had significantly reduced protein kinase activity in vitro. These findings indicate that MPSV, like other Moloney murine sarcoma virus strains, also encodes a functional env-mos protein.
Naturally occurring point mutation in the C terminus of the polymerase gene prevents duck hepatitis B virus RNA packaging
Authors: Chen Y , Robinson W S , Marion P L
Published: January 2020

A duck hepatitis B virus (DHBV) genome cloned from a domestic duck from the People's Republic of China has been sequenced and exhibits no variation in sequences known to be important in viral replication or generation of gene products. Intrahepatic transfection of a dimer of this viral genome into ducklings did not result in viremia or any sign of virus infection, indicating that the genome was defective. Functional analysis of this mutant genome, performed by transfecting the DNA into a chicken hepatoma cell line capable of replicating wild-type virus, indicated that viral RNA is not encapsidated. However, virus core protein is made and can assemble into particles in the absence of encapsidation of viral nucleic acid. Using genetic approaches, it was determined that a change of cysteine to tyrosine in position 711 in the polymerase (P) gene C terminus led to this RNA-packaging defect. By site-directed mutagenesis, it was found that while substitution of Cys-711 with tryptophan also abolished packaging, substitution with methionine did not affect packaging or viral replication. Therefore, Cys-711, which is conserved in all published sequences of DHBV, may not be involved in a disulfide bridge structure essential to viral RNA packaging or replication. Our results, showing that a missense mutation in the region of the DHBV polymerase protein thought to be primarily the RNase H domain results in packaging deficiency, support the previous findings that multiple regions of the complex hepadnaviral polymerase protein may be required for viral RNA packaging.
The requirement for a 5' stem-loop structure in brome mosaic virus replication supports a new model for viral positive-strand RNA initiation
Authors: Pogue G P , Hall T C
Published: January 2020

Sequences with strong similarity to internal control regions 1 and 2 (ICR1 and -2; A and B boxes) of tRNA genes are found at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other plant viruses. The functionality of these motifs was studied by introducing point mutations into the ICR2-like sequence of pRNA-2 M/S, a BMV RNA-2 deletion mutant that replicates in the presence of RNAs-1 and -2 but does not encode a functional viral protein. The accumulation of positive-strand progeny from pRNAs bearing single and double base substitutions was 70 to 91% lower than that of the wild type, while the addition of single bases at position 8 of this motif reduced replication by 80%. These dramatic decreases in positive-strand synthesis paralleled decreases in transcription seen (C. Traboni, G. Ciliberto, and R. Cortese, Cell 36:179-187, 1984) from a tRNAPro gene containing similar mutations, suggesting comparable functions for the ICR regions in protein factor binding and demonstrating that a wild-type composition of the virus ICR2-like motif is required for proper RNA replication. Substitutions introduced at bases surrounding the ICR2 motif yielded levels of pRNA replication that differed, depending on the maintenance of a putative 5' stem-loop structure in the positive strand of the viral genome. Mutations disrupting this positive-strand stem-loop while maintaining the integrity of the complementary negative-strand structure reduced pRNA replication by 85 to 97%. In contrast, disruption of the negative-strand structure while maintaining the positive-strand stem-loop did not reduce pRNA replication. Similar positive-strand structures can be predicted to form from 5' sequences of cucumber mosaic virus (strain Q) and cowpea chlorotic mottle virus RNAs-1 and -2, supporting the concept that such structures comprise an integral part of virus genomic positive-strand promoters. The requirement of a stem-loop structure present on the positive-strand provided the basis for a new model describing how these sequence and structural elements act in the production of virus positive-strand RNA.
Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus
Authors: Tzeng T H , Tu C L , Bruenn J A
Published: January 2020

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.
Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population
Authors: Schuitemaker H , Koot M , Kootstra N A , Dercksen M W , de Goede R E , van Steenwijk R P , Lange J M , Schattenkerk J K , Miedema F , Tersmette M
Published: January 2020

The composition of human immunodeficiency virus type 1 (HIV-1) clonal populations at different stages of infection and in different compartments was analyzed. Biological HIV-1 clones were obtained by primary isolation from patient peripheral blood mononuclear cells under limiting dilution conditions, with either blood donor peripheral blood lymphocytes or monocyte-derived macrophages (MDM) as target cells, and the biological phenotype of the clones was analyzed. In asymptomatic individuals, low frequencies of HIV-1 clones were observed. These clones were non-syncytium inducing and preferentially monocytotropic. In individuals progressing to disease, a 100-fold increase in frequencies of productively HIV-1-infected cells was observed as a result of a selective expansion of nonmonocytotropic clones. In a person progressing to AIDS within 19 months after infection, only syncytium-inducing clones were detected, shifting from MDM-tropic to non-MDM-tropic over time. From his virus donor, a patient with wasting syndrome, only syncytium-inducing clones, mostly non-MDM-tropic, were recovered. Parallel clonal analysis of HIV-1 populations in cells present in bronchoalveolar lavage fluid and peripheral blood from an AIDS patient revealed a qualitatively and quantitatively more monocytotropic virus population in the lung compartment than in peripheral blood at the same time point. These findings indicate that monocytotropic HIV-1 clones, probably generated in the tissues, are responsible for the persistence of HIV-1 infection and that progression of HIV-1 infection is associated with a selective increase of T-cell-tropic, nonmonocytotropic HIV-1 variants in peripheral blood.
Tacaribe arenavirus RNA synthesis in vitro is primer dependent and suggests an unusual model for the initiation of genome replication
Authors: Garcin D , Kolakofsky D
Published: January 2020

A Tacaribe virus in vitro system for RNA synthesis was established and found in large part to faithfully reproduce RNA synthesis in vivo. Similar to influenza virus and bunyavirus in vitro systems, this system was also highly dependent on added oligonucleotides. Of the eight tested, only three were active, in the order GpC greater than CpG greater than ApApC. Determination of the 5' ends of the transcripts suggested that the oligonucleotides were acting as primers. In particular, whereas stimulation with CpG (complementary to positions +1 and +2 of the template) led to RNAs whose 5' ends were at position +1 as expected, GpC stimulation led to transcripts whose 5' ends were at position -1 rather than at position +2, as GpC is complementary to positions +2 and +3 of the template. This finding suggests a model for the initiation of genome replication in which pppGpC is first made on the template at positions +2 and +3 but slips backwards on the template so that the 5' end is at position -1 before elongation can continue.
Functional characterization of a U5 ribozyme: intracellular suppression of human immunodeficiency virus type 1 expression
Authors: Dropulić B , Lin N H , Martin M A , Jeang K T
Published: January 2020

We have designed a ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in U5 (at nucleotide +115). This ribozyme was tested in vitro and was found to give efficient and specific digestion of RNA containing the HIV-1 U5 sequence. When the U5 ribozyme was placed into the HIV-1 genome, virus replication was suppressed in tissue culture. Introduction of this ribozyme into cells by using an amphotropic retrovirus vector significantly reduced expression of U5-containing RNA in cells chronically infected with HIV-1. Naive T cells were cocultivated with packaging cells that produce defective amphotropic retroviruses containing the U5 ribozyme. These lymphocytes were found to be partially protected from HIV-1 infection.
High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay
Authors: Tchenio T , Heidmann T
Published: January 2020

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.
Binding of a host cell nuclear protein to the stem region of human immunodeficiency virus type 1 trans-activation-responsive RNA
Authors: Rounseville M P , Kumar A
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by both viral and host cell factors. Although the viral trans-activator protein, Tat, and its cis-responsive element, trans-activation-responsive (TAR) RNA, have been identified and characterized, the mechanism of HIV-1 transcriptional regulation has not been satisfactorily described. Whereas Tat is necessary to activate transcription, additional factors, derived from the host cell, are important in regulating HIV-1 transcription. To identify such host cell-specific factors, we used an RNase protection mobility shift assay and UV cross-linking to detect a 140-kDa HeLa cell nuclear protein that binds specifically to TAR RNA. By extensive mutational analysis, we determined that the binding of this protein is dependent on both the sequence and the structure of the TAR RNA stem region. Other groups have shown that the production of prematurely terminated transcripts from the HIV-1 promoter is also dependent on the sequence and structure of the TAR RNA stem. This correlation with our results suggests that the TAR RNA stem-binding protein is involved in the production of prematurely terminated transcripts from the HIV-1 promoter and in the regulation of HIV-1 gene expression.
Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis
Authors: Percy N , Belsham G J , Brangwyn J K , Sullivan M , Stone D M , Almond J W
Published: January 2020

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle.
Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle
Authors: Zack J A , Haislip A M , Krogstad P , Chen I S
Published: January 2020

Using a quantitative polymerase chain reaction (PCR) method, we have previously shown that a molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1) can efficiently enter quiescent primary lymphocytes; however, the reverse transcription process is not completed in these cells. In this study, we further characterized the reverse transcription of HIV-1 in quiescent cells, and our results indicate that while initiation of reverse transcription occurs simultaneously in both activated and quiescent lymphocytes, it not only ends prematurely but also proceeds more slowly in quiescent cells. We also performed experiments to address the role of partial reverse transcripts as intermediates in the viral life cycle. We used azidothymidine either before or after infection with HIV-1 to prevent formation of and further DNA synthesis by partial reverse transcripts, respectively. Decreases in virus production from these cells following mitogenic stimulation indicated that partial reverse transcripts can contribute significantly to virus rescue from infected quiescent cells stimulated subsequent to infection. Furthermore, we established that mitogenic stimulation of infected quiescent cells induces reinitiation of DNA synthesis from partial reverse transcripts. However, the virus rescue is inefficient relative to the initial multiplicity of infection, and this is explained by inefficient completion of DNA synthesis from the partial reverse transcript. Thus, the arrest of reverse transcription in quiescent cells may play an important role in HIV-1 pathogenesis by contributing to the inefficient infection of potential target cells in the peripheral blood of HIV-1-infected individuals.
Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I
Authors: Ciminale V , Pavlakis G N , Derse D , Cunningham C P , Felber B K
Published: January 2020

Novel cytoplasmic mRNA species produced by human T-cell leukemia virus type I (HTLV-I) were cloned by using the polymerase chain reaction technique. Five novel 3' splice sites located in the X region and upstream of the env gene were identified. Splicing to the 3' splice sites in the X region generates mRNAs that express two previously unidentified viral proteins, named Rof and Tof. Tof accumulates in the nucleoli of transfected cells. The other viruses of the HTLV family, such as HTLV-II and bovine leukemia virus, also have a complex splicing pattern and are capable of producing additional proteins encoded in the X region. These results suggest that HTLV-I and other members of the HTLV family produce novel proteins, which may contribute to the biological properties of these viruses.
Herpesvirus saimiri oncogene STP-C488 encodes a phosphoprotein
Authors: Jung J U , Desrosiers R C
Published: January 2020

The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-1 cells by STP-C488.
Use of suppressor analysis to identify DNA polymerase mutations in herpes simplex virus which affect deoxynucleoside triphosphate substrate specificity
Authors: Wang Y S , Woodward S , Hall J D
Published: January 2020

Herpes simplex virus DNA polymerase mutations which map in the N-terminal part of the protein and appear to alter deoxynucleoside triphosphate (dNTP) substrate specificity are described. These mutations suppress a drug hypersensitivity associated with the downstream mutation, Aphr10. We suggest that the mutant residues form part of the dNTP-binding site, a site previously thought to be confined to the C terminus.