bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence of the fourth gene of human rotaviruses recovered from asymptomatic or symptomatic infections
Authors: Gorziglia M , Green K , Nishikawa K , Taniguchi K , Jones R , Kapikian A Z , Chanock R M
Published: January 2020

The complete nucleotide sequence of the fourth gene of symptomatic (Wa, DS-1, P, and VA70) and asymptomatic (M37, 1076, McN13, and ST3) rotaviruses of serotype 1, 2, 3, or 4 was determined by the dideoxy chain termination method. In each strain, the fourth gene, which encodes the outer capsid protein VP3, is 2,359 base pairs in length and has 5'- and 3'-noncoding regions of 9 and 25 nucleotides, respectively. The gene has a single long open reading frame of 2,325 base pairs that is capable of coding for a protein of 775 amino acids. A total of 14 N-terminal and 12 C-terminal amino acids are completely conserved or almost completely conserved, respectively, among nine human rotavirus VP3 genes that have been sequenced. In addition, there is conservation of arginine at the two trypsin cleavage sites as well as conservation of clusters of amino acids in different regions of the two VP3 cleavage products, VP8 and VP5. Three distinct forms of VP3 were identified among the nine human rotavirus strains analyzed. Three symptomatic rotaviruses (serotypes 1, 3, and 4) possess highly related VP3 genes (92.2 to 97% nucleotide identity). Two symptomatic serotype 2 rotaviruses possess VP3 genes which are even more closely related to each other (98.6% nucleotide identity) and only moderately related to the aforementioned VP3 genes of serotypes 1, 3, and 4 (87.4 to 88.2% nucleotide identity). The four asymptomatic rotaviruses, which constitute the third group, possess highly related VP3 genes (95.5 to 97.5% nucleotide identity) which are distinct from those of the virulent rotaviruses (73 to 74.8% nucleotide identity). At 91 positions in the protein sequence of VP3, an amino acid is conserved among the asymptomatic rotaviruses, while a different amino acid is conserved among the symptomatic rotaviruses. Notably, five regions are conserved among the symptomatic rotaviruses, while a different set of sequences are conserved among the asymptomatic rotaviruses. It is possible that some or all of these regions of sequence dimorphism may be responsible for the difference in virulence of these two groups of human rotaviruses. There are 13 regions in the VP3 protein sequence which exhibit the greatest variability; the majority of these variable regions are observed between amino acids 106 to 192. These regions may represent potential antigenic sites related to heterotypic rotavirus neutralization.
Translational control of synthesis of simian virus 40 late proteins from polycistronic 19S late mRNA
Authors: Dabrowski C , Alwine J C
Published: January 2020

The simian virus 40 (SV40) 19S late mRNA is polycistronic, encoding multiple late proteins: agnoprotein, VP2, and VP3. We constructed a chloramphenicol acetyltransferase (CAT) transient expression vector in which the SV40 sequences between nucleotides 5171 and 1046 (via the SV40 origin of replication and including the late promoter) were inserted 5' to the cat gene; therefore, the AUG for CAT expression occurs after the AUGs for agnoprotein, VP2, and VP3. CAT enzyme activity assayed after transfection of these constructions indicates the level of CAT AUG utilization and, therefore, can be used as a measure of the ability of prior AUGs to intercept scanning ribosomes. Specifically, deletions and point mutations of the viral AUGs resulted in increased CAT enzyme activity owing to increased utilization of the downstream CAT AUG. To compare a variety of mutants, we used the levels of increase to calculate the translational efficiency of the viral AUGs. Some of our data agree with predictions of the modified scanning model (MSM). Little variation in downstream CAT AUG utilization was noted regardless of whether the VP2 AUG (in a weak MSM sequence context) was intact or removed. Hence, a scanning ribosome may easily bypass it. Similar analysis of the VP3 AUG (in a favorable MSM sequence context) demonstrated that it could efficiently intercept ribosomes prior to the downstream AUG. Overall, these data indicate that the structure of the 19S late mRNA and the relative efficiency of translational start codon utilization can account for the VP3/VP2 ratio found in infected cells. The agnoprotein reading frame, depending on how the mRNA precursor is spliced, is either not contained in the mRNA or is terminated near the VP2 AUG. Under these conditions, the ability of the agnoprotein AUG to block downstream CAT AUG utilization was found to be minimal in our assay. However, we directly tested the blocking ability of the agnoprotein AUG under conditions in which the reading frame terminated well after the CAT AUG. Although the agnoprotein AUG lies in a very good sequence context, this direct analysis showed that it interfered minimally with utilization of the CAT AUG when under the control of the SV40 late promoter. However, expected high levels of interference were regained when the late promoter was replaced with the Rous sarcoma virus long terminal repeat.(ABSTRACT TRUNCATED AT 400 WORDS)
Insufficiency of transformation by simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes for conversion of ethanolamine-responsive mammary cells to ethanolamine-nonresponsive cells
Authors: Kano-Sueoka T , King D M
Published: January 2020

Normal mammary epithelial cells (ethanolamine responsive) require ethanolamine to enable them to grow in defined culture medium because they cannot synthesize de novo a sufficient amount of phosphatidylethanolamine. Mammary tumor cells which retain properties of the normal tissue are also likely to be ethanolamine responsive, whereas dedifferentiated, highly tumorigenic mammary tumor cells are ethanolamine nonresponsive. The nonresponsive tumor cells are able to synthesize the necessary amount of phosphatidylethanolamine to sustain growth. Therefore, the progression of malignancy seems to convert ethanolamine-responsive mammary cells to ethanolamine-nonresponsive ones. In an attempt to prove the above assumption and to understand the mechanism responsible for the conversion during the progression of malignant transformation, mammary tumor cell line 64-24, which is typically ethanolamine responsive, was transfected with simian virus 40, polyomavirus, EJ-ras, or v-myc oncogenes, and the resulting transfectants were examined for their growth response to ethanolamine. Many of the transfectants exhibited typical transformed phenotypes; however, none of the transfectants converted to ethanolamine-nonresponsive cells. Some of the SV40 and polyomavirus transformants were able to grow in the absence of ethanolamine, although they grew better in the presence of ethanolamine, unlike typical ethanolamine-nonresponsive cells. These cells could grow in the absence of ethanolamine, even though their membrane phospholipid was phosphatidylethanolamine deficient. The present study indicates that the expression of any one of the four oncogenes tested, which allows the cells to exhibit transformed phenotypes in 64-24 cells, is not sufficient for the conversion of ethanolamine-responsive cells to -nonresponsive cells.
Alterations in the regulatory region of the human papillomavirus type 6 genome are generated during propagation in Escherichia coli
Authors: Kasher M S , Roman A
Published: January 2020

We analyzed the long control regions (LCRs) of seven human papillomavirus type 6b (HPV-6b) clones, which contained prototype HPV-6b sequences recloned into various plasmid vectors and propagated in different strains of Escherichia coli. Southern blot analysis and DNA sequencing demonstrated three different sequences, each distinct from the published prototype HPV-6b sequence. Two of the plasmids contained insertions of 24 and 94 base pairs (bp) and a 1-bp deletion. Four plasmids contained insertions of 24 and 58 bp and a deletion of 49 bp. One plasmid contained a single insertion of 77 bp. The 94-, and 58-bp insertions occurred at the same site and had 100% positional identity across their shared lengths. All changes were located in the purine-thymidine-rich region of the LCR (nucleotides 7292 to 7400). Two additional LCR sequences were detected by restriction analysis of two other HPV-6b clones. We conclude that the purine-thymidine-rich region of the LCR is a hot spot for recombination in E. coli and that the alterations are the result of recA-independent events. These results emphasize the need to rigorously prove that a cloned isolate is an authentic copy of the genomic DNA present in the original lesion. In addition, the data indicate that the HPV-6b LCR sequences employed in different laboratories may be different, even if their parental DNAs were identical. Finally, we discuss the need for caution in assigning biological significance to alterations in this region, in view of the limited data available on the true identity of the HPV-6b LCR.
Induction of complement-dependent and -independent neutralizing antibodies by recombinant-derived human cytomegalovirus gp55-116 (gB)
Authors: Britt W J , Vugler L , Stephens E B
Published: January 2020

The human cytomegalovirus (HCMV) envelope glycoprotein complex gp55-116 was expressed in both Escherichia coli and cells infected with a recombinant vaccinia virus. E. coli produced a single protein of Mr 100,000 which approximated the size of the nonglycosylated gp55-116 precursor found in HCMV-infected cells. Cells infected with the recombinant vaccinia virus contained three intracellular forms of Mr 160,000, 150,000, and 55,000 which were detected by a monoclonal antibody reactive with gp55. Comparison of the immunological properties of these recombinant proteins indicated that several of the HCMV gp55-116 monoclonal antibodies and sera from patients infected with HCMV reacted with the vaccinia virus-derived proteins whereas a more restricted group of monoclonal antibodies recognized the E. coli-produced protein. Immunization of mice with either E. coli or vaccinia virus recombinant HCMV gp55-116 resulted in production of virus-neutralizing antibodies. In contrast to the almost exclusive production of complement-dependent neutralizing antibodies following immunization with recombinant vaccinia virus, the E. coli-derived protein induced complement-independent neutralizing antibodies.
The 89,000-Mr murine cytomegalovirus immediate-early protein stimulates c-fos expression and cellular DNA synthesis
Authors: Schickedanz J , Philipson L , Ansorge W , Pepperkok R , Klein R , Koszinowski U H
Published: January 2020

The immediate-early (IE) genes of murine cytomegalovirus (MCMV) are expressed in the absence of prior viral protein synthesis and regulate the transcription of MCMV early genes. The effect of MCMV IE genes on growth induction was studied. Different plasmids containing MCMV IE genes were microinjected into arrested NIH 3T3 mouse fibroblasts. Plasmids containing the ieI gene coding for the 89,000-Mr major IE protein pp89 were found to stimulate the expression of the c-fos protooncogene. Synthesis of pp89 and its transport to the nucleus appeared to be required for c-fos expression. DNA synthesis occurred in cells that were injected with MCMV IE genes and in neighboring cells that were not injected. The results suggest that the phosphoprotein pp89 stimulates cells to enter the cell cycle.
In vivo and in vitro models of demyelinating disease: efficiency of virus spread and formation of infectious centers among glial cells is genetically determined by the murine host
Authors: Wilson G A , Dales S
Published: January 2020

Resistance or susceptibility of various mouse strains to central nervous system disease caused by different strains of coronavirus is well known. Data from the present study draw attention to an additional, genetically determined mechanism controlling CV infections. The resistance to A59 and JHM virus (JHMV) associated with SJL mice was maintained in explanted glial cultures which, by contrast, fully supported a productive infection by the serorelated mouse hepatitis virus type 3. A comparative analysis of the infectious process in glial cell explants from SJL and CD.1 mice helped to define the stage at which restriction is manifested. Cultures of oligodendrocytes and astrocytes from these strains of mice were challenged with JHMV or mouse hepatitis virus type 3, and cell-virus interactions were monitored, including adsorption, uptake of inoculum, transcription, and cell-to-cell dissemination. The sequence of early events from adsorption to genome activation occurred with about equal efficiency with both viruses and genetically different cells, indicating that SJL resistance is not due to any deficiency in specific receptors or penetration of the inoculum or general expression of viral functions. However, intercellular spread of the infection was restricted in SJL glial cells owing to an as yet undefined component. Since cells from (SJL x CD.1)F1 mice were fully susceptible to JHMV, resistance to virus spread must be due to a deficiency in some factor, perhaps a proteolytic activity necessary for dissemination.
Role of the adenovirus E1B 19,000-dalton tumor antigen in regulating early gene expression
Authors: White E , Denton A , Stillman B
Published: January 2020

Mutations in the adenovirus gene encoding the E1B 19-kilodalton protein (the 19K protein) result in pleiotropic phenotypes that affect the host cell and virus growth. Examination of viral gene expression in HeLa cells infected with E1B 19K mutant viruses revealed synthesis and accumulation of E1A proteins to higher steady-state levels than those proteins synthesized during infection with the wild-type virus. As a consequence of elevated E1A levels, another early gene product, the 72K DNA-binding protein, accumulated earlier in mutant-infected cells. In a 12S E1A cDNA virus background, E1B 19K gene mutations had a more profound effect. Larger amounts of the 12S E1A product were present in E1B mutant-infected cells. A deletion mutation that eliminated expression of the 19K protein was also responsible for a 200-fold increased plaque-forming efficiency of the 12S cDNA virus in HeLa cells and an increased rate of virus production. Therefore, the E1B 19K tumor antigen may function to down-regulate virus replication by repressing E1A-dependent gene transcription. Eliminating expression of the E1A 13S and 12S gene products by substitution of an E1A 9S cDNA gene, however, uncovered a stimulatory effect of the E1B 19K protein on early gene expression and virus replication. An E1A 9S virus with a wild-type gene encoding the E1B 19K protein displayed increased early gene transcription, synthesized more 72K DNA-binding protein, and replicated more efficiently than an E1A 9S virus containing a mutation that eliminated expression of the 19K protein. Therefore, the E1B 19K protein has both positive and negative effects on early gene expression and virus replication. In the presence of functional E1A gene products, the 19K protein repressed E1A-dependent gene expression, but in the absence of E1A, the 19K protein stimulated viral gene expression and DNA synthesis. This raises the possibility that the E1B 19K protein functions to repress transcription by modifying the activity of the E1A proteins. Independent of E1A, however, the E1B 19K protein can increase viral gene expression and DNA synthesis, which then leads to increased virus replication.
Asp-286----Asn-286 in polyomavirus large T antigen relaxes the specificity of binding to the polyomavirus origin
Authors: Tang W J , Folk W R
Published: January 2020

We isolated revertants of a polyomavirus whose origin of DNA replication contains a point mutation in the palindrome to which large T antigen binds. Four independent second-site revertants contain an Asp-286----Asn-286 substitution in large T antigen. This mutant large T antigen activates replication of DNAs containing the mutant polyomavirus origin as well as replication of DNAs containing the wild-type origin; however, replication of DNAs with enhancer mutations is not activated by this large T antigen. The Asn-286 mutation occurs in a positively charge region of large T antigen near the location of several mutations which inactivate DNA replication. We suggest that this region of large T antigen is responsible for recognition of specific DNA sequences at the origin and that ionic forces are important for this interaction.
The ets sequence is required for induction of erythroblastosis in chickens by avian retrovirus E26
Authors: Nunn M F , Hunter T
Published: January 2020

E26 is a replication-defective avian retrovirus that causes an erythroblastic leukemia in vivo and transforms hematopoietic precursor cells of both the erythroid and the myeloid lineages in vitro. The E26 genome contains two sets of cell-derived sequences, ets and myb. myb sequences are also present in avian myeloblastosis virus, which transforms myeloblasts exclusively. To determine whether the ets sequence is responsible for the erythroid specificity of E26, we analyzed the transforming activities of several viruses carrying mutations in the ets sequence constructed in vitro. The mutant viruses retained the ability to transform myeloid cells in vitro, indicating that the myb oncogene is sufficient for this viral function. However, the ets-deficient viruses did not cause an overt leukemia in chickens. The results indicate that the ets sequence is required for the induction of erythroblastosis by E26.
Expression in recombinant vaccinia virus of the equine herpesvirus 1 gene encoding glycoprotein gp13 and protection of immunized animals
Authors: Guo P X , Goebel S , Davis S , Perkus M E , Languet B , Desmettre P , Allen G , Paoletti E
Published: January 2020

The equine herpesvirus 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was cloned into the hemagglutinin (HA) locus of vaccinia virus (Copenhagen strain). Expression of the gp13 gene was driven by the early/late vaccinia virus H6 promoter. Metabolically radiolabeled polypeptides of approximately 47 and 44 kilodaltons and 90 kilodaltons (glycosylated form) were precipitated with both polyclonal and gp13-specific monoclonal antibodies. Presentation of gp13 on the cytoplasmic membrane of cells infected with the recombinant gp13 vaccinia virus was demonstrated by immunofluorescence of unfixed cells. Inoculation of the recombinant gp13 vaccinia virus into guinea pigs induced neutralizing antibodies to both EHV-1 and vaccinia virus. Hamsters vaccinated with the recombinant gp13 vaccinia virus survived a lethal challenge with the hamster-adapted Kentucky strain of EHV-1. These results indicate that expression in vaccinia virus vectors of EHV-1 gp13, the glycoprotein homolog of herpes simplex virus gC-1 and gC-2, pseudorabies virus gIII, and the varicella-zoster virus gpV may provide useful vaccine candidates for equine herpesvirus infections.
Both excision and replication of cloned autonomous parvovirus DNA require the NS1 (rep) protein
Authors: Rhode S L
Published: January 2020

When a bacterial plasmid containing the entire genome of LuIII virus except for the terminal 18 nucleotides from the right end is transfected into HeLa cells, the viral DNA is rescued and replicated, with production of infectious virus. This experimental system was used to examine the viral proteins and cis elements required for the excision and replication of viral DNA. The deletion of the entire NS1 gene provided a viral genome that was excised from the plasmid and replicated only when an NS1 gene was provided in trans. A frameshift mutation in the NS2 intron that truncates NS1 prevented excision and replication. Deletion of the left-end terminal inverted repeat or the right-end inverted repeat prevented excision of viral DNA from that end but not from the wild-type terminus. The viral terminus excised from the plasmid was protected from a processive degradation process, which began on the vector portion of the plasmid. The inhibitor of DNA polymerases alpha and delta, aphidicolin, blocked the excision reaction.
Characterization of the translational defect to fiber synthesis in monkey cells abortively infected with human adenovirus: role of ancillary leaders
Authors: Silverman L , Klessig D F
Published: January 2020

Synthesis of the fiber protein of human adenovirus serotype 2 (Ad2) is 100-fold lower in abortively infected monkey cells, compared with productive infections, despite only a 5- to 10-fold reduction in fiber mRNA levels. Previously Anderson and Klessig (Proc. Natl. Acad Sci. USA 81:4023-4027, 1984) demonstrated a direct correlation between the productive nature of the infection, efficient synthesis of the fiber protein in vivo, and the presence of the x or y ancillary leaders on 10 to 25% of fiber messages. To determine at what level in translation these leaders might be important, the relative rate of initiation and elongation of each class of fiber message was assessed. The presence of the y ancillary leader in productively infected cells increased the rate of initiation about twofold, although translational elongation was similar on all fiber messages. However, the rate of elongation of all fiber messages was threefold slower in abortively infected than in productively infected cells. This reduced elongation rate in abortive infections was specific for fiber. The similar distribution of fiber mRNAs on polysomes in both infections suggests that initiation must also be partially blocked in abortive infections. Since the majority of the fiber mRNA even in productive infections did not contain the ancillary leaders, the initiation and elongation defects in the abortive infection cannot be fully explained by the absence of these leaders. Therefore, other factors in the infected cell must be influencing the rate of translation.
Simian virus 40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm
Authors: Ryu W S , Mertz J E
Published: January 2020

Little or no simian virus 40 (SV40) late mRNA accumulates in the cytoplasm when the primary transcript lacks an excisable intervening sequence. To begin to understand why, we analyzed the synthesis, processing, transport, and stability of SV40 late transcripts accumulated in the nucleus and cytoplasm of monkey cells cotransfected with the DNAs of wild-type and mutants of SV40 lacking precisely various introns. The data from these experiments indicated that (i) the presence of excisable intervening sequences in SV40 late transcripts is necessary for efficient accumulation in the cytoplasm of any of the SV40 late RNA species and (ii) SV40 late transcripts lacking excisable intervening sequences are defective in both stability in the nucleus and transport to the cytoplasm but not in stability in the cytoplasm. We hypothesize that SV40 late transcripts need to be processed via a pathway that couples stabilization of the primary transcript within the nucleus, excision of intervening sequences, proper 5'- and 3'-end formation, and transport to the cytoplasm.
Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals
Authors: Psallidopoulos M C , Schnittman S M , Thompson L M , Baseler M , Fauci A S , Lane H C , Salzman N P
Published: January 2020

Evidence of a latent human immunodeficiency virus type 1 (HIV-1) infection in healthy, seropositive individuals who do not have viral antigens in their sera and from whom virions cannot be rescued in cocultivation experiments was examined. Proviral DNA was detected by amplification by the polymerase chain reaction procedure. In each of 10 seropositive individuals, the presence of HIV-1 proviral sequences was demonstrated in their peripheral blood mononuclear cells. By using fluorescence-activated cell sorting, we obtained highly enriched subpopulations of peripheral blood mononuclear cells and found that the CD4+ T-cell subset is the cell subset that consistently harbors the HIV-1 proviral sequences. The number of HIV-1-infected CD4+ T cells was variable among the 10 healthy individuals, ranging from 1 in 100 to 1 in 40,000. While in vitro infection of CD4+ T cells causes down regulation and eventual loss of CD4 surface molecules, this is not true in vivo where it is only the CD4+ population that harbors the virus. This disparity may reflect differences between a latent infection in vivo with the lytic response of cells infected in vitro.
A mutation in the short 5'-proximal open reading frame on Rous sarcoma virus RNA alters virus production
Authors: Petersen R B , Moustakas A , Hackett P B
Published: January 2020

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.
Human papillomavirus type 48
Authors: Müller M , Kelly G , Fiedler M , Gissmann L
Published: January 2020

The cloning and partial characterization of the genome of human papillomavirus (HPV) type 48 is presented. Hybridization and short DNA sequence analyses permitted the alignment of the genome to the HPV genetic map.
Human papillomavirus (HPV) type 50, a type associated with epidermodysplasia verruciformis (EV) and only weakly related to other EV-specific HPVs
Authors: Favre M , Obalek S , Jablonska S , Orth G
Published: January 2020

The cloning and partial characterization of the genome of human papillomavirus type 50 is presented. Alignment of the genomic map with that of human papillomavirus type 5, with which it is only weakly related, was permitted by partial DNA sequence analysis.
Relative rates of RNA synthesis across the genome of Epstein-Barr virus are highest near oriP and oriLyt
Authors: Metzenberg S
Published: January 2020

The rates of Epstein-Barr virus transcription were measured in isolated nuclei from marmoset and human lymphoblasts transformed in vitro. In B95-8, a marmoset B-lymphoid cell line, the most frequently transcribed viral genes are the EBERs (small nuclear RNAs) and BHLF-1 (encoding a lytic-phase gene product). The EBERs and BHLF-1 genes are separated by nearly 50 kilobase pairs on the Epstein-Barr virus genome and lie adjacent to (less than 300 base pairs from) oriP and oriLyt, respectively. oriP and oriLyt are putative origins of viral DNA replication, and each is associated with a transcriptional enhancer element. Among the human B-lymphoblastoid cell lines tested, only the transcription of EBERs predominates.
cis-acting sequences that control the level of viral DNA synthesis in the polyomavirus late region
Authors: Melucci-Vigo G , Ciotta C , Risuleo G
Published: January 2020

A deletion in the polyomavirus late region results in a drastic reduction of viral replication, as shown after transfection of viral DNA into 3T6 cells. This mutation is cis acting, since cotransfection with wild-type DNA did not restore the normal phenotype. Viral DNA synthesis returned to normal levels only after reintroduction of the authentic sequences in either orientation. The data presented here suggest that these sequences are involved in the binding of a factor(s) that controls the level of viral replication.
Effects of alterations in the leader sequence of Rous sarcoma virus RNA on initiation of translation
Authors: Hensel C H , Petersen R B , Hackett P B
Published: January 2020

The 372-nucleotide leader sequence of Rous sarcoma virus RNA contains three conserved short open reading frames and other sequences responsible for a variety of life cycle functions. We have investigated several aspects of the leader RNA which may influence the translation of the major coding regions to which the leader is juxtaposed. We found that small perturbations of the leader length do not affect the binding and scanning of ribosomal subunits by more than about 10%, that the length and/or structure of the RSV RNA leader is near optimal for translation of the major coding regions of the viral RNA, that inclusion or deletion of open reading frames influences downstream initiation in a manner that is not strictly additive, and that reinitiation of translation at the gag gene is very efficient.
Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein
Authors: Gao M , Knipe D M
Published: January 2020

We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protein 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8 gene so that an ICP8-beta-galactosidase fusion protein was expressed. This virus formed blue plaques on ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Mutated ICP8 gene plasmids cotransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinants were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed ICP8 that neither bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen.
High-frequency leader sequence switching during coronavirus defective interfering RNA replication
Authors: Makino S , Lai M M
Published: January 2020

A system was developed that exploited defective interfering (DI) RNAs of coronavirus to study the role of free leader RNA in RNA replication. A cDNA copy of mouse hepatitis virus DI RNA was placed downstream of the T7 RNA polymerase promoter to generate DI RNAs capable of extremely efficient replication in the presence of a helper virus. We demonstrated that, in the DI RNA-transfected cells, the leader sequence of these DI RNAs was switched to that of the helper virus during one round of replication. This high-frequency leader sequence exchange was not observed if a nine-nucleotide stretch of sequence (UUUAUAAAC) at the junction between the leader and the remaining DI sequence was deleted. This observation suggests that a free leader RNA generated from the genomic RNA of mouse hepatitis virus may participate in the replication of DI RNA.
Polyprotein processing of Theiler's murine encephalomyelitis virus
Authors: Roos R P , Kong W P , Semler B L
Published: January 2020

To investigate polyprotein processing of Theiler's murine encephalomyelitis viruses, we analyzed in vitro translation reactions programmed by in vitro-derived transcripts from an infectious full-length cDNA clone of the DA strain of Theiler's virus. To help identify the proteinases that carried out the processing, we modified the DA cDNA clone transcription template by linearization with different restriction endonucleases that generate templates of different lengths or by constructing linker insertion or deletion mutations or both in putative proteinase-coding regions. Protein 3C carried out most of the cleavages of the polyprotein, as is true for the other picornaviruses that have been studied. A second proteinase also appeared active at the LP12A-2B junction. A protein of slightly faster mobility than the leader protein was seen with translation of transcripts derived from DA cDNA but not GDVII cDNA. This protein may be synthesized from an alternative initiation site in the DA leader-coding region out of phase with the polyprotein reading frame. Our findings are relevant to ongoing investigations of the abnormal virus expression seen in DA virus late demyelinating disease, since polyprotein processing is critical in regulating picornaviral gene expression.
Requirements for species-specific papovavirus DNA replication
Authors: Bennett E R , Naujokas M , Hassell J A
Published: January 2020

Replication of papovavirus DNA requires a functional replication origin, a virus-encoded protein, large T antigen, and species-specific permissive factors. How these components interact to initiate and sustain viral DNA replication is not known. Toward that end, we have attempted to identify the viral target(s) of permissive factors. The functionally defined replication origins of polyomavirus and simian virus 40, two papovaviruses that replicate in different species (mice and monkeys, respectively), are composed of two functionally distinct domains: a core domain and an auxiliary domain. The origin cores of the two viruses are remarkably similar in primary structure and have common binding sites for large T antigen. By contrast, their auxiliary domains share few sequences and serve as binding sites for cellular proteins. It seemed plausible, therefore, that if cellular permissive factors interacted with the replication origin, their targets were likely to be in the auxiliary domain. To test this hypothesis we constructed hybrid origins for DNA replication that were composed of the auxiliary domain of one virus and the origin core of the other and assessed their capacity to replicate in a number of mouse and monkey cell lines, which express the large T antigen of one or the other virus. The results of this analysis showed that the auxiliary domains of the viral replication origins could substitute for one another in DNA replication, provided that the viral origin core and its cognate large T antigen were present in a permissive cellular milieu. Surprisingly, the large T antigens of the viruses could not substitute for one another, regardless of the species of origin of the host cell, even though the two large T antigens bind to the same sequence motif in vitro. These results suggest that species-specific permissive factors do not interact with the origin-auxiliary domains but, rather, with either the origin core or the large T antigen or with both components to effect DNA replication.
In vitro construction of poliovirus defective interfering particles
Authors: Hagino-Yamagishi K , Nomoto A
Published: January 2020

To construct poliovirus defective interfering (DI) particles in vitro, we synthesized an RNA from a cloned poliovirus cDNA, pSM1(T7)1, which carried a deletion in the genome region corresponding to nucleotide positions 1663 to 2478 encoding viral capsid proteins, by using bacteriophage T7 RNA polymerase. The RNA was designed to retain the correct reading frame in nucleotide sequence downstream of the deletion. HeLa S3 monolayer cells were transfected with the deletion RNA and then superinfected with standard virus as a helper. The DI RNA was observed in the infected cells after three passages at high multiplicity of infection. The sequence analysis of RNA extracted from the purified DI particle clearly showed that this DI RNA had the same deletion in size and location as that in the RNA used for the transfection. Thus, we succeeded in construction of a poliovirus DI particle in vitro. To gain insight into the mechanism for DI generation, we constructed poliovirus cDNAs pSM1(T7)1a and pSM1(T7)1b that, in addition to the same deletion as that in pSM1(T7)1, had insertion sequences of 4 bases and 12 bases, respectively, at the corresponding nucleotide position, 2978. The RNA transcribed from pSM1(T7)1a was not a template for synthesis of poliovirus nonstructural proteins and therefore was inactive as an RNA replicon. On the other hand, the RNA from pSM1(T7)1b replicated properly in the transfected cells. Superinfection of the transfected cells with standard virus resulted in production of DI particles derived from pSM1(T7)1b and not from pSM1(T7)1a. These observations indicate that deletion RNAs that are inactive replicons have little or no possibility of being genomes of DI particles suggesting the existence of a nonstructural protein(s) that has an inclination to function as a cis-acting protein(s). The method described here will provide a useful technique to investigate genetic information essential for poliovirus replication.
The hepatitis B virus-associated reverse transcriptase is encoded by the viral pol gene
Authors: Bavand M , Feitelson M , Laub O
Published: January 2020

We have used activity gel analysis and immunoblotting to provide evidence linking the hepatitis B virus (HBV) reverse transcriptase with its longest unassigned open reading frame (polymerase [Pol]-ORF). Activity gel analysis demonstrated that infectious HBV particles secreted by the Hep 2.2.15 cell line contain major (approximately 70 kilodaltons [kDa]) and minor (approximately 90 kDa) reverse transcriptase activities. By Western immunoblotting, we detected in both HBV particles and Hep 2.2.15 cell extract a approximately 70-kDa Pol-specific peptide. This approximately 70-kDa peptide reacted with antisera directed against the carboxy terminus of the pol gene product. No such immunoreactivity was observed with antisera against the amino terminus of the Pol peptide. The reverse transcriptase protein which was eluted from the major approximately 70-kDa region detected on an activity gel reacted with Pol-specific antisera. Furthermore, reverse transcriptase activity was immunoprecipitated from dissociated HBV particles by using Pol-specific antisera. On the basis of our results, we suggest that HBV encodes its reverse transcriptase from the Pol-ORF.
Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester
Authors: Gius D , Laimins L A
Published: January 2020

Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICP0 activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICP0 stimulated expression in a wide variety of cells. The element activated by both TIF and ICP0 was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICP0 further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.
Model for intracellular folding of the human immunodeficiency virus type 1 gp120
Authors: Fennie C , Lasky L A
Published: January 2020

The intracellular folding of the human immunodeficiency virus type 1 gp120 has been assessed by analyzing the ability of the glycoprotein to bind to the viral receptor CD4. Pulse-chase experiments revealed that the glycoprotein was initially produced in a conformation that was unable to bind to CD4 and that the protein attained the appropriate tertiary structure for binding with a half-life of approximately 30 min. The protein appears to fold within the rough endoplasmic reticulum, since blocking of transport to the Golgi apparatus by the oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone did not appear to perturb the folding kinetics of the molecule. The relatively lengthy folding time was not due to modification of the large number of N-linked glycosylation sites on gp120, since inhibition of the first steps in oligosaccharide modification by the inhibitors deoxynojirimycin or deoxymannojirimycin did not impair the CD4-binding activity of the glycoprotein. However, production of the glycoprotein in the presence of tunicamycin and removal of the N-linked sugars by endoglycosidase H treatment both resulted in deglycosylated proteins that were unable to bind to CD4, suggesting in agreement with previous results, that glycosylation contributes to the ability of gp120 to bind to CD4. Interestingly, incomplete endoglycosidase H treatment revealed that a partially glycosylated glycoprotein could bind to the receptor, implying that a subset of glycosylation sites, perhaps some of those conserved in different isolates of human immunodeficiency virus type 1, might be important for binding of the viral glycoprotein to the CD4 receptor.
Infection of human peripheral blood mononuclear cells with a temperature-sensitive mutant of measles virus
Authors: Vydelingum S , Ilonen J , Salonen R , Marusyk R , Salmi A
Published: January 2020

A stable temperature-sensitive mutant of measles virus (MV ts38) was used to study the mechanism of virus-mediated immune suppression of peripheral blood mononuclear cells in vitro. Both unstimulated and phytohemagglutinin-stimulated cultures released infectious virus at 32 degrees C, whereas no virus was released at 37 degrees C, although both viral RNA and viral proteins were synthesized. However, the response of the lymphoid cells to phytohemagglutinin, concanavalin A, and herpes simplex virus antigen was decreased in the presence of MV ts38 at 37 degrees C. The viability of infected cells was not diminished, therefore excluding cell death as a reason for immunosuppression. Interleukin 2 did not play a role in the inhibitory effect of MV ts38. Antibodies to alpha interferon partially reversed the inhibitory effect of the virus infection on lymphocyte mitogenesis, thus implying that alpha interferon plays a role in the immunosuppression. Depletion experiments indicated that adherent cells play a greater role in the measles virus-induced immunosuppression than nonadherent cells. However, monocyte maturation to macrophages had no effect on the degree of immunosuppression.
Helicase, DNA-binding, and immunological properties of replication-defective simian virus 40 mutant T antigens
Authors: Auborn K , Guo M , Prives C
Published: January 2020

Simian virus 40 T antigen (TAg) exhibits nonspecific and origin-specific DNA binding (ori binding) and ATPase and helicase activities, all of which are related to its roles in viral DNA replication. We have characterized some of the properties of four replication-defective but transformation-competent mutant TAgs, C6-2, T22, C11, and C8A. C6-2 and T22 TAgs were each previously determined to lack ori-binding properties, while C11 TAg was reported to lack ATPase activity. The C8A TAg did not exhibit defects in either ori-binding or ATPase functions. We have analyzed additional aspects of these mutant TAgs pertaining to their helicase, DNA-binding, and immunological properties. With the exception of the C11 TAg, all the other TAgs exhibited helicase activity. The lack of helicase activity by C11 TAg was consistent with its previously shown inability to hydrolyze ATP or to replicate viral DNA. These results therefore show that ori-binding and helicase activities are separate functions of TAg. Wild-type and mutant TAgs bound with similar efficiency to either native or denatured calf thymus DNA-cellulose, indicating no marked differences in their nonspecific DNA-binding properties. We also tested the binding of wild-type and mutant TAgs to a monoclonal antibody, PAb 100, that was previously shown to recognize an extremely small class of TAg that may represent a unique conformational form of the protein. Interestingly, while less than 10% of the wild-type, C6-2, C11, and T22 mutant TAgs were recognized by PAb 100, more than 60% of the C8A mutant TAg was bound by this antibody. Therefore, although no defect in biochemical function was observed with the C8A TAg, its deficiency in viral DNA replication may be related to an unusual conformation, as detected by its dramatically increased recognition by PAb 100. These results show that the helicase activity of TAg is not required for its transformation function.
Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins
Authors: García-Barreno B , Palomo C , Peñas C , Delgado T , Perez-Breña P , Melero J A
Published: January 2020

Monoclonal antibodies directed against the glycoproteins of human respiratory syncytial virus were used in competitive enzyme-linked immunosorbent assays for topological mapping of epitopes. Whereas epitopes of the F glycoprotein could be ascribed to five nonoverlapping antigenic sites, anti-G antibodies recognized unique epitopes, many of whose competition profiles overlapped extensively. Variant viruses selected with a neutralizing (47F) anti-F antibody lost the binding for only 47F and 49F antibodies, which mapped in the same antigenic area. In contrast, viruses selected with an anti-G antibody lost the capacity to bind most of the anti-G antibodies, and their G protein was not recognized by an anti-virus antiserum, indicating major changes in the antigenic structure of the G molecule. Finally, we found great antigenic variation of the G protein among viral isolates. This occurred even within viruses of the same subtype with only limited divergence of amino acid sequence between strains. All of these data indicate marked differences in the antigenic organization of the G and F glycoproteins of respiratory syncytial virus; we discuss these differences in terms of the chemical structure of the glycoproteins.
Modulation of ppp(A2'p)nA-dependent RNase by a temperature-sensitive mutant of vaccinia virus
Authors: Cohrs R J , Condit R C , Pacha R F , Thompson C L , Sharma O K
Published: January 2020

Activation of the ppp(A2'p)nA (2-5A)-dependent RNase was investigated during the abortive infection of BSC40 cells by a temperature-sensitive mutant of vaccinia virus, ts22. At the nonpermissive temperature, ts22 has an abortive late phenotype. At the onset of late-viral-gene expression, viral mRNA is degraded and rRNA is cleaved into discrete fragments in the absence of prior interferon treatment (R. F. Pacha and R. C. Condit, J. Virol. 56:395-403, 1985). Concomitant with rRNA cleavage, an increase in 2-5A occurred late during infection. Discrete 18S- and 28S-rRNA degradation products from BSC40 cells infected with ts22 at the nonpermissive temperature comigrated in denaturing agarose gels with rRNA cleaved fragments produced by the activation of 2-5A-dependent RNase in uninfected cells transfected with exogenous 2-5A. An increase in 2-5A levels and a similar discrete and characteristic degradation of rRNA were observed in BSC40 cells infected with wild-type vaccinia virus in the presence of isatin-beta-thiosemicarbazone. The results show that the ts22 lesion and the action of isatin-beta-thiosemicarbazone may affect the same pathway, leading to the activation of latent 2-5A-dependent RNase and resulting in indiscriminate RNA degradation and inhibition of viral replication.
Mutations located on both F1 and F2 subunits of the Newcastle disease virus fusion protein confer resistance to neutralization with monoclonal antibodies
Authors: Neyt C , Geliebter J , Slaoui M , Morales D , Meulemans G , Burny A
Published: January 2020

The fusion gene sequence of six Newcastle disease virus escape mutants revealed that residues important for the integrity of antigenic site 1 and antigenic site 2 were located, respectively, on the F2 subunit and within the cysteine-rich domain of the F1 subunit. We further report the antibody-binding capacity of these mutants.