bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly and polarized release of Punta Toro virus and effects of brefeldin A
Authors: Chen S Y , Matsuoka Y , Compans R W
Published: January 2020

Punta Toro virus (PTV), a member of the sandfly fever group of bunyaviruses, is assembled by budding at intracellular membranes of the Golgi complex. We have examined PTV glycoprotein transport, assembly, and release and the effects of brefeldin A (BFA) on these processes. Both the G1 and G2 proteins were transported out of the endoplasmic reticulum (ER) and retained in the Golgi complex in a stable structure, either during PTV infection or when expressed from a vaccinia virus recombinant. BFA treatment causes a rapid and dramatic change in the distribution of the G1 and G2 proteins, from a Golgi pattern to an ER pattern. The G1 and G2 proteins were found to be modified by medial but not trans Golgi network enzymes, in the presence or absence of BFA. We found that BFA blocks PTV release from cells but does not interfere with the intracellular assembly of infectious virions. Further, the BFA block of virus release is fully reversible, with high levels of virus release occurring upon removal of the inhibitor. It was also found that the release of PTV virions is polarized, occurring exclusively from the basolateral surfaces of the polarized Vero C1008 epithelial cell line.
Solubilization and immunoprecipitation of alphavirus replication complexes
Authors: Barton D J , Sawicki S G , Sawicki D L
Published: January 2020

Alphavirus replication complexes that are located in the mitochondrial fraction of infected cells which pellets at 15,000 x g (P15 fraction) were used for the in vitro synthesis of viral 49S genome RNA, subgenomic 26S mRNA, and replicative intermediates (RIs). Comparison of the polymerase activity in P15 fractions from Sindbis virus (SIN)- and Semliki Forest virus (SFV)-infected cells indicated that both had similar kinetics of viral RNA synthesis in vitro but the SFV fraction was twice as active and produced more labeled RIs than SIN. When assayed in vitro under conditions of high specific activity, which limits incorporation into RIs, at least 70% of the polymerase activity was recovered after detergent treatment. Treatment with Triton X-100 or with Triton X-100 plus deoxycholate (DOC) solubilized some prelabeled SFV RIs but little if any SFV or SIN RNA polymerase activity from large structures that also contained cytoskeletal components. Treatment with concentrations of DOC greater than 0.25% or with 1% Triton X-100-0.5% DOC in the presence of 0.5 M NaCl released the polymerase activity in a soluble form, i.e., it no longer pelleted at 15,000 x g. The DOC-solubilized replication complexes, identified by their polymerase activity in vitro and by the presence of prelabeled RI RNA, had a density of 1.25 g/ml, were 20S to 100S in size, and contained viral nsP1, nsP2, phosphorylated nsP3, nsP4, and possibly nsP34 proteins. Immunoprecipitation of the solubilized structures indicated that the nonstructural proteins were complexed together and that a presumed cellular protein of approximately 120 kDa may be part of the complex. Antibodies specific for nsP3, and to a lesser extent antibodies to nsP1, precipitated native replication complexes that retained prelabeled RIs and were active in vitro in viral RNA synthesis. Thus, antibodies to nsP3 bound but did not disrupt or inhibit the polymerase activity of replication complexes in vitro.
Mechanism of altered Sindbis virus neurovirulence associated with a single-amino-acid change in the E2 Glycoprotein
Authors: Tucker P C , Griffin D E
Published: January 2020

The mechanism by which amino acid changes in the E1 and E2 surface glycoproteins of Sindbis virus affect neurovirulence is unknown. We have studied two recombinant viruses which differ in virulence. One (TE) contains Gly and the other (TES) contains Arg at position 172 in E2. TE causes more rapid death than TES in newborn mice. Both viruses replicate similarly in nonneuronal cells, but TE replicates more rapidly in the brains of newborn mice and in neuroblastoma cells. TE also induces earlier viral RNA synthesis in neuroblastoma cells. 35S-labeled TE binds more efficiently to brain and neuroblastoma cells, but not to nonneuronal cells, than TES. We propose that a region of the E2 glycoprotein affected by the amino acid occupying position 172 is important for binding to an alphavirus receptor on neurons and influences neurovirulence by this mechanism.
Variable expression of latent membrane protein in nasopharyngeal carcinoma can be related to methylation status of the Epstein-Barr virus BNLF-1 5'-flanking region
Authors: Hu L F , Minarovits J , Cao S L , Contreras-Salazar B , Rymo L , Falk K , Klein G , Ernberg I
Published: January 2020

Seven virus-coded proteins, the nuclear proteins EBNA-1 to EBNA-6 and the latent membrane protein (LMP), are regularly expressed in Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines. In nasopharyngeal carcinoma (NPC), only EBNA-1 is regularly expressed; LMP is detected in about 65% of the tumors. In Burkitt's lymphoma tumors only EBNA-1 is expressed. We have recently shown that the methylation patterns of the EBV genome varied between these cell types. In virally transformed lymphoblastoid cell lines of normal origin, the EBV DNA is completely unmethylated. In contrast, in the Burkitt's lymphoma-derived cell line Rael and in a nude mouse-passaged NPC tumor, C15, there was an extensive methylation of CpG pairs. The methylation extended into the coding regions of the two expressed genes, EBNA-1 (in both tumor types) and LMP (in C15). Two presumptive control regions were exempted from this overall methylation: the oriP that contains both an origin of DNA replication and an EBNA-1-dependent enhancer and the 5'-flanking region of the BNLF-1 open reading frame that codes for LMP. The latter was only exempted in the LMP expressing NPC. We have now investigated the relation between expression of LMP and methylation of DNA in the 5'-flanking 1 kb region of BNLF-1, coding for LMP. LMP was methylated in 3 of 12 NPC biopsies that did not express LMP but was partially or totally unmethylated in the remaining 9 that expressed the protein. The three BNLF-1 exons were highly methylated in all the tumors. The oriP region was unmethylated in all the tumors, as in the previously studied Rael cell line and nude mouse-passaged NPC. Also, the BamHI W enhancer region involved in the expression of EBNA nuclear proteins was methylated. None of the biopsies expressed EBNA-2. Our data show that the EBV genomes are highly methylated in NPC tumors. The strong reverse correlation between the methylation of the putative control region of the LMP gene and the expression of LMP suggests that methylation has a role in the regulation of this gene.
Isolation of a Marek's disease virus (MDV) recombinant containing the lacZ gene of Escherichia coli stably inserted within the MDV US2 gene
Authors: Cantello J L , Anderson A S , Francesconi A , Morgan R W
Published: January 2020

We have isolated a stable, recombinant Marek's disease virus (MDV) containing the lacZ gene of Escherichia coli inserted into the unique short region of the genome. The nucleotide sequence of the insertion site indicates that it lies within a sequence homologous to the US2 gene of herpes simplex virus. Stable insertion of the lacZ gene into the MDV US2 gene indicates that the site is nonessential for MDV growth in cell culture.
Autonomous parvovirus DNA replication requires topoisomerase I and its activity is increased during infection
Authors: Gu M L , Rhode S L
Published: January 2020

Topoisomerases I and II (topo I and topo II) are nuclear enzymes functioning to resolve DNA topological problems during replication, transcription, and other DNA processes. We tested the effects of camptothecin and VP16, specific inhibitors of topo I and II, respectively, on the DNA replication of parvoviruses LuIII and H-1 and found that viral DNA synthesis was suppressed by camptothecin but not by VP16. Transcription of H-1 virus was measured by a nuclear runoff assay and showed no inhibition by camptothecin. Interestingly, topo I in the LuIII virus-infected cell nuclear extract appears to have more activity for covalently binding to viral DNA than that in mock-infected cell nuclear extracts. Our data suggested that this activity was not due to an increased transcription of the topo I gene or to greater amounts of topo I.
Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus
Authors: Gribaudo G , Lembo D , Cavallo G , Landolfo S , Lengyel P
Published: January 2020

The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.
Altered pathogenesis in herpes simplex virus type 1 infection due to a syncytial mutation mapping to the carboxy terminus of glycoprotein B
Authors: Goodman J L , Engel J P
Published: January 2020

A syncytial (syn) variant of herpes simplex virus type 1 strain 17 syn+ was selected by serial passage in heparin, a glycosaminoglycan which potently inhibits herpes simplex virus infectivity. This virus, 17 hep syn, is sixfold more heparin resistant than its parent. By using marker transfer techniques, its syn phenotype, but not heparin resistance, was mapped first to the BamHI G fragment (0.343 to 0.415 map units) and then to a 670-bp KpnI-PstI subclone (0.345 to 0.351 map units) encoding the carboxy terminus of glycoprotein B (gB). Three cloned syncytial recombinants were generated from cotransfections of 17 syn+ with either 17 hep syn BamHI-G or the 670-bp subclone. After footpad inoculation of mice, 17 hep syn was as virulent as its parent, despite reaching lower titers in feet, sciatic nerves, dorsal root ganglia, spinal cords, and brains. Animals infected with 17 hep syn or the gB recombinant viruses developed a unique pattern of disease that was strikingly different than that seen with wild-type virus: severe inflammation and edema of the inoculated limb and death without antecedent paralysis. Histopathologic examination revealed limitation of spinal involvement by 17 hep syn to the dorsal aspect of the cord and decreased virus-induced damage in the central nervous system. The genetically unrelated syn variant MP, in contrast, was avirulent and did not cause severe local inflammation. After intracerebral inoculation, 17 hep syn was highly virulent and replicated to high titers in the brain. Yet, unlike the parental virus, it resulted in an altered distribution of herpes simplex virus antigens, which were limited to the ependymal and subependymal regions surrounding the lateral ventricles. Despite their syncytial phenotype and pathogenic properties, the recombinant viruses, unlike 17 hep syn, were not heparin resistant. We conclude that a transferable alteration in the 670-bp carboxy-terminal portion of the glycoprotein gB gene of 17 hep syn results in both its syncytial phenotype and the unique pattern of disease that it causes but does not result in heparin resistance. These observations provide direct biological evidence for an important role for herpes simplex virus gB in pathogenic events both at the peripheral site of infection and within the nervous system.
Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence
Authors: Salvato M , Borrow P , Shimomaye E , Oldstone M B
Published: January 2020

Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype.
Absent or rare human immunodeficiency virus infection of bone marrow stem/progenitor cells in vivo
Authors: Davis B R , Schwartz D H , Marx J C , Johnson C E , Berry J M , Lyding J , Merigan T C , Zander A
Published: January 2020

An important question in human immunodeficiency virus (HIV) pathogenesis is whether HIV-infected bone marrow CD34+ stem/progenitor cells serve as a significant reservoir of virus in HIV-infected individuals. Our data indicate that infection of bone marrow stem/progenitor cells with HIV occurs rarely, if ever, in vivo. In the present study, CD34+ cells were immunomagnetically purified from the bone marrow of HIV-seropositive individuals, and purified cells or colony-forming cells of the granulocyte/macrophage lineage were analyzed for HIV proviral DNA by the polymerase chain reaction. No HIV DNA was detected in colony-forming cells of the granulocyte/macrophage lineage from HIV-positive patients. Furthermore, no virus was found in CD34(+)-enriched cells from six of seven samples from asymptomatic HIV-infected individuals and four of four samples from patients with AIDS-related complex or AIDS. Thus, infected stem cells are not a major source of persistent HIV and do not account for hematopoietic suppression. These findings have positive implications for the concept of marrow reconstitution with autologous stem cells, genetically engineered for HIV resistance, following marrow-ablative antiviral therapy.
Strain-specific neutralizing determinant in the transmembrane protein of simian immunodeficiency virus
Authors: Kodama T , Burns D P , Silva D P , Veronese F D , Desrosiers R C
Published: January 2020

Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn-Asp) determined the strain specificity of the monoclonal antibody. This strain-specific neutralizing determinant is located within a variable region of SIVmac and human immunodeficiency virus type 2 (HIV-2) which includes conserved, clustered sites for N-linked glycosylation. The determinant corresponds exactly to a variable, weak neutralizing epitope in HIV-1 TMP which also includes conserved, clustered sites for N-linked glycosylation. Thus, the location of at least one neutralizing epitope appears to be common to both SIVmac and HIV-1. Our results suggest a role for this determinant in the viral entry process. Genetic variation was observed in this neutralizing determinant following infection of a rhesus monkey with molecularly cloned SIVmac239; variant forms of the strain-specific, neutralizing determinant accumulated during persistent infection in vivo. Selective pressure from the host immune response in vivo may result in sequence variation in this neutralizing determinant.
Human transformed trophoblast-derived cells lacking CD4 receptor exhibit restricted permissiveness for human immunodeficiency virus type 1
Authors: Zachar V , Spire B , Hirsch I , Chermann J C , Ebbesen P
Published: January 2020

We investigated the nature of interaction of the malignantly transformed cell lines of trophoblast origin BeWo, JAR, and JEG-3 with three different human immunodeficiency virus type 1 (HIV-1) isolates (RF, 3B, and NDK). After inoculation with cell-free virus, the persistence of infection was determined for 1 month by monitoring the presence of viral DNA in the cells by the polymerase chain reaction (PCR). Furthermore, the infectious virus in the culture supernatant was assayed with CEM-SS cells, and attempts to rescue the virus by cocultivation with CEM-SS cells were made. Appraised on the basis of the relative amount of viral DNA and the frequency of positive cocultivation. JEG-3 was the most permissive and BeWo was the least permissive cell line. However, when the cells were transfected with two biologically active molecular clones of HIV-1, the BRU and NDK isolates, all three cell lines turned out to support the production of mature virus progeny to the same extent. The abundance of viral DNA sequences in the infected cells varied with the isolate, showing an overall decline from RF to NDK. The amount of viral DNA in the cells and its expression decreased during the period of observation; this decrease was mirrored in an erosion of the virus recovery rate at cocultivation from 71% recovery on day 8 to failure of isolation on day 32. None of the cell lines expressed detectable amounts of cell surface CD4 molecules when assayed by flow microfluorometry and direct radioimmunoassay. Northern (RNA) blot hybridization analysis of both the total RNA and the mRNA did not reveal any CD4-specific message: nonetheless, by using the PCR, sequences specifically related to the CD4 gene were uncovered. The data demonstrate that the trophoblast-derived cell lines are susceptible to infection with HIV and that they support transient viral replication in the initial phases of infection. However, the latent form of infection may persist over a period of several weeks.
Antigenic and protein sequence homology between VP13/14, a herpes simplex virus type 1 tegument protein, and gp10, a glycoprotein of equine herpesvirus 1 and 4
Authors: Whittaker G R , Riggio M P , Halliburton I W , Killington R A , Allen G P , Meredith D M
Published: January 2020

Monospecific polyclonal antisera raised against VP13/14, a major tegument protein of herpes simplex virus type 1 cross-reacted with structural equine herpesvirus 1 and 4 proteins of Mr 120,000 and 123,000, respectively; these proteins are identical in molecular weight to the corresponding glycoprotein 10 (gp10) of each virus. Using a combination of immune precipitation and Western immunoblotting techniques, we confirmed that anti-VP13/14 and a monoclonal antibody to gp10 reacted with the same protein. Sequence analysis of a lambda gt11 insert of equine herpesvirus 1 gp10 identified an open reading frame in equine herpesvirus 4 with which it showed strong homology; this open reading frame also shared homology with gene UL47 of herpes simplex virus type 1 and gene 11 of varicella-zoster virus. This showed that, in addition to immunological cross-reactivity, VP13/14 and gp10 have protein sequence homology; it also allowed identification of VP13/14 as the gene product of UL47.
Mutual functional antagonism of the simian virus 40 T antigen and the hepatitis B virus trans activator
Authors: Seto E , Yen T S
Published: January 2020

The hepatitis B virus X protein (pX) trans activates transcription of a wide variety of viral and cellular genes, apparently by interacting with multiple cellular transcription factors. It has been shown previously that the simian virus 40 early-region gene products (large-T and small-t antigens) prevent trans activation by pX. We show here that this phenomenon can be ascribed solely to the large-T antigen and that T antigen binds to pX in vitro. Mapping studies reveal that the region of large-T antigen around residues 125 and 126 is critical for this binding and also for the ability of T antigen to prevent trans activation by pX. Furthermore, pX in turn interferes with two of the known functions of T antigen, transcriptional trans activation and simian virus 40 DNA replication. We propose that pX and T antigen inactivate each other by forming a nonfunctional complex in vivo.
Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles
Authors: Almela M J , González M E , Carrasco L
Published: January 2020

The entry of animal viruses into cells is associated with permeabilization of the infected cells to protein toxins such as alpha-sarcin (C. Fernández-Puentes and L. Carrasco, Cell 20:769-775, 1980). This phenomenon has been referred to as "the early permeabilization by animal viruses" (L. Carrasco, Virology 113:623-629, 1981). A number of inhibitors of poliovirus growth such as WIN 51711 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DEPC) and Ro 09-0410 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabilization induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabilization of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalized along with heparin in endosomes in the presence of WIN 51711, DEPC, or Ro 09-0410. We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered.
Structural topography of simian virus 40 DNA replication
Authors: Schirmbeck R , Deppert W
Published: January 2020

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.
Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function
Authors: Wong S W , Schaffer P A
Published: January 2020

Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication are flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays.
Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis
Authors: Giachetti C , Semler B L
Published: January 2020

A molecular genetic analysis has been combined with an in vitro biochemical approach to define the functional interactions required for nucleotidyl protein formation during poliovirus RNA synthesis. A site-directed lesion into the hydrophobic domain of a viral membrane protein produced a mutant virus that is defective in RNA synthesis at 39 degrees C. The phenotypic expression of this lesion affects initiation of RNA synthesis, in vitro uridylylation of the genome-linked protein (VPg), and the in vivo synthesis of plus-strand viral RNAs. Our results support a model that employs a viral membrane protein as carrier for VPg in the initiation of plus-strand RNA synthesis. Our data also suggest that a separate mechanism could be used in the initiation of minus-strand RNA synthesis, thereby providing a means for strand-specific regulation of picornavirus RNA replication.
Formation and selection of intergenogroup reassortants during cell culture adaptation of rotaviruses from dually infected subjects
Authors: Ward R L , Nakagomi O , Knowlton D R , McNeal M M , Nakagomi T , Huda N , Clemens J D , Sack D A
Published: January 2020

A previous study showed that intergenogroup reassortants of human rotaviruses can persist in nature (R.L. Ward, O. Nakagomi, D.R. Knowlton, M.M. McNeal, T. Nakagomi, J.D. Clemens, D.A. Sack, and G.M. Schiff, J. Virol. 64:3219-3225, 1990), but the mechanisms involved in their formation and selection had not been determined. In this study it was shown that, during cell culture adaptation of rotaviruses belonging to different genogroups from stools of dually infected subjects, intergenogroup reassortants were formed and selected, presumably mimicking the processes that occur in nature.
Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins
Authors: Brown C S , Van Lent J W , Vlak J M , Spaan W J
Published: January 2020

Empty parvovirus B19 capsids were isolated from insect cells infected with a recombinant baculovirus expressing parvovirus B19 VP2 alone and also with a double-recombinant baculovirus expressing both VP1 and VP2. That VP2 alone can assemble to form capsids is a phenomenon not previously observed in parvoviruses. The stoichiometry of the capsids containing both VP1 and VP2 was similar to that previously observed in parvovirus B19-infected cells. The capsids were similar to native capsids in size and appearance, and their antigenicity was demonstrated by immunoprecipitation and enzyme-linked immunosorbent assay with B19-specific antibodies.
Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2
Authors: Equestre M , Genovese D , Cavalieri F , Fiore L , Santoro R , Perez Bercoff R
Published: January 2020

Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.
Direct entry of rabies virus into the central nervous system without prior local replication
Authors: Shankar V , Dietzschold B , Koprowski H
Published: January 2020

Rabies virus pathogenesis was studied in a mouse model by inoculation of the masseter muscle. At different intervals, the masseter muscle, trigeminal ganglia, and brain were analyzed for virus-specific RNA with a polymerase chain reaction assay, which revealed that as early as 18 h postinfection (p.i.), virus-specific RNA was present in the trigeminal ganglia, and at 24 h p.i., viral RNA was identified in the brain stem. Analysis of the masseter muscle demonstrated virus at 1 h p.i. but no virus-specific RNA between 6 and 30 h p.i., indicating that virus invaded the nerve ending directly, without prior replication in the muscle. At 36 h p.i., viral RNA was detected again in the masseter muscle. Selective amplification of plus- and minus-strand RNA isolated from the masseter muscle at 96 h p.i. revealed that the majority of the rabies virus-specific RNA was in the positive sense, suggesting virus replication in muscle tissue during late stages of infection.
Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera
Authors: van Zijl M , Wensvoort G , de Kluyver E , Hulst M , van der Gulden H , Gielkens A , Berns A , Moormann R
Published: January 2020

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).
Chicken ovalbumin upstream promoter transcription factor binds to a negative regulatory region in the human immunodeficiency virus type 1 long terminal repeat
Authors: Cooney A J , Tsai S Y , O'Malley B W , Tsai M J
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains a negative regulatory element (NRE) which downregulates the rate of LTR-directed transcription and HIV-1 replication. Within the NRE is a GGTCA palindrome, which binds a possible member of the steroid/thyroid hormone receptor superfamily. Mutation of this site leads to an increase in LTR-directed transcriptional activity compared with the wild type, consistent with the element's being a functional part of the NRE. The palindrome contains significant identity to the chicken ovalbumin upstream promoter (COUP) element to which COUP transcription factors (COUP-TFs), members of the steroid/thyroid hormone receptor superfamily, bind. We demonstrate here that human COUP-TFs can bind specifically to this HIV-1 COUP-like element in a manner identical to binding to ovalbumin COUP. We show that the predominant COUP-TF family member synthesized in T cells is the 68-kDa form, which is likely to be responsible for any in vivo function of the HIV-1 COUP-like element in these cells. Finally, we have identified three HIV-1 variant strains that contain mutations in the HIV-1 COUP-like element which affect the binding affinity of COUP-TF for these variant COUP elements.
Differentiation-associated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia
Authors: Young L S , Lau R , Rowe M , Niedobitek G , Packham G , Shanahan F , Rowe D T , Greenspan D , Greenspan J S , Rickinson A B
Published: January 2020

The BZLF1 protein of Epstein-Barr virus (EBV) is a key immediate-early protein which has been shown to disrupt virus latency in EBV-infected B cells. We have generated a monoclonal antibody, BZ1, to BZLF1 which reacts in immunohistology, immunoblotting, and immunoprecipitation and which recognizes both the active, dimeric form and the inactive, monomeric form of the protein. Biopsies of oral hairy leukoplakia, an AIDS-associated lesion characterized by high-level EBV replication, were examined by immunohistochemistry using the BZ1 monoclonal antibody. A differentiation-associated pattern of BZLF1 expression was observed, BZ1 reacting with nuclei of the upper spinous layer of the lesion. This finding suggests that the BZLF1 promoter may be regulated by the degree of squamous differentiation. A comparison of in situ hybridization to EBV DNA and viral capsid antigen staining with BZ1 reactivity suggested that BZLF1 expression precedes rampant virus replication. The inability to detect EBV in the lower epithelial layers of oral hairy leukoplakia raises questions concerning the nature of EBV latency and persistence in stratified squamous epithelium.
Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses
Authors: Holland J J , de la Torre J C , Clarke D K , Duarte E
Published: January 2020

We describe a sensitive, internally controlled method for comparing the genetic adaptability and relative fitness of virus populations in constant or changing host environments. Certain monoclonal antibody-resistant mutants of vesicular stomatitis virus can compete equally during serial passages in mixtures with the parental wild-type clone from which they were derived. These genetically marked "surrogate wild-type" neutral mutants, when mixed with wild-type virus, allow reliable measurement of changes in virus fitness and of virus adaptation to different host environments. Quantitative fitness vector plots demonstrate graphically that even clones of an RNA virus are composed of complex variant populations (quasispecies). Variants of greater fitness (competitive replication ability) were selected within very few passages of virus clones in new host cells or animals. Even clones which were well adapted to BHK21 cells gained further fitness during repeated passages in BHK21 cells.
Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59
Authors: Denison M R , Zoltick P W , Leibowitz J L , Pachuk C J , Weiss S R
Published: January 2020

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.
vRel is an inactive member of the Rel family of transcriptional activating proteins
Authors: Richardson P M , Gilmore T D
Published: January 2020

The vRel oncoprotein is member of a family of related proteins that also includes cRel, NF-kappa B, and Dorsal. We investigated the transcriptional regulatory properties of several Rel proteins in cotransfection assays with chicken embryo fibroblasts (CEF). Retroviral vectors expressing hybrid proteins that contain the DNA-binding domain of LexA fused to portions of the viral oncoprotein vRel or chicken, mouse, human, or Drosophila melanogaster (Dorsal) cRel proteins were cotransfected with a reporter plasmid that contains the DNA sequence recognized by LexA, a promoter, and the assayable gene for chloramphenicol acetyltransferase. In transient assays, a LexA-vRel protein did not activate transcription in CEF. Full-length chicken cRel, mouse cRel, and Dorsal fusion proteins all activated transcription weakly; however, deletion of N-terminal Rel sequences from each of these proto-oncogene encoded proteins resulted in strong activation by LexA fusion proteins containing only C-terminal sequences. Inhibition of the C-terminal chicken cRel gene activation domain by N-terminal sequences was seen in CEF and mouse and monkey fibroblasts. These results show that cRel proteins from different species have the same general organization: an N-terminal inhibitory domain and a C-terminal activation domain. Sequence comparison suggests that the inhibitory domain is conserved but the activation domain is species specific. In contrast, vRel lacks a strong C-terminal gene activation function, since a LexA fusion protein containing C-terminal vRel sequences alone only weakly activated transcription. In addition, the wild-type vRel protein (lacking LexA sequences) repressed transcription from reporter plasmids containing NF-kappa B target sequences; nontransforming vRel mutants did not repress transcription from these plasmids. Our results suggest that vRel transforms cells by interfering with transcriptional activation by cellular Rel proteins.
A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs
Authors: van der Most R G , Bredenbeek P J , Spaan W J
Published: January 2020

Two murine hepatitis virus strain A59 defective interfering (DI) RNAs were generated by undiluted virus passages. The DI RNAs were encapsidated efficiently. The smallest DI particle, DI-a, contained a 5.5-kb RNA consisting of the following three noncontiguous regions from the MHV-A59 genome, which were joined in frame: the 5'-terminal 3.9 kb, a 798-nucleotide fragment from the 3' end of the polymerase gene, and the 3'-terminal 805 nucleotides. A full-length cDNA clone of the DI-a genome was constructed and cloned downstream of the bacteriophage T7 promoter. Transcripts derived from this clone, pMIDI, were used for transfection of MHV-A59-infected cells and found to be amplified and packaged. Deletion analysis of pMIDI allowed us to identify a 650-nucleotide region derived from the 3' end of the second open reading frame of the polymerase gene that was required for efficient encapsidation.
Human parainfluenza virus type 3 transcription in vitro: role of cellular actin in mRNA synthesis
Authors: De B P , Lesoon A , Banerjee A K
Published: January 2020

Purified ribonucleoprotein complexes of human parainfluenza virus type 3 (HPIV-3) virions required, in addition to the viral proteins, soluble cytoplasmic proteins from uninfected cells for the synthesis of mRNAs in vitro. In contrast to Sendai virus transcription, in vitro RNA synthesis from HPIV-3 ribonucleoprotein complexes was not stimulated significantly by purified tubulin. Moreover, cytoplasmic extract depleted of tubulin by immunoprecipitation stimulated HPIV-3 transcription effectively, suggesting involvement of a host protein(s) other than tubulin in the HPIV-3 transcription process. The transcription stimulatory factor was purified from uninfected cell extract by conventional chromatography and was found to contain a major 43-kDa polypeptide. In Western blot (immunoblot) analysis, this protein reacted with antiactin antibody, suggesting that the 43-kDa polypeptide is actin. This possibility was further supported by its polymerization activity and properties of binding to blue-Sepharose and heparin-Sepharose columns. Furthermore, when the cell extract was depleted of actin by immunoprecipitation by antiactin antibody, the stimulatory activity was abolished, indicating an involvement of actin in the stimulation of HPIV-3 transcription. After purification from RNAses, similar stimulatory activity associated with the 43-kDa protein was detected in other cell lines as well, including CV-1, HeLa, and BHK.
Premature stop codons in the G glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies
Authors: Rueda P , Delgado T , Portela A , Melero J A , García-Barreno B
Published: January 2020

Mutants of human respiratory syncytial (RS) virus which escaped neutralization by monoclonal antibodies directed against the G glycoprotein were selected from the Long strain. Most mutants showed drastic antigenic changes, reflected in the lack of reactivity with several anti-G antibodies, including the one used for selection. Sequence analysis revealed the presence of in-frame premature stop codons in the mutated G genes which shortened the G polypeptide by between 11 and 42 amino acids. In contrast, two mutants selected with monoclonal antibody 25G contained two amino acid substitutions (Phe-265----Leu and Leu-274----Pro) and had lost only the capacity to bind the antibody used in their selection. These results demonstrate that the carboxy-terminal end of the G molecule is dispensable for infectivity in tissue culture and indicate the importance of this part of the G protein in determining its antigenicity.
Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells
Authors: Novak J E , Kirkegaard K
Published: January 2020

We have developed a ribonuclease protection method suitable for sensitive detection of an RNA species in the presence of a large excess of its complementary strand, as for the detection of negative strands of positive-strand RNA viruses. By using this method to probe for poliovirus negative strands in virions, we found that positive strands are present in at least 40,000-fold excess over negative strands. Thus, we have confirmed that poliovirus encapsidation is highly specific for positive strands and have demonstrated that the genome-linked protein VPg, which is covalently attached to the 5' ends of both positive and negative strands, cannot be the sole determinant of RNA packaging. We tested the ratios of viral positive strands to negative strands in cells at different times during infection; this value ranged from approximately 40/1 to 70/1, being highest at 4 h and lower at 2 and 6 h postinfection.
Role of CD4+ and CD8+ T cells in murine resistance to street rabies virus
Authors: Perry L L , Lodmell D L
Published: January 2020

Mice of the SJL/J and BALB/cByJ inbred strains are naturally resistant to street rabies virus (SRV) injected via the intraperitoneal route. To determine the cellular mechanism of resistance, monoclonal antibodies specific for CD4+ or CD8+ subsets of T cells were used to deplete the respective cell population in SRV-infected animals. Elimination of CD4+ T-helper cells abrogated the production of immunoglobulin G (IgG) neutralizing antibodies in response to rabies virus infection and reversed the resistant status of SJL/J and BALB/cByJ mice. In contrast, in vivo depletion of CD8+ cytotoxic T cells had no measurable effect on host resistance to SRV. These results indicate that serum neutralizing antibodies of the IgG class are a primary immunological mechanism of defense against rabies virus infection in this murine model of disease. CD8+ cytotoxic T lymphocytes, which have been shown to transfer protection in other rabies virus systems, appear to have no role in protecting mice against intraperitoneally injected SRV.
Biosynthesis of the secretory core protein of duck hepatitis B virus: intracellular transport, proteolytic processing, and membrane expression of the precore protein
Authors: Schlicht H J
Published: January 2020

The biosynthesis of the secretory core gene product of the duck hepatitis B virus (DHBe protein) was examined. Recombinant vaccinia viruses were constructed encoding either the full-length or C-terminally truncated forms of the DHBe precursor protein (precore protein) and used to express these proteins in the human hepatoma cell line HepG2. Western immunoblot analysis of core gene products isolated from cells producing the full-length precore protein revealed the presence of DHBe precursor proteins containing the strongly basic C-terminal sequence which is lacking in the mature DHBe protein. These proteins were not secreted, suggesting that C-terminal proteolytic processing of the precore protein represents an obligatory step for DHBe biosynthesis. Pulse-chase experiments showed that this cleavage reaction occurs late during DHBe synthesis. Interestingly, when mutated precore proteins were expressed which lacked the basic C-terminal domain, proteins were produced which were glycosylated but not secreted. This shows that the transient presence of this region is essential for intracellular transport of the precore protein. Cell sorter analyses revealed that production of a cell surface-expressed variant of the secretory core protein is a feature conserved between the duck and the human hepatitis B viruses. Surprisingly, the C terminus of the membrane-expressed DHBe protein was accessible from the outside, showing that the topology of this interesting protein is more complicated than expected.