bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribozymes which cleave arenavirus RNAs: identification of susceptible target sites and inhibition by target site secondary structure
Authors: Xing Z , Whitton J L
Published: January 2020

The development of safe and effective antiviral agents has been a slow process, largely because of the difficulty in distinguishing between virus and host functions; materials toxic to the virus are frequently harmful also to the host in which the agent resides. Recently, techniques which target nucleic acid sequences as a means of reducing gene expression have emerged. This antisense armamentarium includes ribozymes, RNA enzymes which cleave other RNA molecules in a sequence-specific manner. We wish to assess the ability of ribozymes to control animal virus infection. Reasoning that the viruses most vulnerable to ribozyme intervention will be those whose complete life cycle is based on RNA (with no DNA stage), we have begun to develop ribozymes directed toward lymphocytic choriomeningitis virus (LCMV), the prototype of the arenavirus family. Using ribozymes of the hammerhead variety, we have identified several sites on the LCMV genome which can be efficiently cleaved in trans. The efficiency of cleavage is site dependent, and we demonstrate that secondary structure at the target site can abolish ribozyme cleavage. Computer-assisted analysis indicates that much of the LCMV genome may be involved in base pairing, which may render it similarly resistant to ribozyme attack. The few remaining open regions of LCMV lack a GUC target site, on which most studies to date have relied. Here we show that AUC, CUC, and AUU are alternative sites which can be cleaved by trans-acting ribozymes. This finding is important given the aforementioned restriction of available sites, imposed by secondary structure.
Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement
Authors: Jilbert A R , Wu T T , England J M , Hall P M , Carp N Z , O'Connell A P , Mason W S
Published: January 2020

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.
Ty3 GAG3 and POL3 genes encode the components of intracellular particles
Authors: Hansen L J , Chalker D L , Orlinsky K J , Sandmeyer S B
Published: January 2020

Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes. It is distinct from the copialike Ty1 and Ty2 retrotransposons of S. cerevisiae in both the sequences of encoded proteins and gene order. It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses. This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon. Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S. These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins. In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins. These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins. Ty3 integrase proteins of 61 and 58 kDa were identified previously (L. J. Hansen and S. B. Sandmeyer, J. Virol. 64:2599-2607, 1990). Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates. Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins. Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter. Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses.
Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation
Authors: Duke G M , Hoffman M A , Palmenberg A C
Published: January 2020

The nucleotide sequence of the 5' nontranslated region of encephalomyocarditis virus (EMCV-Rueckert) was determined, and a consensus RNA structural model for this sequence (850 bases) and three other poly(C)-containing cardioviruses (mengovirus, EMCV-B, and EMCV-D) was created through reiterative use of a minimum-free-energy folding algorithm. The RNA elements within this region which contribute to translation of EMCV proteins were mapped in cell-free reactions programmed with cDNA-derived RNA transcripts. The data provide evidence that stem-loop motifs I, J and K, formed by viral bases 451 to 785, are important components of cap-independent translation. In contrast to other reports, a minimal role for stem-loop H (bases 406 to 444) in translational activity is indicated. Small 5' nontranslated region fragments (bases 667 to 797) containing the J and K motifs proved strong competitive inhibitors when added to cell-free reactions programmed with exogenous capped or uncapped mRNAs. The putative sequestering of required translational factors by this segment clearly contributes to translational activity, but also suggests a possible competitive mechanism for the down regulation of host protein synthesis during viral infection.
Identification and characterization of an extracellular envelope glycoprotein affecting vaccinia virus egress
Authors: Duncan S A , Smith G L
Published: January 2020

Sequence analysis of the vaccinia virus strain Western Reserve genome revealed the presence of an open reading frame (ORF), SalL4R, which has the potential to encode a transmembrane glycoprotein with homology to C-type animal lectins (G. L. Smith, Y. S. Chan, and S. T. Howard, J. Gen. Virol. 72:1349-1376, 1991). Here we show that the SalL4R gene is transcribed late during infection from a TAAATG motif at the beginning of the ORF. Antisera raised against a TrpE-SalL4R fusion protein identified three glycoprotein species of Mr 22,000 to 24,000 in infected cells. Immunogold electron microscopy demonstrated that SalL4R protein is present in purified extracellular enveloped virus particles but not in intracellular naked virus (INV). A mutant virus was constructed by placing a copy of the SalL4R ORF downstream of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible vaccinia virus promoter within the thymidine kinase locus and subsequently deleting the endogenous SalL4R gene. The growth kinetics of this virus demonstrated that SalL4R was nonessential for the production of infectious INV but was required for virus dissemination. Consistent with this finding, the formation of wild-type-size plaques by this mutant was dependent on the presence of IPTG. Electron microscopy showed that without SalL4R expression, the inability of the virus to spread is due to a lack of envelopment of INV virions by Golgi-derived membrane, a morphogenic event required for virus egress.
Protective immune response against foot-and-mouth disease
Authors: McCullough K C , De Simone F , Brocchi E , Capucci L , Crowther J R , Kihm U
Published: January 2020

The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of the Aphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Th-lymphocyte epitopes (4, 5, 7, 8), although a Th-lymphocyte-independent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter work was performed in mice, and it is not certain that a similar situation would be found in cattle. As for the major effector immune defense, this relies on the interaction between antibody-virus complexes and the phagocytic cells of the reticuloendothelial system (17, 19).
Human antibodies recognize multiple distinct type-specific and cross-reactive regions of the minor capsid proteins of human papillomavirus types 6 and 11
Authors: Yaegashi N , Jenison S A , Batra M , Galloway D A
Published: January 2020

Human serum samples derived from a case-control study of patients with cervical carcinoma (n = 174) or condyloma acuminatum (n = 25) were tested for the presence of immunoglobulin G antibodies to human papillomavirus type 6 (HPV6) L2 and HPV11 L2 recombinant proteins in a Western immunoblot assay. Thirty-six samples (18%) were positive for HPV6 L2 antibodies alone, 25 (13%) were positive for HPV11 L2 antibodies alone, and 34 (17%) were positive for both HPV6 L2 and HPV11 L2 antibodies. Thirty samples that were positive for both antibodies were tested for the presence of HPV6-HPV11 L2 cross-reactive antibodies. Fifteen (50%) serum samples contained HPV6-HPV11 L2 cross-reactive antibodies, and 15 (50%) contained independent, type-specific HPV6 L2 and HPV11 L2 antibodies. Altogether, 82% of the HPV6 L2 and HPV11 L2 antibody reactivities were type specific and 18% were HPV6-HPV11 cross-reactive. There was no significant difference in the prevalence of antibody reactivities between samples from patients with cervical carcinoma and those with condyloma acuminatum. Deletion mapping identified five HPV6 L2 regions that reacted with HPV6 type-specific antibodies: 6U1 (amino acids [aa] 152 to 173), 6U2 (aa 175 to 191), 6U3 (aa 187 to 199), 6U4 (aa 201 to 217), and 6U5 (aa 351 to 367). Five HPV11 L2 regions that reacted with HPV11 type-specific antibodies were identified: 11U1 (aa 49 to 84), 11U2 (aa 147 to 162), 11U3 (aa 179 to 188), 11U4 (aa 180 to 200), and 11U5 (aa 355 to 367). Two HPV6-HPV11 cross-reactive regions were identified: 6CR1 (HPV6 L2 aa 106 to 128)/11CR1 (HPV11 L2 aa 103 to 127) and 6CR2 (HPV6 L2 aa 187 to 199)/11CR2 (HPV11 L2 aa 180 to 200).
Random mutagenesis of the thymidine kinase gene of varicella-zoster virus
Authors: Suzutani T , Lacey S F , Powell K L , Purifoy D J , Honess R W
Published: January 2020

To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.21), we established rapid screening and phenotypic selection of mutant VZV TK genes in TK-deficient Escherichia coli C600 by using a constitutive pKK223-3 expression plasmid. In this screening system, mutant TK genes generated by random mutagenesis were identified by the sensitivity of E. coli-expressing VZV TKs to 5-bromo-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil. Twenty-four mutant clones with amino acid substitutions were isolated, and their nucleotide sequence and enzymatic activities were determined. Of the 24 clones, 20 had single amino acid substitutions, 2 clones had double amino acid substitutions, and 1 clone had triple amino acid substitutions. In 17 cases of single amino acid substitution, six mutations led to lost enzyme activity, and four of these six mutations centered in the ATP-binding site. The other 11 mutations resulted in reduction of both TK and thymidylate kinase activities or only thymidylate kinase activity and were located in scattered positions in the VZV TK gene, although 5 mutations showed a tendency to cluster in the region between positions 251 and 260.
Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia
Authors: Sawtell N M , Thompson R L
Published: January 2020

A rapid and physiologically relevant hyperthermia-based induction procedure has been utilized to develop an in vivo model of induced herpes simplex virus (HSV) reactivation in outbred Swiss Webster mice. This procedure was found to efficiently reactivate latent virus from both trigeminal and lumbosacral ganglia. Examination of the time between hyperthermia and virus production demonstrated that detectable levels of infectious virus were present in ganglia as soon as 14 h posttreatment, with peak percent recoveries at 24 h. These data indicated that the switch from latent to active viral gene transcription occurred rapidly following treatment. Immunohistochemical staining for HSV type 1 antigens revealed rare antigen-positive ganglionic neurons 24 h postinduction. HSV antigens were not detected in any other cell type, and lateral spread of the infection was not observed. This is the first report of the detection of HSV antigens in vivo following induced reactivation in the intact nervous system and demonstrates that the neuron is the site of infectious virus production. In addition, our data strongly suggest that at least some neurons in which HSV antigens are detected during reactivation do not survive. Because the temporal and spatial characteristics of HSV reactivation have been clearly defined, this model is uniquely suited for the molecular dissection of the reactivation process.
Human immunodeficiency virus type 1 major neutralizing determinant exposed on hepatitis B surface antigen particles is highly immunogenic in primates
Authors: Schlienger K , Mancini M , Rivière Y , Dormont D , Tiollais P , Michel M L
Published: January 2020

Hepatitis B surface antigen (HBsAg) produced by recombinant DNA technology is now widely and safely used worldwide for hepatitis B vaccination. We used the HBsAg particle as a carrier molecule for presentation of selected human immunodeficiency virus type 1 (HIV-1) determinants to the immune system. Immunization of rhesus monkeys with an HBsAg chimera carrying the HIV-1 envelope major neutralizing determinant allowed us to generate proliferative T-cell responses and, in some cases, neutralizing antibodies and antibody-dependent cellular cytotoxicity. Since there is an overlap between populations at risk for hepatitis B virus and HIV, HBsAg recombinant particles may be relevant carriers for HIV-1 epitopes and could offer a new approach to the development of an AIDS vaccine.
In vitro packaging and replication of individual genomic segments of bacteriophage phi 6 RNA
Authors: Gottlieb P , Strassman J , Qiao X , Frilander M , Frucht A , Mindich L
Published: January 2020

The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.
ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, can infect both CD4+ and CD8+ T cells but requires CD4+ T cells in order to cause paralysis and immunodeficiency
Authors: Saha K , Wong P K
Published: January 2020

When neonatal FVB/N mice were inoculated with ts1, a temperature-sensitive mutant of Moloney murine leukemia virus TB, they developed a progressive bilateral hindlimb paralysis and immunodeficiency leading to death 4 to 6 weeks after inoculation. T lymphocytes have been shown to be primarily responsible for this ts1-induced syndrome. Here we compare the role played by each subset of T lymphocytes, i.e., CD4+ and CD8+ T cells, in disease development. Mice were depleted of a specific subset for the first 10 days of their lives by using either anti-CD4 or anti-CD8 monoclonal antibodies in vivo. Disease development in these mice was then monitored. Depletion of CD4+ T cells significantly attenuated the ts1-induced syndrome: virus replication was decreased, disease latency was extended, and death was prevented in 60% of the mice. Similar treatment with anti-CD8 antibody had almost no effect on disease progression. However, when depletion was begun 2 weeks after neonatal ts1 inoculation, CD4+ T cell depletion did not affect disease development. ts1 infected CD4+ and CD8+ T lymphocytes equally well in vivo, as shown by flow cytometric analysis, but virus replication was restricted primarily to the CD4+ subset of T cells, as found by in vitro assay. Hence, CD4+ T lymphocytes play an important role in the development of ts1-induced paralysis and immunodeficiency. The mechanism of this CD4+ T-cell-mediated disease production by ts1 is not clear; however, increased replication of ts1 in the CD4+ T cells, especially in the early stages of the disease, seems to play a crucial role.
Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones
Authors: Yakushijin Y , Yasukawa M , Kobayashi Y
Published: January 2020

In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.
Insertional mutagenesis of flvi-2 in tumors induced by infection with LC-FeLV, a myc-containing strain of feline leukemia virus
Authors: Levy L S , Lobelle-Rich P A
Published: January 2020

LC-FeLV is a myc-containing strain of feline leukemia virus (FeLV) which exhibits only partial transforming activity in vitro and in vivo. LC-FeLV infection in kittens may induce, but does not necessarily induce, thymic lymphosarcoma in viremic animals after a short latency. These observations suggest that infection with LC-FeLV is not sufficient to induce complete transformation and that another genetic event(s) is required. One possibility for such an event is that the integrating provirus acts as an insertional mutagen and thereby disrupts the structure or function of another proto-oncogene. Using a strategy of transposon tagging, this possibility was examined in eight feline T-cell lymphosarcomas, including four induced by experimental infection with LC-FeLV, three induced by natural infection with FeLV, and one FeLV-negative tumor. The analysis demonstrated one locus, termed flvi-2, to be structurally altered in six of the tumors examined, including three induced by LC-FeLV and three in which no activated myc oncogene is apparent. Inverse polymerase chain reaction was used to demonstrate the presence and transcriptional orientation of proviruses integrated at flvi-2 in five of these tumors. The flvi-2 locus does not hybridize to cloned probes representing 21 previously identified proto-oncogenes or common domains of retroviral integration. Thus, the data suggest that interruption of the flvi-2 locus cooperates with the myc oncogene in the induction of T-cell lymphomas by LC-FeLV; indeed, the observations indicate that the insertional mutagenesis of flvi-2 plays a role in T-cell lymphomagenesis even in the absence of feline v-myc.
Herpes simplex virus type 1 ICP0 regulates expression of immediate-early, early, and late genes in productively infected cells
Authors: Cai W , Schaffer P A
Published: January 2020

The herpes simplex virus type 1 protein, ICP0, can activate expression of all kinetic classes of viral promoters in transient expression assays. To examine the role of ICP0 in the regulation of viral gene expression during productive infection, we characterized the wild-type virus, an ICP0 null mutant (7134), and several ICP0 nonsense mutant viruses with regard to virus replication and protein synthesis in Vero cells. Relative to wild-type virus, 7134 was severely deficient in viral growth and protein synthesis at low multiplicities of infection but exhibited a nearly wild-type phenotype at high multiplicities. The phenotypes of the ICP0 nonsense mutants were intermediate between those of the wild-type virus and 7134 in that the more ICP0-coding sequence expressed by a given nonsense mutant, the more wild type-like was its phenotype. The location of the ICP0 domain responsible for transactivation during productive infection was confirmed to be within the N-terminal portion of the protein, as previously shown in transient expression assays. Immunoprecipitation and immunofluorescence tests were used to detect low-level expression of selected immediate-early (IE), early (E), and late (L) proteins by mutant and wild-type viruses following low-multiplicity infection. The 7134 deletion mutant and several nonsense mutants expressed markedly reduced levels of E and L proteins but wild-type levels of the IE protein, ICP4. Because the latency-associated transcripts (LATs) are specified by the strand opposite that which encodes ICP0, the ICP0 deletion and nonsense mutants are by definition ICP0-LAT double mutants. The ability of a LAT- ICP0+ mutant to replicate as efficiently as wild-type virus at low multiplicities and the ability of ICP0-expressing 0-28 cells to complement the defects of the mutants in E and L protein synthesis indicates that the phenotypes of the mutants are caused by mutations in ICP0 and not the LATs. Thus, we conclude that ICP0 up-regulates E and L but not necessarily IE gene expression during productive infection. The activation of IE gene expression by ICP0 during productive infection is likely overshadowed by the activity of the virion-associated protein, VP16. This hypothesis was tested by transfection of Vero cells with infectious mutant and wild-type viral DNAs. In such tests, no VP16 is present at early times posttransfection. Significantly fewer cells transfected with infectious 7134 DNA expressed ICP4 than cells transfected with KOS DNA. This reduction was fully reversed by cotransfection with an ICP0-expressing plasmid.(ABSTRACT TRUNCATED AT 400 WORDS)
Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines
Authors: Guzo D , Rathburn H , Guthrie K , Dougherty E
Published: January 2020

Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed.
Expression of Aleutian mink disease parvovirus capsid proteins by a recombinant vaccinia virus: self-assembly of capsid proteins into particles
Authors: Clemens D L , Wolfinbarger J B , Mori S , Berry B D , Hayes S F , Bloom M E
Published: January 2020

A portion of a cDNA clone containing coding sequences for both structural proteins (VP1 and VP2) of Aleutian mink disease parvovirus (ADV) was inserted into recombinant vaccinia viruses, VV:ADSP. Immunohistochemical staining of VV:ADSP-infected cells revealed that the ADV antigen was readily detected and localized in the nuclei of infected cells. Analysis of VV:ADSP-infected cell lystates indicated that both VP1 and VP2 were produced and comigrated with authentic VP1 and VP2 from ADV-infected Crandell feline kidney cells. These results suggested, therefore, that both VP1 and VP2 were synthesized from a single cloned transcript. CsCl density gradient centrifugation of partially purified VV:ADSP-infected cell lysates indicated that the majority of the antigen was located in a fraction with a density near 1.33 g/ml, indicative of empty ADV particles. Subsequent electron microscopic examination revealed the presence of 27-nm icosahedral virion-like structures at the same density, suggesting that the proteins self-assembled into empty virions. Furthermore, sera from eight of eight mice inoculated with VV:ADSP contained ADV-specific antibodies and two of these eight serum samples had neutralizing activity, indicating that the particles produced in VV:ADSP-infected cells were immunogenic. Finally, when lysates from VV:ADSP-infected cells were compared with standard ADV antigens in counterimmunoelectrophoresis assays, a similar pattern of specific reactivity was observed for sera from normal and infected mink.
Human herpesvirus 7 is a constitutive inhabitant of adult human saliva
Authors: Wyatt L S , Frenkel N
Published: January 2020

We report the frequent isolation of human herpesvirus 7 from the saliva of healthy adults. Virus isolates recovered from different individuals exhibited minimal restriction enzyme polymorphism, which was mostly confined to heterogeneous (het) sequences in the genome. DNAs of isolates recovered from the same individual over a period of several months showed the same characteristic het fragments, indicating the stability of the het sequences upon virus replication and shedding in vivo. In contrast to the results of previous reports, human herpesvirus 6, the causative agent of roseola infantum, could not be isolated from the saliva specimens, raising questions regarding oral transmission of human herpesvirus 6 and human herpesvirus 7 to young children.
Murine retrovirus-induced spongiform encephalomyelopathy: host and viral factors which determine the length of the incubation period
Authors: Czub M , McAtee F J , Portis J L
Published: January 2020

A molecular clone of wild mouse ecotropic retrovirus CasBrE (clone 15-1) causes a spongiform neurodegenerative disease with a long incubation period, greater than or equal to 6 months. This virus infects the central nervous system (CNS) at low levels. In contrast, a chimeric virus, FrCasE, containing env and 3' pol sequences of 15-1 in a Friend murine leukemia virus background, infects the CNS at high levels and causes a rapid neurodegenerative disease with an incubation period of only 16 days. With both viruses, the induction of neurologic disease is dependent on inoculation during the perinatal period. Since the length of the incubation period of this disease appears to be a function of the relative level of CNS infection, we have attempted to identify the viral and host factors which determine the relative level of virus infection of the CNS. It was previously shown that the CNS is susceptible to infection only during the perinatal period (M. Czub, S. Czub, F. J. McAtee, and J. L. Portis, J. Virol. 65:2539-2544, 1991). Here we have found that the susceptibility of the CNS wanes progressively or gradually as a function of the age of the host, this age-dependent resistance being complete by 12 to 14 days of age. Utilizing a group of chimeric viruses, we found that the relative level of CNS infection achieved after inoculation of mice at 1 day of age was a function of the kinetics of virus replication and spread in peripheral organs. Viruses which reached peak viremia titers early (5 to 7 days of age) infected the CNS at high levels, and viruses which reached peak titers later infected the CNS at lower levels. Among the group of viruses examined in the current study, the kinetics of peripheral virus replication and spread appeared to be influenced primarily by sequences within the R-U5-5' leader region of the viral genome. These results suggested that the relative level of CNS infection was determined very early in life and appeared to be a function of a dynamic balance between the kinetics of virus replication in the periphery and a progressively developing restriction of virus replication in the CNS.
Preventive effects of early anti-CD4 or anti-CD8 treatment on Borna disease in rats
Authors: Stitz L , Sobbe M , Bilzer T
Published: January 2020

Borna disease is a virus-induced, immunopathological encephalomyelitis in which CD4+ cells and macrophages dominate the pathological picture. However, significant numbers of CD8+ cells have been morphologically identified in perivascular infiltrates as well. To determine the contribution of different T-cell subsets to the pathogenesis of Borna disease, virus-infected rats were treated with monoclonal antibodies specific for CD4+ and CD8+ cells. Both types of monoclonal antibodies were able to significantly decrease or even prevent the local inflammatory reaction in the brain if given early during the infection. However, CD8-specific monoclonal antibodies appeared to be more effective than antibodies directed against CD4+ cells. Treatment initiated 4 days postinfection did not result in inhibition of encephalitis and disease. Virus titers in the brain of infected rats treated with T-cell-specific antibodies did not differ from titers in untreated infected control animals. The results indicate an important functional role of CD8+ cells, in addition to CD4+ cells, in the pathogenesis of Borna disease.
Determinants of substrate recognition by poliovirus 2A proteinase
Authors: Hellen C U , Lee C K , Wimmer E
Published: January 2020

Poliovirus proteinase 2A (2Apro) is autocatalytically released from the viral polyprotein by cleavage in cis of a Tyr-Gly dipeptide at its own amino terminus, resulting in separation of the P1 structural and P2-P3 nonstructural protein precursors. A second Ty-Gly dipeptide within 3D polymerase is cleaved by 2Apro in trans, but this is not essential for viral proliferation. The mechanism which limits cleavage to only 2 of the 10 Tyr-Gly dipeptides within the poliovirus polyprotein has not been characterized. We have therefore undertaken a systematic mutational analysis of the VP1-2A site to elucidate determinants of substrate recognition by 2Apro. The P2 and P1' positions are important determinants for cis cleavage of this site, whereas a variety of substituents could be tolerated at the P2', P1, and P3 positions. The requirements for trans cleavage of this site were more stringent. We found that the 2Apro of coxsackievirus type A21 and rhinoviruses 2 and 14 have stringent requirements similar to those of poliovirus 2Apro for cleavage in trans.
Epstein-Barr virus enters B cells and epithelial cells by different routes
Authors: Miller N , Hutt-Fletcher L M
Published: January 2020

Epstein-Barr virus (EBV) infects two cell types, B lymphocytes and epithelial cells. Electron microscopic studies have shown that the virus fuses with the lymphoblastoid cell line Raji but is endocytosed into thin-walled non-clathrin-coated vesicles in normal B cells before fusion takes place. To compare early interactions of EBV with epithelial cells and B cells, a fluorescence dequenching assay of fusion was employed, using virus labeled either with the pH-insensitive probe octadecyl rhodamine B chloride (R18) or with 5(N-octadecanoyl) aminofluorescein (AF), which loses emission intensity at a pH below 7.4. Fusion of virus labeled with R18 could be monitored with B cells, Raji cells, and epithelial cells. Lowering the extracellular pH or pretreatment of cells with ammonium chloride or methylamine had no effect on these measurements. In contrast, fusion of virus labeled with AF could be measured with Raji cells and epithelial cells, but not with normal B cells unless cells were previously treated with ammonium chloride. Fusion of virus with normal B cells was inhibited with chlorpromazine, chloroquine, and sodium azide, but none of these reagents had any effect on fusion with Raji or epithelial cells. These results suggest that entry of EBV into nonpolarized suspensions of epithelial cells occurs by fusion at the cell surface, that EBV may be incapable of fusing with normal B cells unless it has first been endocytosed, and that pH appears to be irrelevant to either event. A combination of the two probes, R18 and AF, may have general use for determining the sites of entry of enveloped viruses that fuse in a pH-independent manner.
The herpes simplex virus 1 RNA binding protein US11 is a virion component and associates with ribosomal 60S subunits
Authors: Roller R J , Roizman B
Published: January 2020

The herpes simplex virus 1 US11 gene encodes a site- and conformation-specific RNA binding regulatory protein. We fused the coding sequence of this protein with that of beta-galactosidase, expressed the chimeric gene in Escherichia coli, and purified a fusion protein which binds RNA in the same way as the infected cell protein. The fusion protein was used to generate anti-US11 monoclonal antibody. Studies with this antibody showed that US11 protein is a viral structural protein estimated to be present in 600 to 1,000 copies per virion. The great majority of cytoplasmic US11 protein was found in association with the 60S subunit of infected cell ribosomes. US11 protein associates with ribosomes both late in infection at the time of its synthesis and at the time of infection after its introduction into the cytoplasm by the virion. US11 protein expressed in an uninfected cell line stably transfected with the US11 gene associates with ribosomal 60S subunits and localizes to nucleoli, suggesting that US11 protein requires no other viral functions for these associations.
Identification of a putative receptor for subgroup A feline leukemia virus on feline T cells
Authors: Ghosh A K , Bachmann M H , Hoover E A , Mullins J I
Published: January 2020

Retrovirus infection is initiated by the binding of virus envelope glycoprotein to a receptor molecule present on cell membranes. To characterize a receptor for feline leukemia virus (FeLV), we extensively purified the viral envelope glycoprotein, gp70, from culture supernatants of FeLV-61E (subgroup A)-infected cells by immunoaffinity chromatography. Binding of purified 125I-labeled gp70 to the feline T-cell line 3201 was specific and saturable, and Scatchard analysis revealed a single class of receptor binding sites with an average number of 1.6 x 10(5) receptors per cell and an apparent affinity constant (Ka) of 1.15 x 10(9) M-1. Cross-linking experiments identified a putative gp70-receptor complex of 135 to 140 kDa. Similarly, coprecipitation of 125I-labeled cell surface proteins with purified gp70 and a neutralizing but noninterfering anti-gp70 monoclonal antibody revealed a single cell surface protein of approximately 70 kDa. These results indicate that FeLV-A binds to feline T cells via a 70-kDa cell surface protein, its presumptive receptor.
Two AP1 sites binding JunB are essential for human papillomavirus type 18 transcription in keratinocytes
Authors: Thierry F , Spyrou G , Yaniv M , Howley P
Published: January 2020

The activity and epithelial tropism of the human papillomavirus type 18 P105 early promoter, which directs the synthesis of the E6 and E7 transforming genes, are controlled by cis elements included in the viral long control region. To identify potential cellular regulators of this promoter, we mutagenized one or both of the 5'-TGACTAA-3' cis elements capable of interacting with the AP1 transcription factor, which is composed either of homodimers or heterodimers of the Jun products or of heterodimers of Jun and Fos. Mutation of both elements completely abolished P105 promoter activity in human keratinocytes. We show that either AP1 site can interact efficiently in vitro with any of the three different Jun products as heterodimers with c-Fos. However, in nuclear extracts prepared from human keratinocytes, JunB was the predominant Jun component bound to the DNA probe containing this cis element. These results implicate JunB as an important factor in human papillomavirus type 18 transcription in keratinocytes and strongly suggest a potential role of this Jun gene product in the tissue-specific transcription of the genital papillomaviruses.
A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress
Authors: Whealy M E , Robbins A K , Tufaro F , Enquist L W
Published: January 2020

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.
Specific NF-kappa B subunits act in concert with Tat to stimulate human immunodeficiency virus type 1 transcription
Authors: Liu J , Perkins N D , Schmid R M , Nabel G J
Published: January 2020

NF-kappa B is a protein complex which functions in concert with the tat-I gene product to stimulate human immunodeficiency virus (HIV) transcription. To determine whether specific members of the NF-kappa B family contribute to this effect, we have examined the abilities of different NF-kappa B subunits to act with Tat-I to stimulate transcription of HIV in Jurkat T-leukemia cells. We have found that the p49(100) DNA binding subunit, together with p65, can act in concert with Tat-I to stimulate the expression of HIV-CAT plasmid. Little effect was observed with 50-kDa forms of p105 NF-kappa B or rel, in combination with p65 or full-length c-rel, which do not stimulate the HIV enhancer in these cells. These findings suggest that the combination of p49(100) and p65 NF-kappa B can act in concert with the tat-I gene product to stimulate the synthesis of HIV RNA.
Synergistic activation of the human immunodeficiency virus type 1 promoter by the viral Tat protein and cellular transcription factor Sp1
Authors: Kamine J , Chinnadurai G
Published: January 2020

We have previously shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can activate a synthetic promoter containing consensus-binding sites for the cellular transcription factor Sp1. In this report, we show that a GAL-Tat fusion protein targeted via GAL4 DNA-binding sites can also trans activate an HIV-1 LTR promoter independently of the trans-activation response region. To show that the trans activation of the promoter by Tat directly involves the Sp1 protein, we have targeted a GAL-Sp1 fusion protein to the long terminal repeat promoter via upstream GAL4-binding sites. In the presence of Tat and GAL-Sp1, the promoter is synergistically trans activated at the transcriptional level, indicating that Tat and Sp1 functionally interact to trans activate the HIV-1 promoter. The Sp1 synergism is relatively specific, since another chimeric transcriptional activator, GAL-VP16, does not appear to be significantly synergistic with Tat.
Mechanism of action of the papillomavirus E2 repressor: repression in the absence of DNA binding
Authors: Barsoum J , Prakash S S , Han P , Androphy E J
Published: January 2020

Repression of papillomavirus E2-dependent gene expression was studied by using transient transfections into mouse embryo fibroblast cells. Cotransfection of a gene corresponding to the naturally occurring repressor E2-TR along with the full-length E2 gene resulted in up to 98% repression of E2-dependent reporter gene expression. A series of E2 DNA-binding domain mutants were transferred into the E2-TR form and characterized for their ability to repress E2-dependent transactivation. All mutants which were defective for DNA binding but were dimerization competent repressed E2 transactivation as well or nearly as well as the wild-type repressor. E2 mutants which lacked dimerization activity repressed transactivation poorly or not at all. These results indicate that the E2 repressor can inhibit transcription, in the absence of DNA binding, by forming heterodimers with full-length E2.
Detection of feline immunodeficiency virus (FIV) nucleic acids in FIV-seronegative cats
Authors: Dandekar S , Beebe A M , Barlough J , Phillips T , Elder J , Torten M , Pedersen N
Published: January 2020

A study was undertaken to determine the rate of viral transmission among naive specific-pathogen-free (SPF) cats living in close contact with feline immunodeficiency virus (FIV)-infected cats. Twenty SPF cats were housed in the same rooms with experimentally FIV-infected seropositive and virus culture-positive cats for 2 to 4 years and were monitored for the presence of FIV nucleic acids and antibodies. Only 1 of the 20 cats became seropositive and virus culture positive and developed signs of disease. Genomic DNA from bone marrow and peripheral blood mononuclear cells (PBMC) of 10 of 19 healthy-appearing seronegative cats became positive for FIV DNA by the polymerase chain reaction. Twenty-eight SPF cats housed as groups in separate quarters and never exposed to FIV-infected cats were uniformly negative for FIV DNA. FIV RNA transcripts were detected in concanavalin A-stimulated PBMC cultures from 4 of 10 FIV DNA-positive, seronegative cats by in situ hybridization. PBMC from three of four naive SPF cats acquired FIV nucleic acids after the cats were transfused with blood and bone marrow from FIV genome-positive, seronegative donors. Three of five FIV-seronegative cats housed for years with naturally FIV-infected cats in a private household were also found to harbor FIV DNA, indicating that the same phenomenon occurred in the field. These findings demonstrate that cats living in close contact with FIV-infected seropositive cats can acquire FIV nucleic acids without developing detectable levels of serum antibodies or disease.
Physical and functional interaction of human cytomegalovirus DNA polymerase and its accessory protein (ICP36) expressed in insect cells
Authors: Ertl P F , Powell K L
Published: January 2020

Expression of the human cytomegalovirus (HCMV) (AD169) DNA polymerase gene under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus in Spodoptera frugiperda (Sf9) cells has provided a source of highly active CMV DNA polymerase. In extracts from CMV-infected cells, the CMV DNA polymerase is found strongly associated with an additional polypeptide, ICP36. This protein has been identified as the CMV homolog of the herpes simplex virus type 1 UL42 gene product and may have a similar function. We have expressed HCMV DNA polymerase and ICP36 in the same system and demonstrated that they interact to form a stable complex. Moreover, ICP36 functions to stimulate the DNA polymerase activity in a template-dependent manner. We have compared the activity of the recombinant DNA polymerase in the presence and absence of ICP36 on a number of DNA templates and measured the effect of the polymerase inhibitors phosphonoformic acid and acyclovir triphosphate.