bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative ability of different bovine leukocyte populations to support active replication of rinderpest virus
Authors: Rey Nores J E , McCullough K C
Published: January 2020

Bovine peripheral blood mononuclear cells (PBMC) were infected with the pathogenic Saudi isolate of rinderpest virus (RPV) in order to identify the cell subpopulation(s) susceptible to active replication of this virus. Flow cytometry analysis, using a monoclonal antibody recognizing the H glycoprotein of RPV, showed that monocytes were the main subpopulation in which the virus replicated, whereas <2% of lymphocytes expressed viral antigen. The activation of PBMC with concanavalin A before infection resulted in an increase in the capacity of lymphocytes to support RPV replication; >90% of CD4+ and CD8+ T lymphocytes expressed viral antigen at 3 days postinfection, although < or = 40% of gamma/delta T cells were productively infected. B-lymphocyte activation with pokeweed mitogen also resulted in increased replication of this virus in these cells, involving up to 40% of B lymphocytes. An enhancement of lymphocyte susceptibility to infection and active replication by RPV was observed upon coculture of RPV-infected PBMC on bovine endothelial cells. Such enhancement was most marked with the B-cell and CD4+ T-cell subpopulations. Contact between lymphocytes and extracellular matrix components did not alter the capacity of RPV to replicate in lymphocytes. This intercellular contact with endothelial cells increased the viability of certain lymphocyte subpopulations, but it alone could not explain the increased sensitivity to RPV. Intercellular signalling, which resulted in interleukin-2 receptor upregulation, probably played a role. In summary, monocytes are the main target for active, productive infection by RPV. Similar replication in lymphocytes depends on their activation state and on contact with accessory cells such as endothelial cells. These characteristics have important implications for virus traffic in vivo and the pathogenesis of this disease.
Inhibition of topoisomerase II by ICRF-193 prevents efficient replication of herpes simplex virus type 1
Authors: Hammarsten O , Yao X , Elias P
Published: January 2020

Cellular topoisomerase II is specifically inactivated by the drug ICRF-193. This compound turns topoisomerase II into a closed clamp that is unable to cleave DNA. We have investigated the effects of this inhibitor on the replication of herpes simplex virus type 1. We show that ICRF-193 at low multiplicities of infection dramatically inhibits viral DNA synthesis and the production of infectious virus. The inhibition is less efficient at high multiplicities of infection. In addition, inhibition of viral DNA synthesis was observed only when ICRF-193 was present during the first 4 h of the infectious cycle. The transient replication of plasmids containing a herpes simplex virus type 1 origin of DNA replication, oriS, was affected by ICRF-193 in the same way. In contrast, neither cellular DNA synthesis nor replication of plasmids containing a simian virus 40 origin of DNA replication was inhibited. The observed effect on herpes simplex virus DNA replication was not caused by a decreased transcription of replication genes inasmuch as the levels of UL8, UL9, UL29, and UL30 rmRNAs were unaffected by the drug. These results suggest that topoisomerase II plays a vital role during the replication of herpes simplex virus type 1 DNA. We speculate that topoisomerase II is involved in the decatenation of newly synthesized daughter molecules.
Effects of modifying the tRNA(3Lys) anticodon on the initiation of human immunodeficiency virus type 1 reverse transcription
Authors: Huang Y , Shalom A , Li Z , Wang J , Mak J , Wainberg M A , Kleiman L
Published: January 2020

tRNA(3Lys) is a primer for reverse transcription in human immunodeficiency virus type 1 (HIV-1), and the anticodon of tRNA(3Lys) has been implicated in playing a role in both its placement onto the HIV-1 genome and its interaction with HIV-1 reverse transcriptase (RT). In this work, the anticodon in a tRNA(3Lys) gene was changed from UUU to CUA (tRNA(3Lys)Su+) or, in addition, G-73 was altered to A (tRNA(3Lys)Su+G73A). COS-7 cells were transfected with either wild-type or mutant tRNA(3Lys) genes, and both the wild-type and mutant tRNA(3Lys) produced were purified by using immobilized tRNA-specific hybridization probes. Each mutant tRNA(3Lys) was tested for its ability to prime reverse transcription in vitro, either alone or in competition with wild-type tRNA(3Lys). Short RT extensions of wild-type and mutant tRNALys could be distinguished from each other by their different mobilities in one-dimensional single-stranded conformation polymorphism polyacrylamide gel electrophoresis. These reverse transcription products show that heat-annealed tRNA(3Lys)Su+ has the same ability as heat-annealed wild-type tRNA(3Lys) to prime RT and competes equally well with wild-type tRNA(3Lys) for priming RT. tRNA(3Lys)Su+G73A has 60% of the wild-type ability to prime RT but competes poorly with wild-type tRNA(3Lys) for priming RT. However, the priming abilities of wild-type and mutant tRNA(3) are quite different when in vivo-placed tRNA is examined. HIV-1 produced in COS cells transfected with a plasmid containing both the HIV-1 proviral DNA and DNA coding for tRNA(3Lys)Su+ contains both endogenous, cellular wild-type tRNA(3Lys) and mutant tRNA(3Lys). When total viral RNA is used as the source of primer tRNA placed onto the genomic RNA in vivo, only wild-type tRNA(3Lys) is used as a primer. If the total viral RNA is first heated and exposed to hybridizing conditions, then both the wild-type and mutant tRNA(3Lys) act as primers for RT. These results indicate that the tRNA(3Lys)Su+ packaged into the virions is unable to act as a primer for RT, and a model is proposed to explain the disparate results between heat-annealed and in vivo-placed primer tRNA.
A bispecific antibody recognizing influenza A virus M2 protein redirects effector cells to inhibit virus replication in vitro
Authors: Fernandez-Sesma A , Schulman J L , Moran T M
Published: January 2020

Bispecific antibodies can be used to redirect cytotoxic T cells to kill virus-infected cells, overriding the need for major histocompatibility complex restriction. We produced a bispecific antibody (3F12) which binds influenza virus M2 protein and the T-cell receptor and can redirect staphylococcal enterotoxin B-activated T cells to kill influenza virus-infected cells and inhibit virus replication in vitro.
Sequence determinants of 3A-mediated resistance to enviroxime in rhinoviruses and enteroviruses
Authors: Heinz B A , Vance L M
Published: January 2020

Using site-directed mutagenesis of the 3A coding region of rhinovirus 14, we have expanded our analysis of resistance to enviroxime. We have observed that high and low levels of drug resistance involve two domains within 3A and that the amino acid at position 30 is critical in determining resistance.
Three retroviral sequences in amphibians are distinct from those in mammals and birds
Authors: Tristem M , Herniou E , Summers K , Cook J
Published: January 2020

We isolated and characterized three endogenous retroviral fragments from the dart-poison frog Dendrobates ventrimaculatus. These are the first retroviral sequences to be identified in amphibians, and consequently retroviruses have now been found in each of the five major vertebrate classes. Comparison of the amphibian retroviral fragments, termed DevI, DevII, and DevIII, with mammalian and avian isolates revealed significant differences between their nucleotide sequences. This suggested that they were only distantly related to the seven currently recognized retroviral genera. Additional analysis by phylogeny reconstruction showed that the amphibian retroviral fragments were approximately equally related to the Moloney leukemia-related viruses, the spumaviruses, and walleye dermal sarcoma virus. Hybridization experiments revealed that viruses closely related to DevI, DevII, and DevIII do not appear to be widespread in other vertebrates and that DevI, DevII, and DevIII are all present at high copy numbers within their amphibian hosts, typically at over 250 copies per genome. The viruses described here, along with two others which have recently been found in a fish and a reptile, indicate that there may be some major differences in the retroviruses harbored by different vertebrate classes. This suggests that further characterization of retroviruses of fish, reptiles, and amphibians will help in understanding the evolution of the whole retroviral family and may well lead to the discovery of retroviruses with novel biological properties.
Human immunodeficiency virus type 1 gp120 induces anergy in human peripheral blood lymphocytes by inducing interleukin-10 production
Authors: Schols D , De Clercq E
Published: January 2020

The effects of recombinant gp120 on the proliferative responses and cytokine production by normal peripheral blood mononuclear cells (PBMC) were investigated. gp120 inhibited in a dose-dependent fashion the anti-CD3 monoclonal antibody (MAb)- and concanavalin A-induced proliferative responses. The production of interleukin-2 (IL-2) and IL-4 was diminished by gp120 in the anti-CD3- and concanavalin A-stimulated cultures. In unstimulated PBMC, gp120 induced the production of considerable amounts of IL-10, gamma interferon, and tumor necrosis factor alpha. The gp120-induced reduction in the proliferative responses of PBMC was at least partially reversed by the addition of IL-2, anti-CD28 MAb, or transfectants expressing CD80, CD86, or CD40 but not with exogenous IL-4. Also, a neutralizing anti-IL-10 MAb reversed the inhibitory effect of gp120 on the proliferative responses whereas exogenous IL-10 further enhanced this inhibitory effect. These findings indicate that IL-10 plays an important role in the inhibitory effect of gp120 on PBMC proliferation. The ratio of CD3+CD4+ to CD3+CD8+ T cells was the same in gp120-treated and untreated cell cultures. No apoptosis in these two T-cell populations was observed. However, the number of activated CD3+CD4+ T cells and CD3+CD8+ T cells, as judged by CD25, CD69, and HLA-DR expression, was consistently reduced. gp120 induced the expression of IL-10 in the monocyte/macrophage population, and therefore gp120 also reduced the proliferative responses of CD4+ T-cell-depleted PBMC. Taken together, our observations point to the importance of the cytokine pattern changes and, in particular, the role of IL-10 (produced by the monocytes) in the inhibitory effect of gp120. This mechanism of gp120-induced immunosuppression, if operative in vivo, could contribute to the depressed immune responses associated with human immunodeficiency virus infection and thus have important implications for immunotherapeutic strategies to slow down disease progression in AIDS.
Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases
Authors: Benn J , Su F , Doria M , Schneider R J
Published: January 2020

The HBx protein of hepatitis B virus is a dual-specificity activator of transcription, stimulating signal transduction pathways in the cytoplasm and transcription factors in the nucleus, when expressed in cell lines in culture. In the cytoplasm, HBx was shown to stimulate the Ras-Raf-mitogen-activated protein kinase (MAP kinase) cascade, which is essential for activation of transcription factor AP-1. Here we show that HBx protein stimulates two independently regulated members of the MAP kinase family when expressed transiently in cells. HBx protein stimulates the extracellular signal-regulated kinases (ERKs) and the c-Jun N-terminal kinases (JNKs). HBx activation of ERKs and JNKs leads to induction and activation of AP-1 DNA binding activity involving transient de novo synthesis of c-Fos protein and prolonged synthesis of c-Jun, mediated by N-terminal phosphorylation of c-Jun carried out by HBx-activated JNK. New c-Jun synthesis was blocked by coexpression with a dominant-negative MAP kinase kinase (MEK kinase, MEKK-1), confirming that HBx stimulates the prolonged synthesis of c-Jun by activating JNK signalling pathways. Activation of the c-fos gene was blocked by coexpression with a Raf-C4 catalytic mutant, confirming that HBx induces c-Fos by acting on Ras-Raf linked pathways. HBx activation of ERK and JNK pathways resulted in prolonged accumulation of AP-1-c-Jun dimer complexes. HBx activation of JNK and sustained activation of c-jun, should they occur in the context of hepatitis B virus infection, might play a role in viral transformation and pathogenesis.
Down-regulation of paramyxovirus hemagglutinin-neuraminidase glycoprotein surface expression by a mutant fusion protein containing a retention signal for the endoplasmic reticulum
Authors: Tanaka Y , Heminway B R , Galinski M S
Published: January 2020

The human parainfluenza virus type 3 (HPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins are the principal components involved in virion receptor binding, membrane penetration, and ultimately, syncytium formation. While the requirement for both F and HN in this process has been determined from recombinant expression studies, stable physical association of these proteins in coimmunoprecipitation studies has not been observed. In addition, coexpression of other heterologous paramyxovirus F or HN glycoproteins with either HPIV3 F or HN does not result in the formation of syncytia, suggesting serotype-specific protein differences. In this study, we report that simian virus 5 and Sendai virus heterologous HN proteins and measles virus hemagglutinin (H) were found to be down-regulated when coexpressed with HPIV3 F. As an alternative to detecting physical associations of these proteins by coimmunoprecipitation, further studies were performed with a mutant HPIV3 F protein (F-KDEL) lacking a transmembrane anchor and cytoplasmic tail and containing a carboxyl-terminal retention signal for the endoplasmic reticulum (ER). F-KDEL was defective for transport to the cell surface and could down-regulate surface expression of HPIV3 HN and heterologous HN/H proteins from simian virus 5, Sendai virus, and measles virus in coexpression experiments. HN/H down-regulation appeared to result, in part, from an early block to HPIV3 HN synthesis, as well as an instability of the heterologous HN/H proteins within the ER. In contrast, coexpression of F-KDEL with HPIV3 wild-type F or the heterologous receptor-binding proteins, respiratory syncytial virus glycoprotein (G) and vesicular stomatitis virus glycoprotein (G), were not affected in transport to the cell surface. Together, these results support the notion that the reported serotype-specific restriction of syncytium formation may involve, in part, down-regulation of heterologous HN expression.
Nonhomologous RNA-RNA recombination events at the 3' nontranslated region of the Sindbis virus genome: hot spots and utilization of nonviral sequences
Authors: Hajjou M , Hill K R , Subramaniam S V , Hu J Y , Raju R
Published: January 2020

The mechanism of RNA-RNA recombination at the 3' nontranslated region (3'NTR) of the Sindbis virus (SIN) genome was studied by using nonreplicative RNA precursors. The 11.7-kb SIN genome was transcribed in vitro as two nonoverlapping RNA fragments. RNA-1 contained the entire 11.4-kb protein coding sequence of SIN and also carried an additional 1.8-kb nonviral sequence at its 3' end. RNA-2 carried the remaining 0.26 or 0.3 kb of the SIN genome containing the 3'NTR. Transfection of these two fragments into BHK cells resulted in vivo RNA-RNA recombination and release of infectious SIN recombinants. Eighteen plaque-purified recombinant viruses were sequenced to precisely map the RNA-RNA crossover sites at the 3'NTR. Sixteen of the 18 recombinants were found to be genetically heterogeneous at the 3'NTR. Two major clustered sites within the 3'NTR of RNA-2 were found to be fused to multiple locations on the nonviral sequence of RNA-1, resulting in insertions of 10 to 1,085 nucleotides at the 3'NTR. Sequence analysis of crossover sites suggested only limited homology and heteroduplex-forming capability between substrate RNAs. Analysis of additional 23 recombinant viruses generated by mutagenized donor and acceptor templates supports the occurrence of recombination hot spots on donor templates. Introduction of a 17-nucleotide rudimentary replicase recognition signal in the acceptor template alone did not induce the polymerase to reinitiate at the 17-nucleotide signal. Interestingly, deletion of a 24-nucleotide hot spot locus on the donor template abolished crossover events at one of the two sites and allowed the polymerase to reinitiate at the 17-nucleotide replicase recognition signal inserted at the acceptor template. The possible roles of RNA-protein and RNA-RNA interactions in the differential regulation of apparent pausing, template selection, and reinitiation are discussed.
Structure of the rat cytomegalovirus genome termini
Authors: Vink C , Beuken E , Bruggeman C A
Published: January 2020

The lytic replication cycle of herpesviruses can be divided into the following three steps: (i) circularization, in which, after infection, the termini of the linear double-stranded viral genome are fused; (ii) replication, in which the circular DNA serves as template for DNA replication, which generates large DNA concatemers; and (iii) maturation, in which the concatemeric viral DNA is processed into unit-length genomes, which are packaged into capsids. Sequences at the termini of the linear virion DNA are thought to play a key role in both genome circularization and maturation. To investigate the mechanism of these processes in the replication of rat cytomegalovirus (RCMV), we cloned, sequenced, and characterized the genomic termini of this betaherpesvirus. Both RCMV genomic termini were found to contain a single copy of a direct terminal repeat (TR). The TR sequence is 504 bp in length, has a high GC content (76%), and is not repeated at internal sites within the RCMV genome. The TR comprises several small internal direct repeats as well as two sequences which are homologous to herpesvirus pac-1 and pac-2 sites, respectively. The organization of the RCMV TR is unique among cytomegaloviruses with respect to the position of the pac sequences: pac-1 is located near the left end of the TR, whereas pac-2 is present near the right end. Both RCMV DNA termini carry an extension of a single nucleotide at the 3' end. Since these nucleotides are complementary, circularization of the viral genome is likely to occur via a simple ligation reaction.
Induction of apoptosis by La Crosse virus infection and role of neuronal differentiation and human bcl-2 expression in its prevention
Authors: Pekosz A , Phillips J , Pleasure D , Merry D , Gonzalez-Scarano F
Published: January 2020

La Crosse virus causes a highly cytopathic infection in cultured cells and in the murine central nervous system (CNS), with widespread neuronal destruction. In some viral infections of the CNS, apoptosis, or programmed cell death, has been proposed as a mechanism for cytopathology (Y. Shen and T. E. Shenk, Curr. Opin. Genet. Dev. 5:105-111, 1995). To determine whether apoptosis plays a role in La Crosse virus-induced cell death, we performed experiments with newborn mice and two neural tissue culture models. Newborn mice infected with La Crosse virus showed evidence of apoptosis with the terminal deoxynucleotidyl transferase-mediated nicked-end labeling (TUNEL) assay and, concomitantly, histopathological suggestion of neuronal dropout. Infection of tissue culture cells also resulted in DNA fragmentation, TUNEL reactivity, and morphological changes in the nuclei characteristic of apoptotic cells. As in one other system (S. Ubol, P. C. Tucker, D. E. Griffin, and J. M. Hardwick, Proc. Natl. Acad. Sci. USA 91:5202-5206, 1994), expression of the human proto-oncogene bcl-2 was able to protect one neuronal cell line, N18-RE-105, from undergoing apoptosis after La Crosse virus infection and prolonged the survival of infected cells. Nevertheless, expression of bcl-2 did not prevent eventual cytopathicity. However, a human neuronal cell line, NT2N, was resistant to both apoptosis and other types of cytopathicity after infection with La Crosse virus, reaffirming the complexity of cell death. Our results show that apoptosis is an important consequence of La Crosse virus infection in vivo and in vitro.
Biosynthesis of glycoproteins E and I of feline herpesvirus: gE-gI interaction is required for intracellular transport
Authors: Mijnes J D , van der Horst L M , van Anken E , Horzinek M C , Rottier P J , de Groot R J
Published: January 2020

The biosynthesis of glycoproteins E and I of feline herpesvirus was studied by using the vaccinia virus vTF7-3 expression system. gE and gI were synthesized as N-glycosylated, endoglycosidase H (EndoH)-sensitive precursors with Mrs of 83,000 and 67,000, respectively. When coexpressed, gE and gI formed sodium dodecyl sulfate-sensitive hetero-oligomeric complexes that were readily transported from the endoplasmic reticulum (ER). Concomitantly, the glycoproteins acquired extensive posttranslational modifications, including O glycosylation, leading to an increase in their apparent molecular weights to 95,000 and 80,000 to 100,000 for gE and gI, respectively. In the absence of gE, most gI remained EndoH sensitive. Only a minor population became EndoH resistant, but these molecules were processed aberrantly as indicated by their Mrs (100,000 to 120,000). By immunofluorescence microscopy, gI was detected primarily in the ER but also at the plasma membrane. gE, when expressed by itself, remained EndoH sensitive and was found only in the ER and the nuclear envelope. These results were corroborated by studying the biosynthesis of gE in feline herpesvirus (FHV)-infected cells. In cells infected with wild-type FHV, gE acquired the same co- and posttranslational modifications as during vTF7-3-driven expression. However, an FHV mutant lacking gI failed to produce mature gE. We conclude that gE is retained in the ER, presumably by associating with molecular chaperones, and becomes transport competent only when in a complex with gI.
The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis
Authors: Kim K H , Hemenway C
Published: January 2020

A tobacco protoplast system was developed to analyze cis-acting sequences required for potato virus X (PVX) replication. Protoplasts inoculated with transcripts derived from a PVX cDNA clone or from clones containing mutations in their 5' nontranslated regions (NTRs) were assayed for RNA production by S1 nuclease protection assays. A time course of plus- and minus-strand-RNA accumulation indicated that both minus- and plus-strand PVX RNAs were detectable at 0.5 h postinoculation. Although minus-strand RNAs accumulated more rapidly than plus-strand RNAs, maximum levels of plus-strand RNAs were 40- to 80-fold higher. On the basis of these data, time points were chosen for determination of RNA levels in protoplasts inoculated with PVX clones containing deletions or an insertion in their 5' NTRs. Deletions of more than 12 nucleotides from the 5' end, internal deletions, and one insertion in the 5' NTR resulted in substantially decreased levels of plus-strand-RNA production. In contrast, all modified transcripts were functional for minus-strand-RNA synthesis, suggesting that elements in the 5' NTR were not essential for minus-strand-RNA synthesis. Further analysis of the 5' NTR deletion mutants indicated that all mutations that decreased genomic plus-strand-RNA synthesis also decreased synthesis of the two major subgenomic RNAs. These data indicate that cis-acting elements from different regions of the 5' NTR are required for plus-strand-RNA synthesis and that this process may be linked to synthesis of subgenomic RNAs.
Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity
Authors: Schultze B , Krempl C , Ballesteros M L , Shaw L , Schauer R , Enjuanes L , Herrler G
Published: January 2020

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.
Properties of the novel herpes simplex virus type 1 origin binding protein, OBPC
Authors: Baradaran K , Hardwicke M A , Dabrowski C E , Schaffer P A
Published: January 2020

We have recently identified a novel 53-kDa herpes simplex virus type 1 (HSV-1) protein encoded by, and in frame with, the 3' half of the UL9 open reading frame, designated OBPC (K. Baradaran, C. Dabrowski and P. A. Schaffer, J. Virol. 68:4251-4261, 1994). Here we show that OBPC is a nuclear protein synthesized at both early and late times postinfection. In gel-shift assays in vitro-synthesized OBPC bound to oriS site I DNA to form a complex identical in mobility to complex A, generated with infected cell extracts and site I DNA. OBPC inhibited both plaque formation and viral DNA replication in transient assays, consistent with its ability to bind to site I DNA and its limited ability to interact with other essential DNA replication proteins. These properties suggest that OBPC may play a role in the initiation, elongation, or packaging of viral DNA.
Identification and characterization of pseudorabies virus glycoprotein gM as a nonessential virion component
Authors: Dijkstra J M , Visser N , Mettenleiter T C , Klupp B G
Published: January 2020

Sequence analysis within BamHI fragment 3 of the pseudorabies virus (PrV) genome revealed an open reading frame homologous to the UL10 gene of herpes simplex virus. A rabbit antiserum directed against a synthetic oligopeptide representing the carboxy-terminal 18 amino acids of the predicted UL10 product recognized a major 45-kDa protein in lysates of purified Pr virions. In addition, a second protein of 90 kDa which could represent a dimeric form was observed. Enzymatic deglycosylation showed that the PrV UL10 protein is N glycosylated. Therefore, it was designated PrV gM according to its homolog in herpes simplex virus. A PrV mutant lacking ca. 60% of UL10 coding sequences was able to productively replicate on noncomplementing cells, demonstrating that PrV gM is not required for viral replication in cell culture. However, infectivity of the mutant virus was reduced and penetration was delayed, indicating a modulatory role of PrV gM in the initiation of infection.
Replication of murine coronavirus defective interfering RNA from negative-strand transcripts
Authors: Joo M , Banerjee S , Makino S
Published: January 2020

The positive-strand defective interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV), when introduced into MHV-infected cells, results in DI RNA replication and accumulation. We studied whether the introduction of negative-strand transcripts of MHV DI RNA would also result in replication. At a location downstream of the T7 promoter and upstream of the human hepatitis delta virus ribozyme domain, we inserted a complete cDNA clone of MHV DI RNA in reverse orientation; in vitro-synthesized RNA from this plasmid yielded a negative-strand RNA copy of the MHV DI RNA. When the negative-strand transcripts of the DI RNA were expressed in MHV-infected cells by a vaccinia virus T7 expression system, positive-strand DI RNAs accumulated in the plasmid-transfected cells. DI RNA replication depended on the expression of T7 polymerase and on the presence of the T7 promoter. Transfection of in vitro-synthesized negative-strand transcripts into MHV-infected cells and serial passage of virus samples from RNA-transfected cells also resulted in accumulation of the DI RNA. Positive-strand DI RNA transcripts were undetectable in sample preparations of the in vitro-synthesized negative-strand DI RNA transcripts, and DI RNA did not accumulate after cotransfection of a small amount of positive-strand DI RNA and truncated-replication-disabled negative-strand transcripts; clearly, the DI RNA replicated from the transfected negative-strand transcripts and not from minute amounts of positive-strand DI RNAs that might be envisioned as artifacts of T7 transcription. Sequence analysis of positive-strand DI RNAs in the cells transfected with negative-strand transcripts showed that DI RNAs maintained the DI-specific unique sequences introduced within the leader sequence. These data indicated that positive-strand DI RNA synthesis occurred from introduced negative-strand transcripts in the MHV-infected cells; this demonstration, using MHV, of DI RNA replication from transfected negative-strand DI RNA transcripts is the first such demonstration among all positive-stranded RNA viruses.
The zinc finger of nucleocapsid protein of Friend murine leukemia virus is critical for proviral DNA synthesis in vivo
Authors: Yu Q , Darlix J L
Published: January 2020

Nucleocapsid protein NCp10 of murine leukemia virus (MuLV) is encoded by the 3' domain of gag and contains a zinc finger of the form Cys-X2-Cys-X4-His-X4-Cys flanked by basic amino acids. In the course of virus assembly, NCp10 is necessary for core formation, and the zinc finger flanked by the basic residues is required for the packaging of the genomic RNA dimer. In vitro, NCp10 exhibits strong nucleic acid binding and annealing activities that appear to be critical for virus infectivity since NCp10 promotes dimerization of the viral RNA containing the E/DLS packaging-dimerization signal and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (PBS). Recent in vitro studies have suggested that NCp10 may also play a role in proviral DNA synthesis. To investigate the function of NCp10 in proviral DNA synthesis in vivo, we developed a simple and convenient genetic packaging system consisting of two DNA constructs expressing the packaging components gag-pol and env of Friend MuLV and a Moloney MuLV-based lacZ vector with either the MuLV E+ or a rat VL30 E packaging signal. This system allowed us to examine the consequences of a set of mutations in NCp10 on a single round of recombinant virus replication. Most mutations in the N- or C-terminal domain of NCp10 do not significantly alter infectivity, while those in the zinc finger drastically impair infectivity. Analysis of the viral RNA content in virions showed that all mutations in the zinc finger decrease but do not abolish packaging of the recombinant genome. Interestingly enough, mutation of Y-28 to S (mutation Y28S) in the zinc finger results in RNA packaging at a level similar to that observed upon deletion of three prolines and three arginines in the C-terminal domain of NCp10 (mutant delta PR3). However, mutant Y28S is noninfectious while mutant delta PR3 is only threefold less infectious than the wild-type virus, which prompted us to examine the role of NCp10 protein in proviral DNA synthesis in vivo using these nucleocapsid mutants. PCR amplification was used to analyze viral DNA synthesized in newly infected cells, and results indicate that the Y28S zinc finger mutation impairs reverse transcription, thus suggesting that the nucleocapsid protein zinc finger plays a key role in proviral DNA synthesis in vivo.
Transcriptional regulation of human polyomavirus JC: evidence for a functional interaction between RelA (p65) and the Y-box-binding protein, YB-1
Authors: Raj G V , Safak M , MacDonald G H , Khalili K
Published: January 2020

The transcriptional control region of the human neurotropic polyomavirus JC virus contains a consensus NF-kappa B site which has been shown to enhance both basal and extracellular stimulus-induced levels of transcription of JC promoters. Here, we show that the expression of JC late promoter constructs containing the NF-kappa B site is decreased by cotransfection with the NF-kappa B/rel subunits, p50 and p52, but enhanced by the p65 subunit. However, JC promoter constructs lacking the NF-kappa B site were activated by p52 and p50 and repressed by p65. This antithetical response of the JC promoter mapped specifically to the D domain, which is a target site for the cellular transcription factor, YB-1. Band shift studies indicated that YB-1 and p65 modulate each other's binding to DNA: YB-1 augments the affinity of p65 for the NF-kappa B site, while p65 reduces the binding of YB-1 to the D domain. Results from coimmunoprecipitation followed by Western blot (immunoblot) analysis suggest an in vivo interaction between p65 and YB-1 in glial cells. Functionally, YB-1 appears to act synergistically with p65 to control transcription from the NF-kappa B site. A converse pattern is seen with the D domain, in which YB-1 acts synergistically with p50 and p52 to regulate transcription. p50 and p52 may function as transcriptional activators on the D domain by removing the repressive effect of p65 on YB-1 binding to the D domain. On the basis of these data, we propose a model in which NF-kappa B/rel subunits functionally interact with consensus NF-kappa B sites or YB-1-binding sites, with disparate effects on eukaryotic gene expression.
Regulation of the herpesvirus saimiri oncogene stpC, similar to that of T-cell activation genes, in growth-transformed human T lymphocytes
Authors: Fickenscher H , Biesinger B , Knappe A , Wittmann S , Fleckenstein B
Published: January 2020

Herpesvirus saimiri strain C488, a T-cell tumor virus of New World primates, transforms human T lymphocytes to stable interleukin-2-dependent growth without need for further stimulation by antigen or mitogen. The transformed cell lines show the phenotype of activated mature T cells and retain many essential features of the primary parental cells, e.g., antigen specificity. In contrast to transformed New World monkey T cells, the human lines do not support lytic growth of the virus, even after chemical stimulation. Here we show that many viral genes remain silent during episomal persistence. However, the viral oncogene stpC is predominantly transcribed and translated to a stable cytoplasmic protein of 20 kDa that is heterogeneously expressed in individual cells. This 1.7-kb mRNA is bicistronic, encoding also Tip, a viral protein interacting with the T-cell-specific tyrosine kinase Lck. stpC/tip transcripts are heavily induced upon stimulation by mitogen or phorbol ester. Block of protein synthesis does not abolish transcription: treatment with cycloheximide greatly induces stpC/tip mRNA levels. Thus, this gene complex is regulated similarly to early T-cell activation genes. Constitutive and induced expression engage different transcription start sites. The T-cell regulation of the viral genes stpC and tip may contribute to the T-cell tropism of growth transformation by herpesvirus saimiri.
Oligomeric structure of glycoproteins in herpes simplex virus type 1
Authors: Handler C G , Eisenberg R J , Cohen G H
Published: January 2020

A number of herpes simplex virus (HSV) glycoproteins are found in oligomeric states: glycoprotein E (gE)-gI and gH-gL form heterodimers, and both gB and gC have been detected as homodimers. We have further explored the organization of glycoproteins in the virion envelope by using both purified virions to quantitate glycoprotein amounts and proportions and chemical cross-linkers to detect oligomers. We purified gB, gC, gD, and gH from cells infected with HSV type 1 and used these as immunological standards. Glycoproteins present in sucrose gradient-purified preparations of two strains of HSV type 1, KOS and NS, were detected with antibodies to each of the purified proteins. From these data, glycoprotein molar ratios of 1:2:11:16 and 1:1:14:9 were calculated for gB/gC/gD/gH in KOS and NS, respectively. gL was also detected in virions, although we lacked a purified gL standard for quantitation. We then asked whether complexes of these glycoproteins could be identified, and if they existed as homo- or hetero-oligomers. Purified KOS was incubated at 4 degrees C with bis (sulfosuccinimidyl) suberate (BS3), an 11.4 A (1A = 0.1 mm) noncleavable, water-soluble cross-linker. Virus extracts were examined by Western blotting (immunoblotting), or immunoprecipitation followed by Western blotting, to assay for homo- and hetero-oligomers. Homodimers of gB, gC, and gD were detected, and hetero-oligomers containing gB cross-linked to gC, gC to gD, and gD to gB were also identified. gH and gL were detected as a hetero-oligomeric pair and could be cross-linked to gD or gC but not to gB. We conclude that these glycoproteins are capable of forming associations with one another. These studies suggest that glycoproteins are closely associated in virions and have the potential to function as oligomeric complexes.
Increased envelope spike density and stability are not required for the neutralization resistance of primary human immunodeficiency viruses
Authors: Karlsson G B , Gao F , Robinson J , Hahn B , Sodroski J
Published: January 2020

Previous observations that the gp120 envelope glycoprotein contents of some primary, clade B human immunodeficiency virus type 1 (HIV-1) isolates were higher than those of laboratory-passaged HIV-1 isolates suggested the hypothesis that increased envelope glycoprotein spike density or stability contributes to the relative neutralization resistance of the primary viruses. To test this, the structural, replicative, and neutralization properties of a panel of recombinant viruses with HIV-1 envelope glycoproteins from divergent clades were examined in an env complementation assay. In this system, although the spike density and stability of envelope glycoproteins from primary HIV-1 isolates were not greater than those from a laboratory-adapted isolate, relative resistance to neutralizing antibodies and soluble CD4 was observed for the viruses with primary envelope glycoproteins. Thus, neither high envelope glycoprotein spike density nor stability is necessary for the relative neutralization resistance of primary HIV-1 viruses.
The structurally diverse intergenic regions of respiratory syncytial virus do not modulate sequential transcription by a dicistronic minigenome
Authors: Kuo L , Fearns R , Collins P L
Published: January 2020

The first nine genes of respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus, are separated by intergenic regions which range in size from 1 to 52 nucleotides for strain A2 and lack obvious consensus elements except that each ends in an A (genome sense). Their significance for gene expression was investigated by using RSV-CAT-LUC RNA, a helper-dependent cDNA-encoded dicistronic analog of RSV genomic RNA in which the viral genes were replaced by a negative-sense copy of the translational open reading frame (ORF) encoding chloramphenicol acetyltransferase (CAT) as the upstream, leader-proximal gene and that encoding luciferase (LUC) as the downstream gene. These foreign ORFs were flanked by the RSV gene-start (GS) and gene-end (GE) transcription signals and separated by the naturally occurring G/F intergenic region. The RSV-CAT-LUC minigenome was synthesized in vitro and transfected into RSV-infected cells, and synthesis of the CAT and LUC mRNAs was monitored by enzyme assay and Northern (RNA) blot hybridization. Surprisingly, substitution of each of the other naturally occurring RSV intergenic regions in turn did not significantly alter the absolute or relative amounts of the two mRNAs. Substitution of a nonnatural 10-nucleotide intergenic region, or elimination of the intergenic region altogether, also had little effect on the level of expression of the two genes. Four of the minigenome variants containing naturally occurring intergenic regions were modified further by replacing part of the LUC ORF with a second copy of the CAT ORF, so that each of the two mRNAs would hybridize equally with a CAT-specific probe and their relative molar amounts could be determined. The level of expression of the downstream gene was 0.30 to 0.36 that of the upstream one. This determined the magnitude of RSV transcriptional polarity across a gene pair and confirmed that this value was very similar among the various intergenic regions. Minigenome transcription also yielded a CAT-LUC readthrough mRNA at a level 0.10 to 0.13 that of the LUC mRNA. In summary, the structurally diverse RSV intergenic regions do not appear to play a role in modulating RSV gene expression.
Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family
Authors: Nunn M F , Marsh J W
Published: January 2020

Although human immunodeficiency virus (HIV) Nef is essential for the induction of AIDS, its biochemical function has remained an enigma. In this study, HIV Nef protein is shown to associate with a serine-threonine kinase that recognizes histone H4 as a substrate, is serologically related to rat p21-activated kinase (PAK), and is specifically activated by Rac and Cdc42. These characteristics define the Nef-associated kinase as belonging to the PAK family. PAKs initiate kinase cascades in response to environmental stimuli, and their identification as a target of Nef implicates these signaling molecules in HIV pathogenesis and provides a novel target for clinical intervention.
Inhibitors of human immunodeficiency virus type 1 zinc fingers prevent normal processing of gag precursors and result in the release of noninfectious virus particles
Authors: Turpin J A , Terpening S J , Schaeffer C A , Yu G , Glover C J , Felsted R L , Sausville E A , Rice W G
Published: January 2020

The Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys zinc fingers of retroviral nucleocapsid proteins are prime antiviral targets because of conservation of the Cys and His chelating residues and the absolute requirement of these fingers in both early and late phases of retroviral replication. We previously reported that certain disulfide-substituted benzamides (DIBAs) chemically modify the Cys residues of the fingers, resulting in inhibition of human immunodeficiency virus type 1 (HIV-1) replication (W. G. Rice, J. G. Supko, L. Malspeis, R. W. Buckheit, Jr., D. Clanton, M. Bu, L. Graham, C. A. Schaeffer, J. A. Turpin, J. Domagala, R. Gogliotti, J. P. Bader, S. M. Halliday, L. Coren, R. C. Sowder II, L. O. Arthur, and L. E. Henderson, Science 270:1194-1197, 1995). We now examine the consequences of the interaction of DIBAs with the zinc fingers of the HIV-1 p7 nucleocapsid protein and its Pr55gag precursor. In HIV-1-infected U1 cells, DIBAs inhibited the release of infectious virions, and even under conditions in which virion particles were produced, the particles were noninfectious. DIBAs caused abnormal processing of Gag precursors, and the inhibitory effect on processing was not due to inhibition of the HIV-1 protease enzyme or Pr55gag myristoylation. Rather, the defect in processing was due to the formation of intermolecular cross-linkages among the zinc fingers of adjacent Gag molecules, rendering the precursors no longer recognizable by HIV-1 protease. Likewise, DIBAs caused intermolecular cross-linkage among recombinant Pr55gag packaged into pseudovirions, thereby generating modified precursors that were resistant to the action of protease. Thus, DIBAs chemically modified the mutationally intolerant retroviral zinc fingers in infected cells, interrupting protease-mediated maturation of virions and leading ultimately to the production of compromised virions.
Localization of human T-cell leukemia virus type 1 tax to subnuclear compartments that overlap with interchromatin speckles
Authors: Semmes O J , Jeang K T
Published: January 2020

Tax, the virally encoded activator of the human T-cell leukemia virus type 1 long terminal repeats, regulates the expression of many cellular genes. This protein has been implicated in transformation events leading to the development of adult T-cell leukemia. Because subcellular localization contributes importantly to protein function, we determined the compartment(s) within the cell in which Tax is found. Using confocal microscopy, we found that Tax localizes to subnuclear domains which overlap with structures previously identified as interchromatin granules or spliceosomal speckles. These Tax speckled structures are coincident with a subset of nuclear transcriptional hot spots. Disruption of the Tax speckled structures by heat shock revealed the existence of different populations of Tax. One population of Tax is tightly associated with nuclear speckles. A second population exists outside of the speckles and is transcriptionally active for some promoters.
The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit is required for viral RNA synthesis in replication of the influenza virus genome
Authors: Nakagawa Y , Oda K , Nakada S
Published: January 2020

We indicated that the PB1 and PA subunits of RNA polymerase and nucleoprotein (NP) can support replication of the influenza virus genome as well as transcription to yield uncapped poly(A)(+)-RNA (Y. Nakagawa, N. Kimura, T. Toyoda, K. Mizumoto, A. Ishihama, K. Oda, and S. Nakada, J. Virol. 69:728-733, 1995). To analyze the functions of the PB1 and PA subunits in replication and transcription, YP1N clones in which the PB1 and NP genes can be expressed in response to dexamethasone were established. cRNA was transcribed from model viral RNA (vRNA), but vRNA synthesis from model cRNA was not detected in YP1N clones. Furthermore, poly(A)(+)-RNA directed from model vRNA was synthesized in YP1N clones. These results indicated that PB1 and NP can support the syntheses of cRNA and poly(A)(+)-RNA and that the PA subunit, in addition to that of PB1 and to NP, is required for vRNA synthesis. In summary, the PB1 subunit is involved in the catalytic activities of nucleotide elongation, and the PA subunit may act as an allosteric modulator and cause a conformational change from a cRNA-to a vRNA-synthesizing form of the PB1 subunit.