bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein
Authors: Wu X , Conway J A , Kim J , Kappes J C
Published: January 2020

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.
The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements
Authors: Smith C D , Baglia L A , Curristin S M , Ruddell A
Published: January 2020

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.
Monocyte maturation controls expression of equine infectious anemia virus
Authors: Maury W
Published: January 2020

In vivo, equine infectious anemia virus (EIAV) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. No viral replication has been detected in circulating peripheral blood monocytes. However, proviral DNA can be detected in these cells, and monocytes may serve as a reservoir for the virus. In this study, an in vitro model was developed to clarify the role of monocyte maturation in regulating EIAV expression. Freshly isolated, nonadherent equine peripheral blood monocytes were infected with a macrophage-tropic strain of EIAV, and expression of EIAV was monitored in cells held as nonadherent monocytes and cells allowed to adhere and differentiate into macrophages. A 2- to 3-day delay in viral antigen expression was observed in the nonadherent cells. This restriction of viral expression in monocytes was supported by nuclear run-on studies demonstrating that on day 5 postinfection, the level of actively transcribed viral messages was 4.7-fold lower in monocyte cultures than in macrophage cultures. Electrophoretic mobility shift assays identified three regions of the U3 enhancer that interacted with nuclear extracts from normal equine macrophages. Each region contained the core binding motif of a family of transcription factors that includes the product of the proto-oncogene ets. Antibodies to the Ets family member PU.1 caused a supershifting of retarded bands in an electrophoretic mobility shift assay. Transfection studies of ets motif mutants demonstrated that the U3 ets sites were important in the regulation of EIAV transcription in macrophages. Interactions between the ets motif and nuclear extracts from freshly isolated, nonadherent monocytes, macrophages adherent for 1 or 2 days, or macrophages adherent for 5 days gave different patterns of retarded bands, although the binding specificities were similar with all three extracts. The different complexes formed by monocyte and macrophage nuclear extracts may explain the enhanced ability of mature macrophages to support EIAV expression.
trans activation by the full-length E2 proteins of human papillomavirus type 16 and bovine papillomavirus type 1 in vitro and in vivo: cooperation with activation domains of cellular transcription factors
Authors: Ushikai M , Lace M J , Yamakawa Y , Kono M , Anson J , Ishiji T , Parkkinen S , Wicker N , Valentine M E , Davidson I
Published: January 2020

Papillomaviral E2 genes encode proteins that regulate viral transcription. While the full-length bovine papillomavirus type 1 (BPV-1) E2 peptide is a strong trans activator, the homologous full-length E2 product of human papillomavirus type 16 (HPV-16) appeared to vary in function in previous studies. Here we show that when expressed from comparable constructs, the full-length E2 products of HPV-16 and BPV-1 trans activate a simple E2- and Sp1-dependent promoter up to approximately 100-fold in human keratinocytes and other epithelial cells as well as human and animal fibroblasts. Vaccinia virus-expressed, purified full-length HPV-16 and BPV-1 E2 proteins bound a consensus E2 site with high specific affinities (Kd = approximately 10(-9) M) and stimulated in vitro transcription up to six- to eightfold. In vivo and in vitro trans activation by either E2 protein required cooperation with another activator, such as Sp1, or other factors that interact with papillomavirus promoters, such as AP-1, Oct-1, nuclear factor 1/CTF, transcriptional enhancer factor 1, or USF. The glutamine-rich domain B of Sp1 or the mutually unrelated activation domains of other transcription factors were necessary and sufficient for cooperation with either E2 factor. We conclude that like BPV-1 E2, the HPV-16 E2 protein has the potential to function as a strong activator of viral gene expression in cooperation with cellular transcription factors.
Inhibition by interleukin-4 of constitutive beta interferon synthesis in mouse macrophages
Authors: Nickolaus P , Zawatzky R
Published: January 2020

We have found that interleukin-4 (IL-4) abrogated constitutive beta interferon (IFN-beta) synthesis in mouse macrophages. Analysis of endogenous IFN mRNA by reverse transcription-PCR clearly documented the presence of IFN-beta but not of IFN-alpha transcripts in RNA from bone marrow-derived macrophages (BMM). Culture of bone marrow cells for 7 days in the presence of macrophage colony-stimulating factor plus IL-4 or treatment of mature BMM with IL-4 for 3 to 4 days resulted in pronounced inhibition of IFN-beta transcript levels. As a consequence, BMM became highly permissive to viral infection and induction of the IFN-responsive 2',5'-oligoadenylate synthetase was inhibited. In view of the evidence for low-level constitutive secretion of IFN in vivo, it is possible that IL-4 influences the host's antiviral defense system.
Human herpesvirus 6B origin: sequence diversity, requirement for two binding sites for origin-binding protein, and enhanced replication from origin multimers
Authors: Dewhurst S , Krenitsky D M , Dykes C
Published: January 2020

A previously identified human herpesvirus 6B (HHV-6B) origin of DNA replication contains two binding sites for the origin-binding protein (OBPH6B). We have investigated the functional significance of these sites by determining the replication efficiencies of mutated origin sequences, using a transient replication assay. The results indicate that both sites are required for DNA replication. In addition, we have tested the functional consequences of linear sequence amplifications in the origin. The data show that tandemized origin elements are more efficiently replicated than single-copy origins. Finally, we have determined the extent of interstrain origin sequence variation that exists among HHV-6 isolates by cloning, sequencing, and analyzing origins from a number of virus isolates, including examples of both HHV-6A and HHV-6B.
Low-affinity E2-binding site mediates downmodulation of E2 transactivation of the human papillomavirus type 8 late promoter
Authors: Stubenrauch F , Pfister H
Published: January 2020

The constitutively active promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is transactivated by the viral E2 protein. The distribution of potential E2-binding sites (ACCN6GGT) in the viral transcription control region is highly conserved among epidermodysplasia verruciformis-associated human papillomaviruses and differs completely from that of other papillomaviruses. To investigate the role of E2-binding sites P0 to P4 in P7535 regulation, we analyzed their binding affinities in gel retardation experiments using a full-length HPV8 E2 protein expressed from a recombinant baculovirus. Binding site P1 within a transcriptional silencer showed the highest affinity, followed by P0 within the L1 gene and P3 downstream of P7535. P2, 33 nucleotides upstream of the mRNA cap site, and P4 were very weak binders. There is some indication that the number of A/T pairs in the nonconserved core of the recognition sequence is critical for the binding of HPV8 E2. Transient transfection experiments were carried out with an HPV8 E2 expression vector and reporter plasmids containing mutated E2-binding sites in the context of the HPV8 regulatory region. The knockout of the strongest binding site P1 sufficed to clearly diminish transactivation. P0, P3, and P4 mutations had little effect on their own, whereas double mutations P01 and P34 strongly reduced E2 inducibility. Both mutations in P2 severely affected constitutive promoter activity but had opposite effects on transactivation. They revealed an inverse correlation between E2-binding strength and the extent of E2 transactivation. This finding suggests that P2 mediates a negative control of P7535 by E2, counteracting E2 transactivation exerted via the four distal E2 target sequences.
Improved self-inactivating retroviral vectors derived from spleen necrosis virus
Authors: Olson P , Nelson S , Dornburg R
Published: January 2020

Self-inactivating (SIN) retroviral vectors contain a deletion spanning most of the right long terminal repeat's (LTR's) U3 region. Reverse transcription copies this deletion to both LTRs. As a result, there is no transcription from the 5' LTR, preventing further replication. Many previously developed SIN vectors, however, had reduced titers or were genetically unstable. Earlier, we reported that certain SIN vectors derived from spleen necrosis virus (SNV) experienced reconstitution of the U3-deleted LTR at high frequencies. This reconstitution occurred on the DNA level and appeared to be dependent on defined vector sequences. To study this phenomenon in more detail, we developed an almost completely U3-free retroviral vector. The promoter and enhancer of the left LTR were replaced with those of the cytomegalovirus immediate-early genes. This promoter swap did not impair the level of transcription or alter its start site. Our data indicate that SNV contains a strong initiator which resembles that of human immunodeficiency virus. We show that the vectors replicate with efficiencies similar to those of vectors possessing two wild-type LTRs. U3-deleted vectors carrying the hygromycin B phosphotransferase gene did not observably undergo LTR reconstitution, even when replicated in helper cells containing SNV-LTR sequences. However, vectors carrying the neomycin resistance gene did undergo LTR reconstitution with the use of homologous helper cell LTR sequences as template. This supports our earlier finding that sequences within the neomycin resistance gene can trigger recombination.
Artificial mosaic protein containing antigenic epitopes of hepatitis E virus
Authors: Khudyakov Y E , Favorov M O , Khudyakova N S , Cong M E , Holloway B P , Padhye N , Lambert S B , Jue D L , Fields H A
Published: January 2020

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.
Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding
Authors: Freund R , Bauer P H , Crissman H A , Bradbury E M , Benjamin T L
Published: January 2020

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.
Virus-induced immunosuppression: kinetic analysis of the selection of a mutation associated with viral persistence
Authors: Evans C F , Borrow P , de la Torre J C , Oldstone M B
Published: January 2020

Infection of neonatal mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (ARM) results in a lifelong persistent infection. Viral variants (cytotoxic T lymphocyte [CTL] negative, persistence positive [CTL- P+]) can be isolated from the lymphoid tissues of such mice. Adult mice inoculated with these CTL- P+ viruses fail to generate sufficient cytotoxic T lymphocytes to clear the acute infection and become persistently infected. By contrast, inoculation of a similar dose of the parental ARM virus (CTL+ P-) into adult mice leads to the generation of a vigorous virus-specific CTL response that clears the infection. Sequence analysis revealed a phenylalanine (Phe)-to-Leucine (Leu) change at amino acid 260 of the viral glycoprotein (GP) as a marker for variant viruses with the CTL- P+ phenotype. An RNA PCR assay that detects the variant GP sequence and thus allows kinetic studies of the selection of the Leu at position 260 was developed. We found that although CTL- P+ viruses are known to be lymphotropic, mature T and B cells were not required for the generation and selection of the Leu at GP amino acid 260. Kinetically, in mice infected at birth with LCMV ARM, as early as 3 weeks postinfection the Phe-to-Leu change was found in virus in the serum. By 5 weeks, viral nucleic acid obtained from peritoneal macrophages, spleen, lymph nodes, and liver showed the Phe-to-Leu change. At 2 months postinfection, the Leu change was detected in virus from the thymus, heart, lung, and kidney. By contrast, virus replicating in the central nervous system showed only minimal levels of the Leu change by 4 months and as long as 1 year postinfection. In vitro studies showed that the parental LCMV ARM CTL+ P- virus replicates more efficiently and outcompetes CTL- P+ virus in a cultured neuronal cell line, indicating that differential growth properties in neurons are likely the basis for the selection of the parental virus over the CTL- P+ variant in the brain.
Sequence requirements for stable binding and function of Rep68 on the adeno-associated virus type 2 inverted terminal repeats
Authors: Chiorini J A , Wiener S M , Owens R A , Kyöstió S R , Kotin R M , Safer B
Published: January 2020

Replication of the palindromic inverted terminal repeats (ITRs) of adeno-associated virus type 2 requires several functions of the viral nonstructural Rep proteins. These include binding to the ITR, nicking of the double-stranded replication intermediate at the terminal resolution site (trs), and then strand displacement and synthesis from the nick. This report demonstrates the ability of both recombinant fusion maltose-binding protein (MBP)-Rep68 delta produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, delta 57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68 delta of approximately 8 x 10(-10) M was determined for the wt ITR and delta 57 ITR probes. Truncation of delta 57 ITR to generate delta 28 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than delta 57 ITR. Extension of delta 28 ITR with nonspecific sequence restored the ability of MBP-Rep68 delta to bind to delta 28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the delta 57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep68 delta. The requirements for stable binding were further defined with a series of oligonucleotide probes which spanned the region protected by MBP-Rep78 in DNase I footprinting. The binding activity of either MBP-Rep68 delta or wt Rep68 to hairpin ITR or delta 57 ITR was indistinguishable. However, the binding activity of MBP-Rep68 delta to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68 delta to the nonhairpin delta 57 ITR was approximately 100-fold less efficient than its linkage to a hairpin-containing ITR. Therefore, although the hairpin portion of the ITR does not appear to play a role in recognition and stabilization of MBP-Rep68 delta binding, its presence does affect the trs cleavage activity of the protein.
Apparent functional role for a cysteine-rich polydnavirus protein in suppression of the insect cellular immune response
Authors: Li X , Webb B A
Published: January 2020

Polydnaviruses suppress the cellular immune response and inhibit growth and development in their lepidopteran host, allowing survival of their endoparasitic hymenopteran host. Characterization of genes disrupting insect physiological systems is a major objective in the study of polydnaviruses. Recently, a cysteine-rich gene family encoding a motif composed of invariable cysteine residues flanking hypervariable intercysteine amino acids was described (S.D. Dib-Hajj, B.A. Webb, and M.D. Summers, Proc. Natl. Acad. Sci. USA 90:3765-3769, 1993). They noted similarities to the positive selection pressure for mutations within the vertebrate major histocompatibility complex (MHC) class II genes and speculated that this class of polydnavirus genes may target and disrupt the insect immune system. To study the functional activity of this family of predicted cysteine-rich proteins, the VHv1.1 gene product was produced from bacterial and baculovirus expression systems. Polyclonal antiserum produced from the bacterial fusion protein reacted with a 30-kDa protein from hemocytes, cell-free plasma, and fat body of parasitized larvae. Immunofluorescence analysis of hemocytes from parasitized insects detected the 30-kDa protein bound to granulocytes and plasmacytes. To assay the functional activity of the 30-kDa VHv1.1 protein, a recombinant baculovirus was constructed allowing in vivo expression of the 30-kDa polydnavirus protein from infected insects. Expression of the VHv1.1 protein from the baculovirus system reduced the encapsulation response to washed wasp eggs relative to controls. The experimental evidence demonstrates that Campoletis sonorensis polydnavirus-infected cells secrete VHv1.1 into the hemolymph, where it binds to hemocytes and is associated with the inhibition of the cellular immune response.
Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin
Authors: Orlich M , Rott R
Published: January 2020

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.
Protein-protein interactions between human cytomegalovirus IE2-580aa and pUL84 in lytically infected cells
Authors: Spector D J , Tevethia M J
Published: January 2020

The human cytomegalovirus immediate-early protein IE2-580aa (ppUL122a) activates transcription of viral and cellular genes and represses its own transcription through sequence-specific binding to the major immediate-early promoter. In lytically infected cells, IE2-580aa interacts with a 75-kDa viral protein (p75), an early protein that is also synthesized at late times after infection. Here we show that p75 is the product of the UL84 gene. Its association with IE2-580aa in infected cells suggests that pUL84 is involved in transcription control.
Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins
Authors: Roden R B , Weissinger E M , Henderson D W , Booy F , Kirnbauer R , Mushinski J F , Lowy D R , Schiller J T
Published: January 2020

We have generated four mouse monoclonal antibodies (MAbs) to bovine papillomavirus virions that bound type-specific, adjacent, and conformationally dependent epitopes on the L1 major capsid protein. All four MAbs were neutralizing at ratios of 1 MAb molecule per 5 to 25 L1 molecules, but only three effectively blocked binding of the virus to the cell surface. Therefore, antibodies can prevent papillomavirus infection by at least two mechanisms: inhibition of cell surface receptor binding and a subsequent step in the infectious pathway. The neutralizing epitopes of the bovine papillomavirus L2 minor capsid protein were mapped to the N-terminal half of L2 by blocking the neutralizing activity of full-length L2 antiserum with bacterially expressed peptides of L2. In addition, rabbit antiserum raised against amino acids 45 to 173 of L2 had a neutralizing titer of 1,000, confirming that at least part of the N terminus of L2 is exposed on the virion surface.
Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins
Authors: Cai X , Chang D , Rottinghaus S , Consigli R A
Published: January 2020

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.
Preferential usage of T-cell receptor V beta 17 by dengue virus-specific human T lymphocytes in a donor with immunity to dengue virus type 4
Authors: Okamoto Y , Gagnon S J , Kurane I , Leporati A M , Ennis F A
Published: January 2020

In order to better understand human T-cell responses to dengue viruses (DV), we analyzed T-cell receptor (TCR) V beta gene usage in DV-specific T lymphocytes. Peripheral blood T lymphocytes from a DV type 4 immune donor were stimulated in vitro with DV type 4 antigen, and TCR usage was examined by reverse transcriptase PCR. TCR V beta 17 was preferentially used (P = 0.020) among T cells stimulated by DV type 4 in bulk culture. Furthermore, 8 of 19 DV type 4-specific CD4+ T-cell clones established from the peripheral blood mononuclear cells expressed V beta 17 (P = 0.008). Preferential usage of TCR V alpha was not found among T cells expressing V beta 17. These results indicate that there is preferential usage of TCR V beta 17 among DV-specific T cells in this donor and suggest that T cells with certain TCRs may be important in immune responses to DV.
A naturally occurring single basic amino acid substitution in the V3 region of the human immunodeficiency virus type 1 env protein alters the cellular host range and antigenic structure of the virus
Authors: Shioda T , Oka S , Ida S , Nokihara K , Toriyoshi H , Mori S , Takebe Y , Kimura S , Shimada K , Nagai Y
Published: January 2020

Human immunodeficiency virus type 1 circulates in vivo as a mixture of heterologous populations (quasispecies). We previously analyzed the quasispecies of the third hypervariable region (V3) in the viral envelope glycoprotein gp120 in an infected individual and found that the species with a basic amino acid substitution (lysine for aspartic acid) at a particular position evolved and became a distinct population within a short period, followed by progression to the typical immunodeficiency stage (S. Oka et al., AIDS Res. Hum. Retroviruses 10:271-277, 1994). In the present study, we examined the biological significance of this amino acid substitution by constructing recombinant viruses with specific point mutations and comparing their replication capabilities in different cell types. The results demonstrated that the single basic amino acid substitution was sufficient to render a virus fully capable of replicating in human T-cell lines under certain conditions. With an acidic amino acid at the position, the virus grew much less fast or did not grow at all in the T-cell lines. Viral neutralization assay and peptide enzyme-linked immunosorbent assays further showed that this amino acid substitution resulted in different recognition by several of the serum specimens from human immunodeficiency virus type 1-infected individuals and thus could alter the antigenic structure. An additional finding worthy of note was that at the terminal stage, the proviral sequences of peripheral blood mononuclear cells and the viral isolates from them were without exception of the late type with the basic amino acid substitution, whereas the early sequence without the substitution was retained as a major subset in the spleen. These results support the notion that basic amino acid substitutions in V3 are a strong predictor of virus tropism and may be relevant to disease progression.
Suppression of apoptosis in insect cells stably transfected with baculovirus p35: dominant interference by N-terminal sequences p35(1-76)
Authors: Cartier J L , Hershberger P A , Friesen P D
Published: January 2020

Expression of p35 from the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) suppresses virus-induced apoptosis and promotes virus replication in Spodoptera frugiperda (SF21) cells. To examine the molecular mechanism by which p35 prevents apoptosis in insects, SF21 cells were stably transfected with p35. Neomycin-resistant cell lines that synthesized protein P35 were identified. Stable transfection with p35 protected SF21 cells from apoptosis induced by actinomycin D concentrations that caused apoptotic death of untransfected cells. Cellular expression of p35 also blocked apoptosis induced by infection with p35 null mutants and restored mutant replication to levels comparable to those of wild-type virus. In contrast, stable expression of the mammalian death suppressor bcl-2 failed to block actinomycin D- or AcMNPV-induced apoptosis. Thus, p35 was sufficient to prevent apoptosis, whereas bcl-2 was not, suggesting that the activities of the two nonhomologous death regulators are functionally distinct. Stable expression of the truncation mutant p35(1-76), containing the N terminus of p35, failed to block apoptosis. However, p35(1-76) interfered with p35 antiapoptotic activity, since stably transfected cells underwent apoptosis upon infection with wild-type AcMNPV. Despite normal levels of viral p35 transcription, P35 levels were selectively reduced during infection. Thus, p35(1-76) acted as a dominant inhibitor by directly or indirectly affecting the synthesis or stability of viral P35. These results suggested that the N terminus of P35 constitutes a functional domain which is required to interact with other proteins, possibly host invertebrate death regulators or P35 itself.
Enhanced responsiveness to nuclear factor kappa B contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14
Authors: Dollard S C , Gummuluru S , Tsang S , Fultz P N , Dewhurst S
Published: January 2020

Infection with a variant of simian immunodeficiency virus, SIVsmmPBj14, leads to severe acute disease in macaques. This study was designed to investigate the functional significance of previously described mutations in the viral long terminal repeat (LTR) and to elucidate their contribution to the unique phenotype of SIVsmmPBj14. LTR-directed transcription was measured by using luciferase reporter constructs that were transiently transfected into cultured cells. In a wide range of cell types, the basal transcriptional activity of the LTR from SIVsmmPBj14 was found to be 2- to 4.5-fold higher than that of an LTR from a non-acutely pathogenic strain. These LTRs differ by five point mutations and a 22-bp duplication in SIVsmmPBj14, which includes a nuclear factor kappa B (NF kappa B) site. Transcriptional differences between these LTRs were further enhanced by two- to threefold upon treatment of cells with phorbol ester or tumor necrosis factor alpha or by cotransfection with plasmids expressing NF kappa B subunits. Mutagenesis studies, and the use of a reporter construct containing an enhancerless promoter, indicate that these transcriptional effects are due principally to the 22-bp sequence duplication and the NF kappa B site contained within it. Finally, infectious virus stocks that were isogenic except for the LTR were generated. The LTR from SIVsmmPBj14 was found to confer an increase in the kinetics of virus replication in cultured cells. Inclusion of this LTR in recombinant SIVs also resulted in a two- to threefold rise in the extent of cellular proliferation that was induced in quiescent simian peripheral blood mononuclear cells. These studies are consistent with the hypothesis that LTR mutations assist SIVsmmPBj14 in responding efficiently to cellular stimulation and allow it to replicate to high titers during the acute phase of viral infection.
Chinese hamster ovary cells contain transcriptionally active full-length type C proviruses
Authors: Lie Y S , Penuel E M , Low M A , Nguyen T P , Mangahas J O , Anderson K P , Petropoulos C J
Published: January 2020

We have isolated a genomic locus from Chinese hamster ovary (CHO) cells that contains a full-length provirus. Nucleotide sequence analysis indicates that it is a defective member of the rodent type C retrovirus family with an env region that is similar to those of mouse amphotropic retrovirus and subgroup B feline leukemia virus. We were able to demonstrate that this provirus is a member of a closely related family of full-length proviruses in CHO cells and Chinese hamster liver. Hybridization probes generated from this genomic clone were used to characterize type C retrovirus RNA expression in CHO cells. Full-length genomic RNA and subgenomic envelope mRNA were detected in CHO cell lines but not in the human-derived 293 cell line. Interestingly, we discovered that the site of retrovirus integration lies within a G repeat sequence belonging to the short interspersed element family of retroposons.
Rapid degradation of the heavy chain of class I major histocompatibility complex antigens in the endoplasmic reticulum of human cytomegalovirus-infected cells
Authors: Yamashita Y , Shimokata K , Saga S , Mizuno S , Tsurumi T , Nishiyama Y
Published: January 2020

Human cytomegalovirus (HCMV) infection results in a marked reduction in the surface expression of class I major histocompatibility complex antigens on the host cells, which is thought to be one of the means for HCMV to evade the host immune system. To clarify the precise mechanism(s) of this phenomenon, we investigated the fate of the heavy chain of class I major histocompatibility complex antigens in HCMV-infected human embryonic lung fibroblasts (HEL) by pulse-chase analysis and immunocytochemical techniques. In HCMV-infected HEL, the heavy chain was synthesized at an increasing rate. However, instead of being transported to the cell surface through the Golgi apparatus, it was retained in the endoplasmic reticulum (ER) without acquisition of a complex-type N-linked oligosaccharide. In addition, it was rapidly degraded, with a half time of 20 min, and the amount of the heavy chain remaining at the end of 3 h of chase was 10% (or less) of that initially synthesized. ER degradation of host glycoproteins in HCMV-infected HEL was selective for the heavy chain, since the posttranslational processing of the transferrin receptor in these cells was not affected. The heavy chain degradation in infected cells was resistant to inhibitors of a lysosomal proteolytic pathway and to metabolic poisons. These observations suggest the presence of an energy-dependent nonlysosomal proteolytic pathway in the ER.
Targeting of promoters for trans activation by a carboxy-terminal domain of the NS-1 protein of the parvovirus minute virus of mice
Authors: Legendre D , Rommelaere J
Published: January 2020

The NS-1 gene of the parvovirus minute virus of mice (MVM) (prototype strain, MVMp) was fused in phase with the sequence coding for the DNA-binding domain of the bacterial LexA repressor. The resulting chimeric protein, LexNS-1, was tested for its transcriptional activity by using various target promoters in which multiple LexA operator sequences had been introduced. Under these conditions, NS-1 was shown to stimulate gene expression driven by the modified long terminal repeat promoters (from the retroviruses mouse mammary tumor virus and Rous sarcoma virus) and P38 promoter (from MVMp), indicating that the NS-1 protein is a potent transcriptional activator. It is noteworthy that in the absence of LexA operator-mediated targeting, the genuine mouse mammary tumor virus and Rous sarcoma virus promoters were inhibited by NS-1. Together these data strongly suggest that NS-1 contains an activating region able to induce promoters with which this protein interacts but also to repress transcription from nonrecognized promoters by a squelching mechanism similar to that described for other activators. Deletion mutant analysis led to the identification of an NS-1 domain that exhibited an activating potential comparable to that of the whole polypeptide when fused to the DNA-binding region of LexA. This domain is localized in the carboxy-terminal part of NS-1 and corresponds to one of the two regions previously found to be responsible for toxicity. These results argue for the involvement of the regulatory functions of NS-1 in the cytopathic effect of this parvovirus product.
Major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein
Authors: Godet M , Grosclaude J , Delmas B , Laude H
Published: January 2020

The spike glycoprotein (S) of coronavirus, the major target for virus-neutralizing antibodies, is assumed to mediate the attachment of virions to the host cell. A 26-kilodalton fragment proteolytically cleaved from transmissible gastroenteritis virus (TGEV) S protein was previously shown to bear two adjacent antigenic sites, A and B, both defined by high-titer neutralizing antibodies. Recombinant baculoviruses expressing C-terminal truncations of the 26-kilodalton region were used to localize functionally important determinants in the S protein primary structure. Two overlapping 223- and 150-amino-acid-long products with serine 506 as a common N terminus expressed all of the site A and B epitopes and induced virus-binding antibodies. Coexpression of one of these truncated protein S derivatives with aminopeptidase N (APN), a cell surface molecule acting as a receptor for TGEV, led to the formation of a complex which could be immunoprecipitated by anti-S antibodies. These data provide evidence that major neutralization-mediating and receptor-binding determinants reside together within a domain of the S protein which behaves like an independent module. In spite of their ability to prevent S-APN interaction, the neutralizing antibodies appeared to recognize a preformed complex, thus indicating that antibody- and receptor-binding determinants should be essentially distinct. Together these findings bring new insight into the molecular mechanism of TGEV neutralization.
Translation of Sindbis virus mRNA: effects of sequences downstream of the initiating codon
Authors: Frolov I , Schlesinger S
Published: January 2020

One incentive for developing the alphavirus Sindbis virus as a vector for the expression of heterologous proteins is the very high level of viral structural proteins that accumulates in infected cells. Although replacement of the structural protein genes by a heterologous gene should lead to an equivalent accumulation of the heterologous protein, the Sindbis virus capsid protein is produced at a level 10- to 20-fold higher than that of any foreign protein. Chimeric mRNAs which contain the first 275 nucleotides of the Sindbis virus 26S mRNA fused to the lacZ gene are also translated at the higher level. The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin-like structure; deletions in this region destroy the activity. These sequences enhance translation in infected cells but have the opposite effect in uninfected cells. Furthermore, translation of this RNA in infected cells is suppressed by a second viral RNA lacking the hairpin-like structure, but translation of the latter RNA is not affected. We propose that the hairpin-like structure presents a barrier to the movement of the ribosomes during translation of mRNA. In infected cells, under conditions in which this mRNA is essentially the only RNA being translated, a slowdown in the transit of the ribosomes gives factors present at low concentrations a chance to bind to the translation complex and permits a high level of functional complexes to be formed. In uninfected cells and in infected cells translating two different viral subgenomic mRNAs, a pause in the movement of the ribosomes along the RNA is no longer an advantage, because the required factors are now usurped by other translation complexes.
Growth control of influenza A virus by M1 protein: analysis of transfectant viruses carrying the chimeric M gene
Authors: Yasuda J , Bucher D J , Ishihama A
Published: January 2020

Analysis of fast-growing reassortants (AWM viruses) of influenza A virus produced by mixed infection with a fast-growing WSN strain and a slowly growing Aichi strain indicated that the M gene plays a role in the regulation of virus growth rate at an early step of infection (J. Yasuda, T. Toyoda, M. Nakayama, and A. Ishihama, Arch. Virol. 133:283-294, 1993). To determine which of the two M gene products, M1 or M2, is responsible for the growth rate control, one recombinant WSN virus (CWA) clone possessing a chimeric M gene (WSN M1-Aichi M2) was generated by using an improved reverse genetics and transfection system. The recombinant CWA virus retained the phenotype of both large plaque formation and early onset of virus growth. This indicates that the WSN M1 protein is responsible for rapid virus growth.
Genetics of mouse hepatitis virus transcription: evidence that subgenomic negative strands are functional templates
Authors: Schaad M C , Baric R S
Published: January 2020

Mouse hepatitis virus (MHV) A59 temperature-sensitive (ts) mutants belonging to complementation group C were characterized and mapped by standard genetic recombination techniques. Temperature shift experiments early in infection suggested that the group C allele can be divided into two phenotypically distinct subgroups, designated C1 and C2. Since previous data indicated that the group C1 mutants probably contained an early defect which affects negative-strand synthesis, RNA synthesis was further examined by analyzing replicative-form (RF) RNA. Full-length as well as subgenomic-length RF RNAs were radiolabeled from 3 to 12 h postinfection (p.i.) and labeled late in infection after shift to the nonpermissive temperature (39.5 degrees C). The relative percent molar ratios of each mRNA and corresponding RF RNA were roughly equivalent throughout infection. Temperature shift experiments at 5.5 or 6.0 h p.i. resulted in an 83 to 92% reduction in the amount of total RF RNA at 39.5 degrees C. Radiolabeling time course experiments after temperature shift to 39.5 degrees C also demonstrated incorporation (6 to 9 h p.i.) into both subgenomic-length and full-length RF RNAs, suggesting that previously transcribed negative strands were functional templates throughout infection. To determine if the reduction in RF RNA was due to a decrease in positive- or negative-strand RNA synthesis, rates of mRNA synthesis were calculated from both full-length and subgenomic-length templates. The rate of mRNA synthesis after the shift was increased at 39.5 degrees C compared with that at 32 degrees C regardless of the template used; however, transcription rates calculated from subgenomic-length templates were similar to those of other viral and eukaryotic polymerases. These findings support the notion that the group C1 allele regulates negative-strand RNA synthesis and strongly suggest that the subgenomic negative-strand RNAs are probably the predominant functional templates for the synthesis of positive-strand RNAs late in infection.
Neurovirulent simian immunodeficiency virus replicates productively in endothelial cells of the central nervous system in vivo and in vitro
Authors: Mankowski J L , Spelman J P , Ressetar H G , Strandberg J D , Laterra J , Carter D L , Clements J E , Zink M C
Published: January 2020

The perivascular location of human immunodeficiency virus-infected cells suggests that the virus enters the central nervous system (CNS) by traversing the blood-brain barrier (BBB). In this study, the simian immunodeficiency virus (SIV) macaque model was used to determine whether SIV infects CNS endothelial cells. SIV RNA was detected in capillary endothelial cells in brain sections from animals parenterally inoculated with a neurovirulent strain of SIV by double immunohistochemistry and in situ hybridization and by reverse transcriptase-in situ PCR. These in vivo observations were extended by examining whether SIV replicated productively in cultured macaque brain endothelial cells (MBEC). A neurovirulent strain, SIVmac239/17E-Br, replicated productively in MBEC as determined by the presence of viral cytopathic effect (syncytia), viral DNA by PCR, viral RNA by in situ hybridization, and viral antigen by immunohistochemistry and by the production of high titers of cell-free virus. Virus replication was confirmed by electron microscopy. In contrast, a nonneurovirulent strain, SIVmac239, did not infect MBEC. Infection of the endothelial cells was not blocked by soluble CD4. Thus, endothelial cells may provide a CD4-independent pathway of virus entry to the CNS. In addition, damage to the BBB as a result of endothelial cell infection may provide a mechanism for amplification of viral load in the CNS and may contribute to the CNS dysfunction that characterizes AIDS dementia and SIV encephalitis. These data suggest that MBEC may serve a selective role in determining virus entry to the CNS.