bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of two temperature-sensitive mutants of coronavirus mouse hepatitis virus strain A59 with maturation defects in the spike protein
Authors: Luytjes W , Gerritsma H , Bos E , Spaan W
Published: January 2020

Two temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59, ts43 and ts379, have been described previously to be ts in infectivity but unaffected in RNA synthesis (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). We present a detailed analysis of the protein synthesis of the mutant viruses at the permissive (31 degrees C) and nonpermissive (39.5 degrees C) temperatures. It was found that synthesis of the nucleocapsid protein N and the membrane protein M of both viruses was insensitive to temperature. However, the surface protein S of both viruses was retained in the endoplasmic reticulum at the nonpermissive temperature. This was shown first by analysis of endoglycosidase H-treated and immunoprecipitated labeled S proteins. The mature Golgi form of S was not present at the nonpermissive temperature for the ts viruses, in contrast to wild-type (wt) virus. Second, gradient purification of immunoprecipitated S after pulse-chase labeling showed that only wt virus S was oligomerized. We conclude that the lack of oligomerization causes the retention of the ts S proteins in the endoplasmic reticulum. As a result, ts virus particles that were devoid of S were produced at the nonpermissive temperature. This result could be confirmed by biochemical analysis of purified virus particles and by electron microscopy.
Construction of adenovirus vectors through Cre-lox recombination
Authors: Hardy S , Kitamura M , Harris-Stansil T , Dai Y , Phipps M L
Published: January 2020

Two barriers prevent adenovirus-based vectors from having wide application. One is the difficulty of making new adenoviruses, and the second is the strong immunological reaction to viral proteins. Here we describe uses of Cre-lox recombination to overcome these problems. First, we demonstrate a simple method for constructing E1-substituted adenoviruses. Second, we demonstrate a method to construct adenovirus vectors carrying recombinant genes in place of all of the viral genes, so-called gutless adenovirus vectors. The pivotal feature in each method is the use of a negatively selected adenovirus named psi5. We engineered a cis-acting selection into psi5 by flanking its packaging site with loxP sites. When psi5 was grown in cells making a high level of Cre recombinase, the packaging site was deleted by recombination and the yield of psi5 was reduced to 5% of the wild-type level. To make a new E1-substituted virus, we used psi5 as a donor virus and recombined it with a shuttle vector via a loxP site. The resulting recombinant virus has a single loxP site next to the packaging site and therefore outgrows psi5 in the presence of Cre recombinase. To make a gutless virus, we used psi5 as a helper virus. The only viral sequences included in the gutless vector are those needed in cis for its replication and packaging. We found that a loxP site next to the packaging site of the gutless virus was necessary to neutralize homologous recombination between psi5 and the gutless viruses within their packaging domains.
Adding an Rb-binding site to an N-terminally truncated simian virus 40 T antigen restores growth to high cell density, and the T common region in trans provides anchorage-independent growth and rapid growth in low serum concentrations
Authors: Tevethia M J , Lacko H A , Kierstead T D , Thompson D L
Published: January 2020

The simian virus 40 large T antigen is sufficient to confer on cells multiple transformed cell growth characteristics, including growth to a high cell density, rapid growth in medium containing low serum concentrations, and anchorage-independent growth. We showed previously that distinct regions of the protein were involved in conferring these properties and that removal of the first 127 amino acids of T antigen abrogated all three activities. At least three large-T-antigen transformation-related activities have been localized to that region: binding of the tumor suppressor gene product Rb and two independent activities contained within the common region shared by large T and small t antigens. The experiments described here were directed toward determining whether these were the only activities from the N terminus that were needed. To do so we reintroduced an Rb-binding region into the N-terminally truncated T antigen (T128-708) and examined the growth properties of cells immortalized by it in the presence and absence of small t antigen, which can provide the T-common-region transformation-related activities in trans. We show that an Rb-binding region consisting of amino acids 101 to 118, when introduced into a heterologous site in T128-708, is capable of physically binding Rb and that binding is sufficient for cells expressing the protein to acquire the ability to grow to a high saturation density. However, in low-serum medium, the growth rate of the cells and maximal cell density are reduced relative to those of wild-type-T-antigen-expressing cells, and the cells cannot divide without anchorage. This result suggests that although Rb binding is sufficient in the context of T128-708 to confer growth to a high density, one or more other N-terminally located T-antigen activities are needed for cells to acquire the additional growth properties. Small t antigen in trans supplied those activities. These results indicate that the T-common-region activities and Rb binding are the only activities from the T-antigen N terminus needed to restore full transforming activity to the N-terminally truncated T antigen.
Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1
Authors: Ho C K , Van Etten J L , Shuman S
Published: January 2020

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap. A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step. These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.
A single 13-kilobase divergent locus in the Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genome contains nine open reading frames that are homologous to or related to cellular proteins
Authors: Nicholas J , Ruvolo V , Zong J , Ciufo D , Guo H G , Reitz M S , Hayward G S
Published: January 2020

Two small fragments of a novel human gammaherpesvirus genome known as Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) have been shown to be present in virtually all AIDS and non-AIDS KS lesions, as well as in body cavity-based lymphomas (BCBL) and in multicentric Castleman's disease. We have extended those studies by identifying and sequencing a third fragment of HHV-8 DNA encoding a viral thymidylate synthetase (TS) gene. Use of this viral TS fragment as a probe led to the identification and mapping of a cluster of overlapping phage lambda clones from a BCBL tumor DNA genomic library that spanned 48 kb on the left-hand side of the HHV-8 genome between the equivalents of open reading frame 6 (ORF6) and ORF31 of herpesvirus saimiri (HVS). DNA sequencing of a 17-kb segment encompassing a gammaherpesvirus divergent locus (DL-B) between ORF11 and ORF17 revealed the presence of nine viral ORFs with predicted gene products related to cellular proteins. These include the complete TS gene and a dihydrofolate reductase (DHFR) gene, four novel cytokine genes (encoding viral interleukin-6, viral MIP-1A, viral MIP-1B, and BCK) that have not previously been found to be encoded by a virus, and a bcl-2 homolog. This region in HHV-8 also contains the T1.1 abundant lytic cycle nuclear RNA gene and encompasses two genes (or exons) encoding proteins with C4HC3 zinc finger domains of the PHD/leukemia-associated protein subtype. The latter are related to the spliced immediate-early IE1 protein of the gamma-2 class herpesvirus bovine herpesvirus type 4 and a similar motif found in HVS ORF12. Although genes for TS and DHFR enzymes are also encoded by HVS (ORF70 and ORF2), both occur at different genomic loci than in HHV-8, and the HHV-8 DHFR protein is much farther diverged from human DHFR than is the HVS version, implying that they were probably acquired as host cell cDNAs by independent evolutionary events. Transcripts from the IE1-A, IE1-B, DHFR, and MIP-1B genes were all detected by Northern blot hybridization analysis in a BCBL cell line at 12 h after induction with butyrate but were not present before induction, indicating that these are all primarily lytic cycle genes. We conclude that the DL-B locus of gammaherpesviruses displays considerably more variability that previously appreciated and that expression of many of these genes is likely to have important implications for HHV-8 biology and therapy.
Double-stranded RNA is a trigger for apoptosis in vaccinia virus-infected cells
Authors: Kibler K V , Shors T , Perkins K B , Zeman C C , Banaszak M P , Biesterfeldt J , Langland J O , Jacobs B L
Published: January 2020

The vaccinia virus E3L gene codes for double-stranded RNA (dsRNA) binding proteins which can prevent activation of the dsRNA-dependent, interferon-induced protein kinase PKR. Activated PKR has been shown to induce apoptosis in HeLa cells. HeLa cells infected with vaccinia virus with the E3L gene deleted have also been shown to undergo apoptosis, whereas HeLa cells infected with wild-type vaccinia virus do not. In this report, using virus recombinants expressing mutant E3L products or alternative dsRNA binding proteins, we show that suppression of induction of apoptosis correlates with functional binding of proteins to dsRNA. Infection of HeLa cells with ts23, which leads to synthesis of increased dsRNA at restrictive temperature, induced apoptosis at restrictive but not permissive temperatures. Treatment of cells with cytosine arabinoside, which blocks the late buildup of dsRNA in vaccinia virus-infected cells, prevented induction of apoptosis by vaccinia virus with E3L deleted. Cells transfected with dsRNA in the absence of virus infection also underwent apoptosis. These results suggest that dsRNA is a trigger that can initiate a suicide response in virus-infected and perhaps uninfected cells.
Repression of host RNA polymerase II transcription by herpes simplex virus type 1
Authors: Spencer C A , Dahmus M E , Rice S A
Published: January 2020

Lytic infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in rapid repression of host gene expression and selective activation of the viral genome. This transformation in gene expression is thought to involve repression of host transcription and diversion of the host RNA polymerase (RNAP II) transcription machinery to the viral genome. However, the extent of virus-induced host transcription repression and the mechanisms responsible for these major shifts in transcription specificities have not been examined. To determine how HSV-1 accomplishes repression of host RNAP II transcription, we assayed transcription patterns on several cellular genes in cells infected with mutant and wild-type HSV-1. Our results suggest that HSV-1 represses RNAP II transcription on most cellular genes. However, each cellular gene we examined responds differently to the transcription repressive effects of virus infection, both quantitatively and with respect to the involvement of viral gene products. Virus-induced shutoff of host RNAP II transcription requires expression of multiple immediate-early genes. In contrast, expression of delayed-early and late genes and viral DNA replication appear to contribute little to repression of host cell RNAP II transcription. Modification of RNAP II to the intermediately phosphorylated (II(I)) form appears unlinked to virus-induced repression of host cell transcription. However, full repression of host transcription is correlated with depletion of the hyperphosphorylated (IIO) form of RNAP II.
Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence
Authors: Poumbourios P , Wilson K A , Center R J , El Ahmar W , Kemp B E
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.
Constitutive expression of p50 homodimer in freshly isolated human monocytes decreases with in vitro and in vivo differentiation: a possible mechanism influencing human immunodeficiency virus replication in monocytes and mature macrophages
Authors: Lewin S R , Lambert P , Deacon N J , Mills J , Crowe S M
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in vitro in differentiated macrophages than in freshly isolated monocytes. We investigated whether this may be partly explained by changes in expression of NF-kappaB with monocyte differentiation. We demonstrated that constitutive expression of NF-kappaB in primary human monocytes changed significantly with differentiation in vitro to monocyte-derived macrophages (MDMs) and differentiation in vivo to alveolar macrophages (AMs). Freshly isolated monocytes constitutively expressed high levels of transcriptionally inactive p50 homodimer which decreased with time in culture in favor of the transcriptionally active p50/p65 and p50/RelB heterodimers. As in MDMs, AMs constitutively expressed p50/p65 and p50/RelB although at lower levels. HIV infection of fresh monocytes failed to induce p50/p65 as seen in MDMs. The replacement of p50 homodimers with transcriptionally active heterodimers following time in culture may partially explain the progressive increase in susceptibility of monocytes to HIV infection during in vitro culture. The change in NF-kappaB components with monocyte differentiation in vivo may also explain the different transcriptional activities of these cell populations in HIV-infected individuals.
Segment-specific noncoding sequences of the influenza virus genome RNA are involved in the specific competition between defective interfering RNA and its progenitor RNA segment at the virion assembly step
Authors: Odagiri T , Tashiro M
Published: January 2020

The generation of influenza A virus defective interfering (DI) particles was studied by using an NS2 mutant which produces, in a single cycle of virus replication, a large amount of DI particles lacking the PA polymerase gene. The decrease in PA gene replication has been shown to occur primarily at the cRNA synthesis step, with preferential amplification of PA DI RNA species present in a marginal amount in the virus stock. In addition, at the assembly step the PA DI RNAs were preferentially incorporated into virions, resulting in selective reduction in the packaging of the PA gene into virions. Similarly, in cells dually infected with the NS2 mutant and wild-type viruses, packaging of the wild-type PA gene was also greatly suppressed. In contrast, incorporation of other RNA segments, i.e., the PB2 and NS genes, was not affected, suggesting that the PA DI RNAs competed only with the PA gene in a segment-specific manner. Experiments involving rescue of recombinant chloramphenicol acetyltransferase (CAT) RNA flanked by the noncoding regions of the PA (PA/CAT RNA) and PB2 (PB2/CAT RNA) genes into viral particles showed that only PA/CAT RNA was not rescued by infection with the NS2 mutant virus containing the PA DI RNAs. However, recombinant PA/CAT RNA in which either the 3' or 5' noncoding region was replaced with that of the PB2 gene was rescued by the NS2 mutant. These results suggest that the noncoding regions of the PA gene are responsible for the competition with PA DI RNA species at the virus assembly step and that coexistence of the both noncoding regions would be a prerequisite for this phenomenon. Decreased packaging of the progenitor RNA by the DI RNA, in addition to the suppression of cRNA synthesis, is likely involved in the production of DI particles.
Study of the dynamics of neutralization escape mutants in a chimpanzee naturally infected with the simian immunodeficiency virus SIVcpz-ant
Authors: Nyambi P N , Lewi P , Peeters M , Janssens W , Heyndrickx L , Fransen K , Andries K , Vanden Haesevelde M , Heeney J , Piot P , van der Groen G
Published: January 2020

Here we report on the use of spectral map analysis of time-paired sequential neutralization data of 11 serum samples of a chimpanzee naturally infected with a simian immunodeficiency virus (SIVcpz-ant) and 8 primary consecutive SIVcpz-ant isolates, taken at about 4-month intervals. The analysis reveals the existence of three SIVcpz-ant isolate and serum neutralization clusters. Each cluster groups virus isolates and/or sera based on similarities of their neutralization spectra. On average, neutralization escape mutants emerged after 15 months and mounted a neutralization response approximately 8 months later. The entire gp160 regions of eight consecutive isolates were sequenced and analyzed by a new statistical method called polygram, which allowed the deduction of amino acid sequence motifs of gp160 which were specific for SIVcpz-ant isolates belonging to the same isolate neutralization clusters. Changes in specific amino acid quadruplets in V1, V2, C3, V4, V5, and CD4 domains of gp120 and gp40 were seen to correlate with the neutralization clusters with most of the specific changes occurring in the V4 region. This method of analysis may facilitate an understanding of the study of the dynamic interplay between human immunodeficiency virus (HIV) and host neutralization responses as well as providing possible insights into mechanisms of persistence of HIV-1-related lentiviruses in their natural hosts.
Induction of phosphorylation of human immunodeficiency virus type 1 Nef and enhancement of CD4 downregulation by phorbol myristate acetate
Authors: Luo T , Downing J R , Garcia J V
Published: January 2020

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) encodes a 27 to 34 kDa myristoylated protein that induces downregulation of CD4 from the cell surface and enhances virus infectivity. As shown by experiments on SIV-infected adult macaques, Nef is important in pathogenesis and disease progression. In vitro, protein kinase C (PKC) phosphorylates Nef, but the role of phosphorylation in the function and expression of this protein has not yet been determined. Here we show that in HIV type 1-infected cells, phosphorylation of Nef increased 8- to 12-fold after treatment with phorbol myristate acetate and phytohemagglutinin (PMA/PHA). Basal and PMA/PHA-induced phosphorylation occurred on serine residues of Nef and was independent of other HIV proteins. The PMA/PHA-induced phosphorylation of Nef was inhibited by bisindolylmaleimide I, a potent and specific inhibitor of PKC, but was unaffected by H89, an inhibitor of protein kinase A. In contrast, treatment with bisindolylmaleimide I did not affect the basal level of Nef phosphorylation, suggesting two different phosphorylation pathways. A PMA-insensitive CD4 mutant in which three serine residues in the cytoplasmic domain have been replaced by alanines was used to determine whether PMA-induced phosphorylation affects Nef-induced CD4 downregulation. In Nef-expressing cells, treatment with PMA enhanced downregulation of the CD4 serine triple mutant from the cell surface, suggesting that phosphorylation is important for Nef function.
Epitope map of human immunodeficiency virus type 1 gp41 derived from 47 monoclonal antibodies produced by immunization with oligomeric envelope protein
Authors: Earl P L , Broder C C , Doms R W , Moss B
Published: January 2020

The biologically relevant form of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein is oligomeric, with the major points of contact between oligomeric partners located in the ectodomain of gp41. To identify and map conserved epitopes and regions in gp41 where structure is influenced by quaternary interactions, we used a panel of 38 conformation-dependent and 9 conformation-independent anti-gp41 monoclonal antibodies (MAbs) produced by immunization of mice with oligomeric Env protein. By cross-competition experiments using these MAbs and several others previously described, six distinct antigenic determinants were identified and mapped. Three of these determinants are conformational in nature and dependent in part on Env oligomeric structure. MAbs to two of these determinants were broadly cross-reactive with Env proteins derived from primary virus strains. The prevalence of antibodies in HIV-1-positive human sera to the antigenic determinants was determined by the ability of such sera to block binding of MAbs to Env protein. Strong blocking activity that correlated with cross-reactivity was found.
Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry
Authors: Chen Z , Zhou P , Ho D D , Landau N R , Marx P A
Published: January 2020

Entry of human immunodeficiency virus type 1 (HIV-1) requires CD4 and one of a family of related seven-transmembrane-domain coreceptors. Macrophage-tropic HIV-1 isolates are generally specific for CCR5, a receptor for the CC chemokines RANTES, MIP-1alpha, and MIP-1beta, while T-cell line-tropic viruses tend to use CXCR4 (also known as fusin, LESTR, or HUMSTR). Like HIV-1, simian immunodeficiency virus (SIV) requires CD4 on the target cell surface; however, whether it also requires a coreceptor is not known. We report here that several genetically divergent SIV isolates, including SIVmac, SIVsmSL92a, SIVsmLib-1, and SIVcpzGAB, can use human and rhesus CCR5 for entry. CXCR4 did not facilitate entry of any of the simian viruses tested, nor did any of the other known chemokine receptors. Moreover, SIVmac251 that had been extensively passaged in a human transformed T-cell line retained its use of CCR5. Rhesus and human CCR5 differed at only eight amino acid residues, four of which were in regions of the receptor that could be exposed, two in the amino-terminal extracellular region and two in the second extracellular loop. The human coreceptor was as active as the simian for SIV entry. In addition, HIV-1 was able to use the rhesus homologs of the human coreceptors, CCR5 and CXCR4. The SIV strains tested were specific for CCR5 regardless of whether they were able to replicate in transformed T-cell lines or macrophages and whether they were phenotypically syncytium inducing or noninducing in MT-2 cells. However, SIV replication was not restricted to cells expressing CCR5. SIV strains replicated efficiently in the human transformed lymphoid cell line CEMx174, which does not express detectable amounts of transcripts of CCR5. SIV also replicated in human peripheral blood mononuclear cells that were genetically deficient in CCR5. These findings indicated that, in addition to CCR5, SIV can use one or more unknown coreceptors that are expressed on human PBMCs and CEMx174 cells.
Genetically engineered live attenuated influenza A virus vaccine candidates
Authors: Parkin N T , Chiu P , Coelingh K
Published: January 2020

We have generated new influenza A virus live attenuated vaccine candidates by site-directed mutagenesis and reverse genetics. By mutating specific amino acids in the PB2 polymerase subunit, two temperature-sensitive (ts) attenuated viruses were obtained. Both candidates have 38 degrees C shutoff temperatures in MDCK cells, are attenuated in the respiratory tracts of mice and ferrets, and have very low reactogenicity in ferrets. Infection of mice or ferrets with either mutant conferred significant protection from challenge with the homologous wild-type virus. Three tests for genetic stability were used to assess the propensity for reversion to virulence: 14 days of replication in nude mice, growth at 37 degrees C in tissue culture, and serial passage in ferrets. One candidate, which contains mutations intended to reduce the ability of PB2 to bind to cap structures, was stable in all three assays, whereas the second candidate, which contains mutations found only in other ts strains of influenza virus, lost its ts phenotype in the last two assays. This approach has therefore enabled the creation of live attenuated influenza A virus vaccine candidates suitable for human testing.
Characterization of a second protein (CM2) encoded by RNA segment 6 of influenza C virus
Authors: Hongo S , Sugawara K , Muraki Y , Kitame F , Nakamura K
Published: January 2020

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.
Generation and neutralization of pseudovirions of human papillomavirus type 33
Authors: Unckell F , Streeck R E , Sapp M
Published: January 2020

Since human papillomaviruses (HPV) cannot be propagated in cell culture, the generation of infectious virions in vitro is a highly desirable goal. Here we report that pseudovirions can be generated by the assembly of virus-like particles (VLPs) in COS-7 cells containing multiple copies of a marker plasmid. Using recombinant vaccinia viruses, we have obtained spherical VLPs of HPV type 33 (HPV-33) which fractionate into heavy and light VLPs in cesium chloride density gradients. VLPs in the heavy fraction (1.31 g/cm3) carry the plasmid in DNase-resistant form and are capable of transferring the genetic marker located on the plasmid to COS-7 cells in a DNase-resistant way (pseudoinfection). The minor capsid protein L2 is not required for encapsidation but is essential for efficient pseudoinfection. Antiserum to HPV-33 VLPs inhibits VLP-mediated DNA transfer with high efficiency. Antisera to VLPs of HPV-18 and HPV-16 are not neutralizing, although the HPV-16 antiserum exhibited some cross-reactivity with HPV-33 VLPs in an enzyme-linked immunosorbent assay. In a cell binding assay, the titer of the HPV-33 VLP antiserum was 1:200 compared to the neutralization titer of 1:10(5). This indicates that neutralization is essentially due to the inhibition of cellular processes after VLP binding to cells. The encapsidation of marker plasmids into VLPs provides a sensitive and fast assay for the evaluation of neutralizing potentials of antisera against papillomavirus infections.
Major linear antibody epitopes and capsid proteins differentially induce protective immunity against Theiler's virus-induced demyelinating disease
Authors: Yahikozawa H , Inoue A , Koh C S , Choe Y K , Kim B S
Published: January 2020

Theiler's murine encephalomyelitis virus-induced immunologically mediated demyelinating disease (TMEV-IDD) in susceptible mice provides a relevant infectious model for multiple sclerosis. Previously, we have identified six major linear antibody epitopes on the viral capsid proteins. In this study, we utilized fusion proteins containing individual capsid proteins and synthetic peptides containing the linear antibody epitopes to determine the potential role of antibody response in the course of virus-induced demyelination. Preimmunization of susceptible mice with VPI and VP2 fusion proteins, but not VP3, resulted in the protection from subsequent development of TMEV-IDD. Mice free of clinical symptoms following preimmunizations with fusion proteins displayed high levels of antibodies to the capsid proteins corresponding to the immunogens. In contrast, the level of antibodies to a particular linear epitope, A1C (VP1(262-276)), capable of efficiently neutralizing virus in vitro increased with the progression of disease. Further immunization with synthetic peptides containing individual antibody epitopes indicated that antibodies to the epitopes are differentially effective in protecting from virus-induced demyelination. Taken together, these results suggest that antibodies to only certain linear epitopes are protective and such protection may be restricted during the early stages of viral infection.
Suppression of human immunodeficiency virus type 1 replication by CD8+ cells: evidence for HLA class I-restricted triggering of cytolytic and noncytolytic mechanisms
Authors: Yang O O , Kalams S A , Trocha A , Cao H , Luster A , Johnson R P , Walker B D
Published: January 2020

Although CD8+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected individuals have been demonstrated to suppress viral replication, the mechanisms of inhibition have not been defined precisely. A large body of evidence indicates that these cells act via soluble inhibitory factors, but the potential role of HLA class I-restricted cytolysis has remained controversial. Here we demonstrate that HIV-1-specific cytotoxic T lymphocytes (CTL) mediate antiviral suppression by both cytolytic and noncytolytic mechanisms. The predominant mechanism requires direct contact of CTL with the infected cells, is HLA class I restricted, and can achieve complete elimination of detectable virus in infected cell cultures. Inhibition occurs even at high multiplicities of infection or at ratios of CTL to CD4 cells as low as 1:1,000. The other mechanism is mediated by soluble inhibitory factors which are triggered in an antigen-specific and HLA-restricted fashion but then act without HLA restriction. These include MIP-1alpha, MIP-1beta, and RANTES, as well as a distinct factor(s) capable of inhibiting HIV-1 strains insensitive to these chemokines. These data indicate that HIV-1-specific CTL are potent mediators of HIV-1 suppression at cell ratios existing in vivo and demonstrate an antigen-specific trigger for CD8+ cell-derived soluble inhibitory factors. These results suggest that CTL play an important role in the observed antiviral activity of CD8+ cells from infected individuals.
Nucleolar and nuclear localization properties of a herpesvirus bZIP oncoprotein, MEQ
Authors: Liu J L , Lee L F , Ye Y , Qian Z , Kung H J
Published: January 2020

Marek's disease virus (MDV) is one of the most oncogenic herpesviruses and induces T lymphomas in chickens within weeks after infection. Only a limited number of viral transcripts are detected in MDV tumor samples and cell lines. One of the major transcripts encodes MEQ, a 339-amino-acid bZIP protein which is homologous to the Jun/Fos family of transcription factors. The C-terminal half of MEQ contains proline-rich repeats and, when fused to the DNA-binding domain of a yeast transcription factor, Gal4 (residues 1 to 147), exhibits transactivation function. MEQ can dimerize with itself and with c-Jun. The MEQ-c-Jun heterodimers bind to an AP-1-like enhancer within the MEQ promoter region with greater affinity than do homodimers of either protein, and they transactivate MEQ expression. Here we show that MEQ is expressed in the nucleus but, interestingly, with a predominant fraction in the nucleoli and coiled bodies. This makes MEQ the first bZIP protein to be identified in the nucleoli. MEQ contains two stretches of basic residues, designated basic region 1 (BR1) and basic region 2 (BR2). Using a series of deletion mutants, we have mapped the primary nuclear localization signal (NLS) and the sole nucleolar localization signal (NoLS) to the BR2 region. BR1 was shown to provide an auxiliary signal in nuclear translocation. To demonstrate that BR2 is an authentic NoLS, BR2 was fused to cytoplasmic v-Raf (delta gag) kinase. The BR2-Raf fusion protein was observed to migrate into the nucleoplasm and the nucleolus. The BR2 region can be further divided into two long arginine-lysine stretches, BR2N and BR2C, which are separated by the five amino acids Asn-Arg-Asp-Ala-Ala (NRDAA). We provide evidence that the requirement for nuclear translocation is less stringent than that for nucleolar translocation, as either BR2N or BR2C alone is sufficient to translocate the cytoplasmic v-Raf (delta gag) into the nucleus, but only in combination can they translocate v-Raf (delta gag) into the nucleolus. Our studies demonstrate that MEQ is both a nuclear and nucleolar protein, adding MEQ to the growing list of transactivators which localize to the nucleolus.
Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death
Authors: Marianneau P , Cardona A , Edelman L , Deubel V , Desprès P
Published: January 2020

The severe outcome of the dengue (DEN) virus infection known as DEN hemorrhagic fever-DEN shock syndrome (DHF-DSS) is, in some cases, accompanied by liver injury. Councilman bodies observed in liver biopsies of DHF-DSS cases may correspond to hepatocytes in apoptosis. We show here that infection of the hepatoma cell line HepG2 with DEN type 1 virus induced cell death typical of apoptosis late in the virus cycle. The transcription factor NF-kappaB was activated concomitantly with viral protein synthesis and thus before the appearance of apoptotic cells. Inhibition of apoptosis was observed when DEN virus-infected cells were treated with NF-kappaB decoys, indicating the involvement of this transcription factor in induction of cell death. Thus, infected hepatocytes appear to be subject to apoptosis in vitro, and this may be a key element in the pathophysiology of hepatic failure associated with DHF-DSS.
Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome
Authors: Balagúe C , Kalla M , Zhang W W
Published: January 2020

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.
Epstein-Barr virus BHRF1 protein protects intestine 407 epithelial cells from apoptosis induced by tumor necrosis factor alpha and anti-Fas antibody
Authors: Kawanishi M
Published: January 2020

Tumor necrosis factor (TNF) and cytotoxic T lymphocytes, which utilize Fas to induce apoptosis in target cells, are known to play a critical role in the host defense against viral infection. In this study, the Epstein-Barr virus BHRF1 protein was stably expressed in intestine 407 cells which were susceptible to cell death mediated through both the TNF receptor and Fas. WST-1 conversion assays and acridine orange staining showed that vector-transfected control cells were killed by TNF-alpha or anti-Fas antibody in a dose-dependent manner, whereas BHRF1-expressing cells were resistant to apoptosis induced by these mediators. DNA fragmentation, a characteristic of apoptosis induced by TNF-alpha and the anti-Fas antibody, was suppressed in BHRF1-expressing cells. These results indicate that the BHRF1 protein protects cells from apoptosis mediated by the TNF receptor and Fas. The role of BHRF1 as an inhibitor of cytokine-induced apoptosis during the Epstein-Barr virus lytic cycle in vivo is discussed.
Initial appearance of the 184Ile variant in lamivudine-treated patients is caused by the mutational bias of human immunodeficiency virus type 1 reverse transcriptase
Authors: Keulen W , Back N K , van Wijk A , Boucher C A , Berkhout B
Published: January 2020

Treatment of human immunodeficiency virus type 1-infected patients with lamivudine (3TC) results in the appearance of drug-resistant virus variants with a mutation at the 184Met codon (ATG) of the reverse transcriptase (RT) gene. The 184Ile (ATA) variant appears first, but subsequently the 184Val (GTG) variant outcompetes the 184Ile variant. We demonstrated previously that the 184Val enzyme and the corresponding virus are more fit than 184Ile, thereby explaining eventual outgrowth of 184Val. In this study, we set out to determine why 184Ile is usually observed first after initiation of 3TC therapy. With a limiting dilution approach during in vitro selection with 3TC, we measured a significantly higher frequency of the G-->A substitution toward the ATA codon (184Ile; 56%) than the A-->G substitution toward GTG (184Val; 12.5%). This result indicates that the initial appearance of the 184Ile variant in patients is a consequence of the mutation bias of the RT enzyme. Interestingly, a novel 3TC-resistant variant which was generated by T-->C substitution (184Thr; 28%) was also observed. The RT enzyme of the 184Thr variant was less than 10% active compared with the wild-type enzyme, and the replication capacity of this variant was severely reduced. Selection of the 184Thr variant illustrates that the limiting dilution approach allows the selection of drug-resistant variants with suboptimal fitness.
Bipartite structure and functional independence of adenovirus type 5 packaging elements
Authors: Schmid S I , Hearing P
Published: January 2020

Selectivity and polarity of adenovirus type 5 DNA packaging are believed to be directed by an interaction of putative packaging factors with the cis-acting adenovirus packaging domain located within the genomic left end (nucleotides 194 to 380). In previous studies, this packaging domain was mutationally dissected into at least seven functional elements called A repeats. These elements, albeit redundant in function, exhibit differences in the ability to support viral packaging, with elements I, II, V, and VI as the most critical repeats. Viral packaging was shown to be sensitive to spatial changes between individual A repeats. To study the importance of spatial constraints in more detail, we performed site-directed mutagenesis of the 21-bp linker regions separating A repeats I and II, as well as A repeats V and VI. The results of our mutational analysis reveal previously unrecognized sequences that are critical for DNA encapsidation in vivo. On the basis of these results, we present a more complex consensus motif for the adenovirus packaging elements which is bipartite in structure. DNA encapsidation is compromised by changes in spacing between the two conserved parts of the consensus motif, rather than between different A repeats. Genetic evidence implicating packaging elements as independent units in viral DNA packaging is derived from the selection of revertants from a packaging-deficient adenovirus: multimerization of packaging repeats is sufficient for the evolution of packaging-competent viruses. Finally, we identify minimally sized segments of the adenovirus packaging domain that can confer viability and packaging activity to viruses carrying gross truncations within their left-end sequences. Coinfection experiments using the revertant as well as the minimal-packaging-domain mutant viruses strengthen existing arguments for the involvement of limiting, trans-acting components in viral DNA packaging.
Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en
Authors: Gubareva L V , Robinson M J , Bethell R C , Webster R G
Published: January 2020

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.
The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable
Authors: Sinclair E , Barbosa P , Feinberg M B
Published: January 2020

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.