bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels
Authors: Kyöstiö S R , Owens R A , Weitzman M D , Antoni B A , Chejanovsky N , Carter B J
Published: January 2020

The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence of the p5 promoter.
Isoprenylation masks a conformational epitope and enhances trans-dominant inhibitory function of the large hepatitis delta antigen
Authors: Hwang S B , Lai M M
Published: January 2020

Hepatitis delta antigen (HDAg) consists of two species, large (LHDAg) and small (SHDAg), which are identical in sequence except that the large form contains 19 extra amino acids at the C terminus. The large form is prenylated on the Cxxx motif. The small form can trans activate HDV RNA replication, while the large form inhibits it. To determine the molecular basis for their differential functions, we examined the effects of prenylation on the conformation and function of HDAg. We show that the presence of prenylates masks a conformational epitope which is present in SHDAg but hidden in wild-type LHDAg; this epitope becomes exposed in all of the nonprenylated mutant LHDAgs. Prenylation also plays a major role in conferring the trans-dominant negative inhibitory activity of LHDAg, since the loss of prenylation in LHDAg reduced its inhibitory activity. The primary amino acids of the C-terminal sequence also contributed to the maintenance of the HDAg protein conformation; a prenylated LHDAg mutant with a five-amino-acid deletion had an exposed C-terminal epitope. By examining LHDAg mutants which have deletions of various extents of C-terminal sequence, with or without the prenylation motif, we have further shown that all of the prenylated mutants have much higher levels of trans-dominant suppressor activities than do the corresponding nonprenylated mutants. Surprisingly, a few nonprenylated LHDAg mutants were able to trans activate HDV RNA replication, while all of the prenylated ones lost this function. These results suggest that isoprenylates cause the masking of a conformational epitope of HDAg and that conformational differences between the large and small HDAgs account for the differences in their trans-activating and trans-dominant inhibitory biological activities.
Characterization of human antibody responses to four corners hantavirus infections among patients with hantavirus pulmonary syndrome
Authors: Jenison S , Yamada T , Morris C , Anderson B , Torrez-Martinez N , Keller N , Hjelle B
Published: January 2020

Hantavirus pulmonary syndrome (HPS) is a human disease caused by a newly identified hantavirus, which we will refer to as Four Corners virus (FCV). FCV is related most closely to Puumala virus (PUU) and to Prospect Hill virus (PHV). Twenty-five acute HPS serum samples were tested for immunoglobulin G (IgG) and IgM antibody reactivities to FCV-encoded recombinant proteins in Western blot (immunoblot) assays. All HPS serum samples contained both IgG and IgM antibodies to the FCV nucleocapsid (N) protein. FCV N antibodies cross-reacted with PUU N and PHV N proteins. A dominant FCV N epitope was mapped to the segment between amino acids 17 and 59 (QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG). All HPS serum samples contained IgG antibodies to the FCV glycoprotein-1 (G1) protein, and 21 of 25 serum samples contained FCV G1 IgM antibodies. The FCV G1 antibodies did not cross-react with PUU G1 and PHV G1 proteins. The FCV G1 type-specific antibody reactivity mapped to a segment between amino acids 59 and 89 (LKIESSCNFDLHVPATTTQKYNQVDWTKKSS). One hundred twenty-eight control serum samples were tested for IgG reactivities to the FCV N and G1 proteins. Nine (7.0%) contained FCV N reactivities, 3 (2.3%) contained FCV G1 reactivities, and one (0.8%) contained both FCV N and FCV G1 reactivities. The epitopes recognized by antibodies present in control serum samples were different from the epitopes recognized by HPS antibodies, suggesting that the control antibody reactivities were unrelated to FCV infections. These reagents constitute a type-specific assay for FCV antibodies.
Native oligomeric human immunodeficiency virus type 1 envelope glycoprotein elicits diverse monoclonal antibody reactivities
Authors: Earl P L , Broder C C , Long D , Lee S A , Peterson J , Chakrabarti S , Doms R W , Moss B
Published: January 2020

We synthesized and purified a recombinant human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein, lacking the gp120/gp41 cleavage site as well as the transmembrane domain, that is secreted principally as a stable oligomer. Mice were immunized with separated monomeric and oligomeric HIV-1 Env glycoproteins to analyze the repertoire of antibody responses to the tertiary and quaternary structure of the protein. Hybridomas were generated and assayed for reactivity by immunoprecipitation of nondenatured Env protein. A total of 138 monoclonal antibodies (MAbs) were generated and cloned, 123 of which were derived from seven animals immunized with oligomeric Env. Within this group, a significant response was obtained against the gp41 ectodomain; 49 MAbs recognized epitopes in gp41, 82% of which were conformational. The influence of conformation on gp120 antigenicity was less pronounced, with 40% of the anti-gp120 MAbs binding to conformational epitopes, many of which blocked CD4 binding. Surprisingly, less than 7% of the MAbs derived from mice immunized with oligomeric Env recognized the V3 loop. In addition, MAbs to linear epitopes in the C-terminal domain of gp120 were not obtained, suggesting that this region of the protein may be partially masked in the oligomeric molecule. A total of 15 MAbs were obtained from two mice immunized with monomeric Env. Nearly half of these recognized the V3 loop, suggesting that this region may be a less predominant epitope in the context of oligomeric Env than in monomeric protein. Thus, immunization with oligomeric Env generates a large proportion of antibodies to conformational epitopes in both gp120 and gp41, many of which may be absent from monomeric Env.
Induction of a major histocompatibility complex class I-restricted cytotoxic T-lymphocyte response to a highly conserved region of human immunodeficiency virus type 1 (HIV-1) gp120 in seronegative humans immunized with a candidate HIV-1 vaccine
Authors: Johnson R P , Hammond S A , Trocha A , Siliciano R F , Walker B D
Published: January 2020

Efforts to induce broadly reactive immunity against human immunodeficiency virus type 1 (HIV-1) have been impaired by the extent of sequence variation exhibited by this lentivirus. Cytotoxic T lymphocytes (CTL) specific for other viruses such as influenza virus have been shown to mediate immunity against divergent viral strains, a property that is related to the ability of CTL to recognize processed antigen derived from conserved viral proteins. A recent candidate HIV-1 vaccine regimen has been described in which subjects receive a primary immunization with a recombinant vaccinia virus expressing gp160 and then a booster immunization with recombinant gp160. Volunteers immunized with this regimen have exhibited augmented humoral responses and have also developed CD4+ and CD8+ CTL specific for gp160. In this report, we have identified the epitopes recognized by CD4+ and CD8+ CTL obtained from two vaccines. An immunodominant CD8+ CTL response was HLA-A3.1 restricted and recognized a 10-amino-acid epitope (gp120/38-47) in a highly conserved region of gp120. CTL specific for the epitope gp120/38-47 were able to lyse targets sensitized with peptides corresponding to all known natural sequence variants in this region. In addition, other HLA class I-restricted CTL epitopes were identified in relatively conserved regions of gp120 and gp41, and CD4+ CTL were shown to recognize two different regions of gp120. Thus, in these two volunteers, immunization with a single strain of HIV-1 induced CD4+ and CD8+ CTL that are specific for multiple conserved regions of HIV-1 and would be expected to recognize a broad range of viral isolates.
Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro
Authors: Heineman T C , Cohen J I
Published: January 2020

Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.
The human T-cell leukemia virus type I Tax protein induces apoptosis which is blocked by the Bcl-2 protein
Authors: Yamada T , Yamaoka S , Goto T , Nakai M , Tsujimoto Y , Hatanaka M
Published: January 2020

The Tax protein of human T-cell leukemia virus type I activates transcription of cellular and viral genes and can immortalize primary T lymphocytes. We have previously reported that the Tax protein transforms Rat-1 cells. Here we show that Tax-transformed Rat-1 cells detach from plates to undergo apoptotic cell death by serum deprivation. These cells exhibit DNA fragmentation into oligonucleosomal fragments and chromatin condensation. Constitutive expression of a proto-oncogene, bcl-2, effectively blocks Tax-mediated apoptosis caused by serum deprivation without affecting the levels of Tax expression and the transformed phenotype of the cells.
Microglial infection by a neurovirulent murine retrovirus results in defective processing of envelope protein and intracellular budding of virus particles
Authors: Lynch W P , Brown W J , Spangrude G J , Portis J L
Published: January 2020

The observation of murine retrovirus infection of microglial cells in brain regions expressing spongiform neurodegenerative changes suggests that these cells may play an important role in pathogenesis. To evaluate this potential in vitro, murine microglial cells were infected in mixed glial cultures with the highly neurovirulent murine retrovirus, FrCasE. The microglia were then isolated from the mixed cultures on the basis of their differential adherence and shown to be approximately 98% pure. The infected microglia expressed viral envelope protein at the plasma membrane, while viral budding was primarily intracellular. Evaluation of the viral envelope protein by immunoblotting indicated that the immunoreactive species produced was exclusively a 90-kDa precursor protein. Very little of the envelope protein was associated with particles released from these cells, and viral titers in the culture supernatant were low. Interestingly, these cells were still capable of infecting permissive target cells when seeded as infectious centers. This partially defective infection of microglial cells suggests a potential cellular means by which a neurovirulent retrovirus could disrupt normal microglia and in turn central nervous system motor system functioning.
Distribution of mouse mammary tumor virus in Asian wild mice
Authors: Imai S , Okumoto M , Iwai M , Haga S , Mori N , Miyashita N , Moriwaki K , Hilgers J , Sarkar N H
Published: January 2020

Several groups of wild mice (Mus musculus) were captured from eight different locations in Asia and bred for several generations in a facility free of any laboratory strains of mice carrying mouse mammary tumor virus (MMTV). The distribution of endogenous MMTV proviral sequences in the liver tissues of these mice was investigated by using Southern blot hybridizations. Four categories of mice were identified. Mice originating from Bogor, Indonesia (Cas-Bgr); He-mei, Taiwan (Cas-Hmi/1); and Malaysia (Cas-Mal) were found to carry an endogenous MMTV provirus consisting of the env, gag-pol, and long terminal repeat sequences. Mice captured from Kojuri, Republic of Korea (Sub-Kjr); Nagoya, Japan (Mol-nag); and three Chinese provinces, Shanghai (Sub-Shh), Beijing (Sub-Bjn), and Jiayuguang (Sub-Jyg/1), appeared to carry defective proviruses. Some mice originating from He-mei (Cas-Hmi/2) and Jiayuguang (Sub-Jyg/2) were found to be completely free of endogenous MMTV. Interestingly, however, the Sub-Jyg/2 mice, after several generations of inbreeding, were found, unlike all of the other subspecies that we examined in the present study, to develop mammary tumors at a high incidence (80 to 90%) with a short period of latency. Electron microscopic examination of the mammary glands and mammary tumors of these mice revealed the presence of numerous intracytoplasmic A, immature, budding, and mature B particles. Furthermore, the mammary tumors were found to contain MMTV proviral sequences. It seems, therefore, that Sub-Jyg/2 mice carry an exogenous MMTV which contributes to their developing mammary tumors.
Analysis of AP-1 function in cellular transformation pathways
Authors: Suzuki T , Murakami M , Onai N , Fukuda E , Hashimoto Y , Sonobe M H , Kameda T , Ichinose M , Miki K , Iba H
Published: January 2020

To understand the role of endogenous AP-1 activity in cellular transformation induced by oncogenes, we have made use of a fos mutant (supfos-1) and a jun mutant (supjun-1), either of which can function as a transdominant inhibitor of AP-1-mediated transcriptional regulation. Chicken embryo fibroblasts (CEF) infected with a series of transforming retroviruses were doubly infected with retrovirus carrying supfos-1 or supjun-1, and suppression of cellular transformation was monitored in terms of reversion to normal cellular morphology or acquisition of anchorage-dependent growth. Cellular transformation induced by several exogenously expressed transforming genes of the fos or jun family was efficiently suppressed, as expected. CEF transformed by v-src, v-yes, v-fps, c-Ha-ras, and N-terminally truncated c-raf were also induced to revert to the normal phenotype by these transdominant mutants, suggesting that functional transcription factor AP-1 activity is essential for the cellular transformation induced by these oncogenes. The suppression is not attributable to nonspecific inhibition of cellular proliferation, because CEF transformed by v-ros or v-myc were not induced to revert to the normal phenotype. We next analyzed changes in all known components of chicken AP-1 induced by v-src, c-Ha-ras, or activated c-raf transformation. The levels of both Fra-2 and c-Jun expression were elevated two- to fourfold, and hyperphosphorylation of Fra-2 was also observed. We further showed that Fra-2-c-Jun heterodimer is mainly responsible for the elevated AP-1 DNA-binding activity in these transformed cells, and we propose that this heterodimer play a crucial role in the transformation induced by these oncogenes.
In vitro activities of purified visna virus integrase
Authors: Katzman M , Sudol M
Published: January 2020

Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.
Transcriptional silencer of the human papillomavirus type 8 late promoter interacts alternatively with the viral trans activator E2 or with a cellular factor
Authors: May M , Grassmann K , Pfister H , Fuchs P G
Published: January 2020

The noncoding region of the highly oncogenic, epidermodysplasia verruciformis-associated human papillomavirus type 8 contains a negative regulatory element (NRE). Quantitative RNase protection analysis confirmed that the NRE sequence acts as a silencer of transcription. A 38-bp sequence upstream of late promoter P7535 down-regulated expression from the homologous P7535 promoter, as well as the heterologous tk gene promoter, independently of its orientation relative to the test promoters. It also reduced gene expression when cloned downstream of the transcription units. Transient expression assays with keratinocytes and fibroblasts of epidermodysplasia verruciformis patients and controls demonstrated that the NRE activity is not cell specific. Gel retardation tests suggested that NRE specifically interacts with only one nuclear factor. Mutational analysis identified three NRE mutants which no longer formed a detectable DNA-protein complex but still repressed transcription, indicating that protein-DNA interaction is not relevant for the silencer function. The NRE contains a binding site of viral trans activator protein E2. It was shown that expression of E2 overrides the inhibitory effect of the NRE sequences. Binding of E2 and that of the cellular factor were mutually exclusive. The bifunctional nature of NRE acting as a silencer and a target site for viral trans activator E2 offers an interesting opportunity to regulate the switch from early to late transcription in the human papillomavirus life cycle.
Subgenomic RNA synthesis directed by a synthetic defective interfering RNA of mouse hepatitis virus: a study of coronavirus transcription initiation
Authors: van der Most R G , de Groot R J , Spaan W J
Published: January 2020

We have used a full-length cDNA clone of a mouse hepatitis virus strain A59 defective interfering (DI) RNA, pMIDI-C, and cassette mutagenesis to study the mechanism of coronavirus subgenomic mRNA synthesis. Promoter sequences closely resembling those of subgenomic mRNAs 3 and 7 were inserted into MIDI-C. Both subgenomic RNA promoters gave rise to the synthesis of a subgenomic DI RNA in virus-infected and DI RNA-transfected cells. From a mutagenic analysis of the promoters we concluded the following. (i) The extent of base pairing between the leader RNA and the intergenic promoter sequence does not control subgenomic RNA abundance. (ii) Promoter recognition does not rely on base pairing only. Presumably, transcription initiation requires recognition of the promoter sequence by the transcriptase. (iii) Fusion of leader and body sequences takes place at multiple--possibly random--sites within the intergenic promoter sequence. A model is presented in which, prior to elongation, the leader RNA is trimmed by a processive 3'-->5' nuclease.
The protease of herpes simplex virus type 1 is essential for functional capsid formation and viral growth
Authors: Gao M , Matusick-Kumar L , Hurlburt W , DiTusa S F , Newcomb W W , Brown J C , McCann P J , Deckman I , Colonno R J
Published: January 2020

The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.
Dissociation of retinoblastoma gene protein hyperphosphorylation and commitment to enter S phase
Authors: Ogryzko V V , Hirai T H , Shih C E , Howard B H
Published: January 2020

Mitogenic activities of simian virus 40 large T and small t antigens were studied in serum-deprived human diploid fibroblasts. Wild-type large T and small t cooperated in stimulating DNA synthesis and in inducing hyperphosphorylation of the Rb gene product (pRb). In contrast, a T antigen mutant defective for pRb binding (Rb- T) possessed no detectable mitogenic activity alone and failed to complement small t in stimulating DNA synthesis. Surprisingly, Rb- T and small t cooperated as strongly as wild-type T and small t with respect to pRb hyperphosphorylation. As a consequence, in two closely related conditions (i.e., stimulation by small t plus wild-type T versus small t plus Rb- T), the fraction of pRb in hyperphosphorylated forms dissociated from the fraction of cells in the S phase. These results indicate that pRb hyperphosphorylation is not always tightly coupled with a commitment to initiate DNA replication.
NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies
Authors: Amberg S M , Nestorowicz A , McCourt D W , Rice C M
Published: January 2020

Several of the cleavages required to generate the mature nonstructural proteins from the flaviviral polyprotein are known to be mediated by a complex consisting of NS2B and a serine proteinase domain located in the N-terminal one-third of NS3. These cleavages typically occur after two basic residues followed by a short side chain residue. Cleavage at a similar dibasic site in the structural region is believed to produce the C terminus of the virion capsid protein. To study this cleavage, we developed a cell-free trans cleavage assay for yellow fever virus (YF)-specific proteolytic activity by using a substrate spanning the C protein dibasic site. Cleavage at the predicted site was observed when the substrate was incubated with detergent-solubilized lysates from YF-infected BHK cells. NS2B and the NS3 proteinase domain were the only YF-specific proteins required for this cleavage. Cell fractionation studies demonstrated that the YF-specific proteolytic activity was membrane associated and that activity could be detected only after detergent solubilization. Previous cell-free studies led to a hypothesis that processing in the C-prM region involves (i) translation of C followed by translocation and core glycosylation of prM by using an internal signal sequence, (ii) signalase cleavage to produce a membrane-anchored form of the C protein (anchC) and the N terminus of prM, and (iii) NS2B-3-mediated cleavage at the anchC dibasic site to produce the C terminus of the virion C protein. However, the results of in vivo transient-expression studies do not support this temporal cleavage order. Rather, expression of a YF polyprotein extending from C through the N-terminal one-third of NS3 revealed that C-prM processing, but not translocation, was dependent on an active NS2B-3 proteinase. This suggests that signalase-mediated cleavage in the lumen of the endoplasmic reticulum may be dependent on prior cleavage at the anchC dibasic site. Possible pathways for processing in the C-prM region are outlined and discussed.
Products of the porcine group C rotavirus NSP3 gene bind specifically to double-stranded RNA and inhibit activation of the interferon-induced protein kinase PKR
Authors: Langland J O , Pettiford S , Jiang B , Jacobs B L
Published: January 2020

The porcine group C rotavirus (Cowden strain) NSP3 protein (the group C equivalent of the group A gene 7 product, formerly called NS34) shares homology with known double-stranded RNA-binding proteins, such as the interferon-induced, double-stranded RNA-dependent protein kinase PKR. A clone of NSP3, expressed both in vitro and in COS-1 cells, led to the synthesis of minor amounts of a product with an M(r) of 45,000 (the expected full-length M(r) of NSP3) and major amounts of products with M(r)s of 38,000 and 8,000. Restriction enzyme digestion analysis prior to expression in vitro and amino-terminal sequence analysis suggest that the products with M(r)s of 38,000 and 8,000 are cleavage products of the protein with an M(r) of 45,000. The full-length protein and the product with an M(r) of 8,000, both of which contain the motif present in double-stranded RNA-binding proteins, bound specifically to double-stranded RNA. The products with M(r)s of 45,000 and 8,000 were also detected in Cowden strain-infected MA104 cells. NSP3 products expressed in COS-1 cells were capable of inhibiting activation of the double-stranded RNA-dependent protein kinase similar to other double-stranded RNA-binding proteins, and NSP3 products expressed in HeLa cells were capable of rescuing the replication of an interferon-sensitive deletion mutant of vaccinia virus.
Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat
Authors: Taylor J P , Pomerantz R J , Raj G V , Kashanchi F , Brady J N , Amini S , Khalili K
Published: January 2020

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.
Deletion mapping of the rotavirus metalloprotein NS53 (NSP1): the conserved cysteine-rich region is essential for virus-specific RNA binding
Authors: Hua J , Chen X , Patton J T
Published: January 2020

NS53 (NSP1), the gene 5 product of the group A rotaviruses, is a minor nonstructural protein of 486 to 495 amino acids which binds zinc and contains an amino-terminal highly conserved cysteine-rich region that may form one or two zinc fingers. To study the structure-function of the gene 5 product, wild-type and mutant forms of NS53 were produced by using a recombinant baculovirus expression system and a recombinant vaccinia virus/T7 (vTF7-3) expression system. Analysis of the RNA-binding activity of the wild-type NS53 immobilized onto protein A-Sepharose beads with NS53-specific antiserum showed that the protein exhibited specific affinity for all 11 rotavirus mRNAs. The use of short virus-specific RNA probes indicated that NS53 specifically recognizes an element located near the 5' ends of viral mRNAs. Analysis of the RNA-binding activity of deletion mutants of NS53 showed that the RNA-binding domain resides within the first 81 amino acids of the protein and that the highly conserved cysteine-rich region within this region of the protein is essential for the activity. Gel electrophoresis and Western immunoblot analyses of intracellular fractions derived from infected cells revealed that large amounts of NS53 were present in the cytosol and in association with the cytoskeletal matrix. Indirect immunofluorescence analysis of cells programmed to transiently express mutant forms of NS53 using vTF7-3 indicated that the intracellular localization domain resides between amino acids 84 and 176 of NS53. Together, these data show that the RNA-binding domain and the intracellular localization domain lie upstream from the region of NS53 previously determined not to be essential for replication of rotaviruses in cell culture (J. Hua and J. T. Patton, Virology 198:567-576, 1994).
Cross-neutralizing activity against divergent human immunodeficiency virus type 1 isolates induced by the gp41 sequence ELDKWAS
Authors: Muster T , Guinea R , Trkola A , Purtscher M , Klima A , Steindl F , Palese P , Katinger H
Published: January 2020

Previously we identified the highly conserved amino acids Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ecto-domain of gp41 as the epitope of a neutralizing monoclonal antibody (2F5) directed against human immunodeficiency virus type 1. In the present study, the sequence defining the epitope was introduced into the loop of antigenic site B of the influenza virus hemagglutinin. The resulting chimeric virus was able to elicit ELDKWA-specific immunoglobulins G and A in antisera of mice. Moreover, the distantly related human immunodeficiency virus type 1 isolates MN, RF, and IIIB were neutralized by these antisera. These data suggest that this conserved B-cell epitope is a promising candidate for inclusion in a vaccine against AIDS. The results also show that influenza virus can be used to effectively present the antigenic structure of this B-cell epitope.
Trichomonas vaginalis with a double-stranded RNA virus has upregulated levels of phenotypically variable immunogen mRNA
Authors: Khoshnan A , Alderete J F
Published: January 2020

Some isolates of Trichomonas vaginalis carry a double-stranded RNA virus (TVV) and undergo phenotypic variation between surface expression and cytoplasmic expression of a prominent immunogen (P270). Only trichomonads with TVV express P270 on the surface. Northern (RNA) blots using a specific cDNA encoding the repeat element of the phenotypically varying P270 immunogen as a probe showed accumulation of P270 transcript only in isolates with TVV (V+) in contrast to isolates without the virus (V-). To test further the influence of virus infection on P270 mRNA expression, V- progeny, derived from the parental V+ isolates, were tested. Trichomonads of V- progeny, like the fresh clinical V- isolates, also showed no accumulation of P270 mRNA. By immunoblotting with a monoclonal antibody to the key repeated epitope of P270, it was found that V- organisms had quantitatively less immunoreactive protein than the parental V+ isolates. Although V+ and V- isolates contained proteins immunoreactive with the monoclonal antibody to P270, it was necessary to test for the presence of the P270 gene among all the isolates. As expected, Southern blots demonstrated that V+ and V- trichomonads possessed the gene encoding P270. These data suggest that the double-stranded RNA virus of T. vaginalis is involved in the regulation of P270 mRNA accumulation.
Constitutive production of alpha and beta interferons in mutant human cell lines
Authors: McKendry R , Pellegrini S , Kerr I M , Stark G R
Published: January 2020

Alpha and beta interferons control expression of a selectable marker in the human hypoxanthine phosphoribosyltransferase-negative cell line 2fTGH, in which transcription of gpt is regulated by the upstream region of an interferon-responsive human gene. Selection of mutagenized 2fTGH cells in hypoxanthine-aminopterin-thymidine medium yielded mutants in one recessive (C1) and two dominant (C2 and C3) complementation groups. The mutants constitutively expressed low levels of beta interferon (C1), alpha interferon (C2), or both (C3).
Inhibition of p53 DNA binding by human papillomavirus E6 proteins
Authors: Lechner M S , Laimins L A
Published: January 2020

Transformation by the human papillomavirus (HPV) early gene products, E6 and E7, involves their interaction with cellular proteins p53 and Rb. Using glutathione S-transferase (GST) fusion proteins, we found that HPV E6 bound human p53 and that the relative efficiency of binding varied such that the GST-HPV type 16 E6 (16E6) protein bound p53 with highest affinity, followed by GST-31E6, GST-18E6, and GST-11E6. The GST-E6 fusion proteins were sufficient for binding p53 purified from a baculovirus expression system as well as in vitro translation sources, while no association was observed with GST-18E7 or a GST-16E6 mutant bearing a five-amino-acid deletion in E6. When the site-specific DNA binding activity of p53 was examined in the presence of GST-E6 proteins, an inhibition of DNA binding was observed. The degree of inhibition correlated with the relative affinity of different E6 proteins for p53; thus, GST-16E6 was the most potent inhibitor of p53 DNA binding activity, and GST-11E6 was the least effective. Prevention of p53 DNA binding is likely to play a role in the abrogation of the transcriptional activity of p53 by HPV E6 and provides a further mechanism for E6 disruption of p53 growth suppressor function in addition to its role in directing specific degradation of p53 through the ubiquitin-mediated pathway. The variation in inhibition of DNA binding seen with the various E6 proteins may thus contribute to the differences in oncogenic potential seen among the HPV types.
Multiple functions of capsid protein phosphorylation in duck hepatitis B virus replication
Authors: Yu M , Summers J
Published: January 2020

We have investigated the role of phosphorylation of the capsid protein of the avian hepadnavirus duck hepatitis B virus in viral replication. We found previously that three serines and one threonine in the C-terminal 24 amino acids of the capsid protein serve as phosphorylation sites and that the pattern of phosphorylation at these sites in intracellular viral capsids is complex. In this study, we present evidence that the phosphorylation state of three of these residues affects distinct steps in viral replication. By substituting these residues with alanine in order to mimic serine, or with aspartic acid in order to mimic phosphoserine, and assaying the effects of these substitutions on various steps in virus replication, we were able to make the following inferences. (i) The presence of phosphoserines at residues 245 and 259 stimulates DNA synthesis within viral nucleocapsids. (ii) The absence of phosphoserine at residue 257 and at residues 257 and 259 stimulates covalently closed circular DNA synthesis and virus production, respectively. (iii) The presence of phosphoserine at position 259 is required for initiation of infection. The results implied that both phosphorylated and nonphosphorylated capsid proteins were necessary for a nucleocapsid particle to carry out all its functions in virus replication, explaining why differential phosphorylation of the capsid protein occurs in hepadnaviruses. Whether these differentially phosphorylated proteins coexist on the same nucleocapsid, or whether the nucleocapsid acquires sequential functions through selective phosphorylation and dephosphorylation, is discussed.
Functional exchange of an oncoretrovirus and a lentivirus matrix protein
Authors: Deminie C A , Emerman M
Published: January 2020

To map functional domains in the retroviral Gag protein we have constructed chimeric viruses where regions of the murine leukemia virus (MuLV) Gag protein have been replaced with analogous sequences from human immunodeficiency virus type 1 (HIV-1). Here we describe the chimeric virus MuLV(MAHIV) which contains the HIV-1 matrix (MA) protein in place of the MuLV MA. MuLV(MAHIV) is infectious but grows at a reduced rate compared with wild-type MuLV. We found that the partial defect in replication of the chimeric virus is at a late stage in the viral life cycle. The MuLV(MAHIV) Gag proteins are distributed aberrantly within cells and are not associated with cellular membranes. Unlike MuLV, HIV-1 is able to integrate into growth-arrested cells. Incorporation of the HIV-1 MA, which is known to play a role in infection of nondividing cells, does not enable MuLV(MAHIV) to be expressed in growth-arrested cells. While it possesses no amino acid homology, we found that the HIV-1 MA can efficiently replace the MuLV matrix protein in infection.
Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus
Authors: Lenhoff R J , Summers J
Published: January 2020

We have used linker scanning and site-directed mutagenesis in an attempt to distinguish among the known functions of the duck hepatitis B virus large envelope protein, p36. We found that linker-encoded amino acid substitutions in at least one region of the pre-S envelope protein p36 produced defects in both the production of enveloped virus and the regulation of covalently closed circular DNA (cccDNA) synthesis. Most linker substitutions, typically in the 5' two-thirds of the pre-S region of the p36 gene did not affect either cccDNA regulation or enveloped virus production but did destroy the infection competence of the enveloped particles produced. Single amino acid substitutions of residues 128 and 131 demonstrated a similar correlation between defects in the ability of p36 to support enveloped virus production and to control cccDNA levels. We concluded from these studies that virus production and cccDNA regulation probably require a common activity of p36.
Serum neutralization of feline immunodeficiency virus is markedly dependent on passage history of the virus and host system
Authors: Baldinotti F , Matteucci D , Mazzetti P , Giannelli C , Bandecchi P , Tozzini F , Bendinelli M
Published: January 2020

Sera from feline immunodeficiency virus (FIV)-infected cats exhibited extremely low levels of neutralizing antibodies against virus passaged a few times in vitro (low passage), when residual infectivity was assayed in the CD3+ CD4- CD8- MBM lymphoid cell line or mitogen-activated peripheral blood mononuclear cells. By sharp contrast, elevated titers of highly efficient neutralizing activity against FIV were measured, by use of high-passage virus, in assays on either the fibroblastoid CrFK or MBM cell line. However, high-passage virus behaved the same as low-passage virus after one in vivo passage in a specific-pathogen-free cat and reisolation. Subneutralizing concentrations of infected cat sera enhanced the production of low-passage virus by MBM cells, an effect not seen with high-passage virus in CrFK cells. These qualitative and quantitative discrepancies could not be attributed to differences in the amount of immunoreactive viral material, to the amount of infectious virus present in the viral stocks, or to the presence of anti-cell antibodies. The observed effects were most likely due to the different passage history of the viral preparations used. The observation that neutralizing antibodies detected with high-passage virus were broadly cross-reactive in assays with CrFK cells but isolate specific in MBM cells suggests also that the cell substrate can influence the result of FIV neutralization assays. This possibility could not be tested directly because FIV adapted to grow in CrFK cells had little infectivity for lymphoid cells and vice versa. In vitro exposure to infected cat sera had little or no effect on the ability of in vivo-passaged FIV to infect cats. These data reveal no obvious relationship between titers against high-passage virus and ability to block infectivity of FIV in cats and suggest caution in the use of such assays to measure vaccine efficacy. In conclusion, by contrast with what has been previously reported for the use of CrFK cells and high-passage virus, both natural and experimental infections of cats with FIV generate poor neutralizing antibody responses with regard to in vivo protection.
Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors
Authors: Valsesia-Wittmann S , Drynda A , Deléage G , Aumailley M , Heard J M , Danos O , Verdier G , Cosset F L
Published: January 2020

On the basis of theoretical structural and comparative studies of various avian leukosis virus SU (surface) envelope proteins, we have identified four small regions (I, II, III, and IV) in their receptor-binding domains that could potentially be involved in binding to receptors. From the envelope gene of an avian leukosis virus of subgroup A, we have constructed a set of SU mutants in which these regions were replaced by the coding sequence of FLA16, a 16-amino-acid RGD-containing peptide known to be the target for several cellular integrin receptors. Helper-free retroviral particles carrying a neo-lacZ retroviral vector were produced with the mutant envelopes. SU mutants in which regions III and IV were substituted yielded normal levels of envelope precursors but were not detectably processed or incorporated in viral particles. In contrast, substitutions in regions I and II did not affect the processing and the viral incorporation of SU mutants. When FLA16 was inserted in region II, it could be detected with antibodies against FLA16 synthetic peptide, but only when viral particles were deglycosylated. Viral particles with envelopes mutated in region I or II were able to infect avian cells through the subgroup A receptor at levels similar to those of the wild type. When viruses with envelopes containing FLA16 peptide in region II were applied to plastic dishes, they were found to promote binding of mammalian cells resistant to infection by subgroup A avian leukosis viruses but expressing the integrins recognized by FLA16. Deglycosylated helper-free viruses obtained by mild treatment with N-glycosidase F have been used to infect these mammalian cells, and infections have been monitored by neomycin selection. No neomycin-resistant clones could be obtained after infection by viruses with wild-type envelopes. Conversely, colonies were obtained after infection by viruses with envelopes bearing FLA16 in region II, and the genome of the retroviral vector was found correctly integrated in cell DNA of these colonies. By using a blocking peptide containing the minimal adhesive RGD sequence contained in FLA16, we have shown that preincubation of target cells could specifically inhibit infection by viruses with FLA16.
Adoptive transfer of polyclonal and cloned cytolytic T lymphocytes (CTL) specific for mouse AIDS-associated tumors is effective in preserving CTL responses: a measure of protection against LP-BM5 retrovirus-induced immunodeficiency
Authors: Green W R , Green K A , Crassi K M
Published: January 2020

Cytolytic T lymphocytes (CTL) can be raised against C57BL/6 B-cell lymphomas from mice with LP-BM5 murine leukemia virus-induced AIDS (MAIDS). Adoptive transfer of polyclonal anti-MAIDS tumor CTL or two CTL clones specific for the B6-1710 MAIDS lymphoma caused preservation of major histocompatibility complex-restricted and allogeneic CTL responses, which may be interpreted as indices of protection from LP-BM5 murine leukemia virus-induced immunodeficiency.