bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure
Authors: Zhou Y J , Burns J W , Morita Y , Tanaka T , Estes M K
Published: January 2020

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.
Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature
Authors: Li Q , Yafal A G , Lee Y M , Hogle J , Chow M
Published: January 2020

Antisera were raised against peptide sequences that are normally internal in the poliovirus virion. These antisera contain neutralizing activity, but this neutralizing activity is dependent on coincubation of the virus and antisera at 37 degrees C. Immunoprecipitation analyses demonstrate that the neutralization is due to exposure of these normally internal sequences at 37 degrees C and subsequent antibody binding. Exposure of these sequences is reversible. These data demonstrate that the poliovirus particle is a dynamic entity that is capable of undergoing conformational alterations at physiological temperatures. This conformational flexibility provides an explanation for earlier observations of virus neutralization by antibodies to internal epitopes which can be accommodated within the framework of existing models for antibody-mediated neutralization of viral infectivity. Analogies between the sequences which are reversibly exposed at 37 degrees C with those which are irreversibly exposed upon receptor binding suggest that the observed conformational dynamics also may play a role in cell entry.
Interactions among the major and minor coat proteins of polyomavirus
Authors: Barouch D H , Harrison S C
Published: January 2020

Murine polyomavirus contains two related minor coat proteins, VP2 and VP3, in addition to the major coat protein, VP1. The sequence of VP3 is identical to that of the carboxy-terminal two-thirds of VP2. VP2 may serve a role in uncoating of the virus, and both minor coat proteins may be important for viral assembly. In this study, we show that VP3 and a series of deletion mutants of VP3 can be expressed in Escherichia coli as fusion proteins to glutathione S-transferase and partially solubilized with a mild detergent. Using an in vitro binding assay, we demonstrate that a 42-amino-acid fragment near the carboxy terminus of VP3 (residues 140 to 181) is sufficient for binding to purified VP1 pentamers. This binding interaction is rapid, saturable, and specific for the common carboxy terminus of VP2 and VP3. The VP1-VP3 complex can be coimmunoprecipitated with an antibody specific to VP1, and a purified VP3 fragment can selectively extract VP1 from a crude cell lysate. The stoichiometry of the binding reaction suggests that each VP1 pentamer in the virus binds either one VP2 or one VP3, with the VP1-VP2/3 complex stabilized by hydrophobic interactions. These results, taken together with studies from other laboratories on the expression of polyomavirus capsid proteins in mouse and insect cells (S. E. Delos, L. Montross, R. B. Moreland, and R. L. Garcea, Virology, 194:393-398, 1993; J. Forstova, N. Krauzewicz, S. Wallace, A. J. Street, S. M. Dilworth, S. Beard, and B. E. Griffin, J. Virol. 67:1405-1413, 1993), support the idea that a VP1-VP2/3 complex forms in the cytoplasm and, after translocation into the nucleus, acts as the unit for viral assembly.
Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow
Authors: Minton E J , Tysoe C , Sinclair J H , Sissons J G
Published: January 2020

Peripheral blood monocytes (PBM) are one site of persistence of human cytomegalovirus (HCMV) in healthy carriers. However, because PBM circulate only briefly before entering the tissues and are difficult to infect with HCMV, it has been suggested that they may acquire HCMV during development in the bone marrow. Consistent with this, we show evidence that bone marrow progenitors from healthy HCMV carriers contain endogenous HCMV DNA as detected by PCR. We also show that bone marrow precursors are readily infected by clinical isolates of HCMV in vitro but that no viral gene expression occurs until these cells become differentiated. In contrast, incubation of these cells at any developmental stage with the laboratory strain AD169 resulted in few cells expressing viral immediate-early genes, and this correlated with a lack of entry of AD169 virus. These observations are consistent with bone marrow progenitors acting as a reservoir for HCMV and transmitting the viral genome to PBM, in the absence of lytic-gene expression, until they leave the circulation and undergo tissue-specific differentiation to macrophages.
T-cell response to cottontail rabbit papillomavirus structural proteins in infected rabbits
Authors: Selvakumar R , Borenstein L A , Lin Y L , Ahmed R , Wettstein F O
Published: January 2020

Cottontail rabbit papillomavirus (CRPV)-induced papillomas progress at a high frequency to carcinomas and thus can serve as a model for high-cancer-risk human papillomavirus infection. Previously, we have shown that antibodies to nonstructural and structural proteins are detected in only a fraction of papilloma-bearing animals. However, the antibody response to structural proteins drastically increases as papillomas progress to carcinoma (Y.-L. Lin, L. A. Borenstein, R. Selvakumar, R. Ahmed, and F. O. Wettstein, J. Virol. 67:382-389, 1993). Here we have monitored the cellular immune response to viral proteins during the course of infection and particularly during progression from papilloma to carcinoma. This was done by measuring the in vitro proliferation response of peripheral blood mononuclear cells (PBMCs) to CRPV structural proteins L1 and L2. The proliferating cells were identified as T cells by selective removal of B or T cells. In general, the T-cell response was low for rabbits at the papilloma stage and none responded to L2. Lymphocytes from animals with carcinomas more frequently and more strongly responded to L1, and more than half also responded to L2. In addition to stimulation of PBMCs, L1- and L2-specific proliferation could also be demonstrated with lymph node and spleen cells. Overall, our data show that progression of papilloma to carcinoma is associated with an increased T-cell response to CRPV structural proteins in addition to an increased humoral response. This greater immune reactivity, however, was not associated with a selectively increased expression of structural proteins, since RNA isolated from papillomas and carcinomas contained similar relative levels of late and early RNA as shown by dot blot analysis. Thus, the heightened immune reactivity seen in carcinoma-bearing rabbits most likely reflects greater stimulation of the immune system owing to dissemination of the tumor. These findings suggest that increased immune responses to papillomavirus proteins may be prognostic of progression to carcinoma and particularly of the development of metastases.
A Friend virus mutant encodes a small glycoprotein that causes erythroleukemia
Authors: Hoatlin M E , Ferro F E , Kozak S L , Kabat D
Published: January 2020

The Pvu delta mutant of Friend spleen focus-forming virus encodes the smallest env glycoprotein (apparent M(r), 41,000) known to activate erythropoietin receptors. In vivo, Pvu delta causes erythroblastosis and the development of erythroleukemia. We isolated two leukemic cell lines that contain Pvu delta; both synthesize hemoglobin in response to dimethyl sulfoxide. The Pvu delta env gene contains a 204-base deletion in the ecotropic-specific region, suggesting that this domain of the glycoprotein is not essential for viral pathogenesis.
Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant
Authors: Sutter G , Ramsey-Ewing A , Rosales R , Moss B
Published: January 2020

Modified vaccinia virus Ankara (MVA), having acquired genomic deletions during passage in chicken embryo fibroblasts, is highly attenuated and unable to productively infect most mammalian cell lines. Multiplication in rabbit kidney-derived RK13 cells, but not other nonpermissive cells, can be restored by insertion of the vaccinia virus K1L gene into the MVA genome. During nonproductive infection of RK13 cells by MVA, transcription of representative viral early genes was revealed by Northern (RNA) blotting, whereas synthesis of an intermediate mRNA and replication of viral DNA could not be detected. Despite the persistence of viral early mRNA for at least several hours, synthesis of virus-induced polypeptides occurred only during the first hour and was followed by abrupt inhibition of all protein synthesis. Transfection of RK13 cells with a eukaryotic expression plasmid that contained the K1L gene allowed MVA infection to proceed to late stages of viral protein synthesis. Moreover, RK13 cell lines that stably expressed the K1L gene were permissive for MVA as well as a K1E deletion mutant of the WR strain of vaccinia virus. This is the first description of the complementation of a poxvirus mutant by cells that stably express a viral gene.
Excision of DNA fragments corresponding to the unit-length a sequence of herpes simplex virus type 1 and terminus variation predominate on one side of the excised fragment
Authors: Umene K
Published: January 2020

DNA fragments corresponding to the unit-length a sequence of herpes simplex virus type 1 (HSV-1) were identified in HSV-1 DNA preparations extracted by the method of Hirt. The DNA fragments were molecularly cloned, and nucleotide sequences were determined. Most termini of the fragments were at sites on DR1 corresponding to the termini of linear HSV-1 DNA generated by the cleavage-packaging system. In one-step growth experiments, DNA fragments of the unit-length a sequence appeared simultaneously with the termini of linear HSV-1 DNAs produced by cleavage of circular and concatemeric DNAs. Therefore, excision of the unit-length a sequence appeared closely related to the cleavage-packaging system. Termini of the excised DNA fragments of the variant a sequence with two DR2 arrays varied on the L-component side, while termini on the S-component side were at the site on DR1 corresponding to the authentic cleavage site. It is thus assumed that the cleavage-packaging system functions adequately on the DR1 second distal from the S component, and cleavages of other DR1 are rare and less accurate. If this notion is tenable, then most termini on the S-component side of the excised DNA fragments are derived from the second DR1 properly cleaved and should be constant, while termini on the L-component side are from regions on and around the DR1 third distal from the S component and may be variable. Cleavage of DR1 is likely to be affected by the topological relationship with the S component.
Human immunodeficiency virus type 1 variants with increased replicative capacity develop during the asymptomatic stage before disease progression
Authors: Connor R I , Ho D D
Published: January 2020

We examined the replicative properties of a series of sequential isolates and biological clones of human immunodeficiency virus type 1 (HIV-1) obtained from an individual who progressed from seroconversion to AIDS in approximately 5 years. HIV-1 isolated soon after seroconversion replicated slowly and to low levels in cultures of peripheral blood mononuclear cells; however, subsequent isolates obtained during asymptomatic infection showed a marked increase in replication kinetics. This was examined in more detail by using a panel of 35 biological clones of HIV-1 generated from sequential patient peripheral blood mononuclear cell samples. Each clone was evaluated for replication in primary macrophages and CD4+ T lymphocytes and for the ability to induce syncytium formation in MT-2 cell cultures. Consistent with earlier observations, we found that all of the clones isolated just after seroconversion were slowly replicating and non-syncytium inducing (NSI). However, NSI variants with increased replication kinetics in macrophages were identified soon thereafter. These variants preceded the appearance of NSI and syncytium-inducing variants, with rapid replication in both macrophages and CD4+ T lymphocytes. To determine whether changes in the rate of replication could be traced to the early stages of the virus life cycle, PCR assays were used to evaluate entry and reverse transcription of selected biological clones in macrophages and CD4+ T lymphocytes. We found there was no inherent block to entry or reverse transcription for the slowly replicating variants; however, this does not preclude the possibility that small differences in the rate of entry may account for larger differences in the replication kinetics over many cycles. Overall, our results demonstrate that rapidly replicating variants of HIV-1 emerge during the asymptomatic period in a patient who subsequently progressed clinically, suggesting that these variants may play an important role in HIV-1 pathogenesis.
The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation
Authors: Goldstein D J , Li W , Wang L M , Heidaran M A , Aaronson S , Shinn R , Schlegel R , Pierce J H
Published: January 2020

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is a highly hydrophobic protein which appears to transform cells through the activation of growth factor receptors. To investigate the specificity of E5-growth factor receptor interactions required for mitogenic signaling, we utilized a nontumorigenic, murine myeloid cell line (32D) which is strictly dependent on interleukin-3 (IL-3) for sustained proliferation in culture. This IL-3 dependence can be functionally substituted by the expression of a variety of surrogate growth factor receptors and the addition of the corresponding ligand. Several receptor cDNAs for the alpha- and beta-type platelet-derived growth factor receptors [alpha PDGFR and beta PDGFR], the epidermal growth factor receptor, and the colony-stimulating factor 1 receptor) were transfected into 32D cells constitutively expressing the E5 protein to test for IL-3-independent growth. Only beta PDGFR was capable of abrogating the IL-3 dependence of 32D cells. The proliferative signal induced by the coexpression of beta PDGFR and E5 was accompanied by stable complex formation between these proteins, constitutive tyrosine phosphorylation of the receptor, and tumorigenicity in nude mice. The lack of cooperative interaction between E5 and the epidermal growth factor receptor, the colony-stimulating factor 1 receptor, and the highly related alpha PDGFR was paralleled by the inability of E5 to bind to these receptors and failure to increase receptor tyrosine phosphorylation. Thus, these data indicate that the ability of E5 to induce sustained proliferation and transformation of 32D cells is a direct consequence of specific interaction between the E5 protein and the beta PDGFR signaling complex and the subsequent stimulation of receptor tyrosine phosphorylation.
A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6
Authors: Thompson J R , Agulnick A D , Ricciardi R P
Published: January 2020

The p41 DNA-binding protein of human herpesvirus 6 is an apparent processivity factor important for viral DNA replication. The p41 promoter was characterized to understand how this processivity factor is regulated. A single transcription start site and a functional TATA box are located 48 and 74 bp, respectively, upstream of the start codon. A reporter construct containing 1,027 bp of the sequence upstream of the p41 start codon was inactive in uninfected T cells but functioned as a strong promoter in human herpesvirus 6-infected cells. Mutational analysis identified a 21-bp element (the EA site) which is located at -73 to -52 bp relative to the transcription start site and is essential for promoter activity. The ability of the EA site to stimulate transcription optimally appears to be strictly dependent upon its distance from the p41 basal promoter. The EA site contains three overlapping sequences, a CAAT-enhancer-binding protein (C/EBP) transcription factor recognition site and two repeat elements. Mobility shift assays using the EA site identified four binding activities (C1 to C4). C1 and C2 are present in both uninfected and infected cells and do not contain C/EBP factors. In infected cells, point mutation of the EA site abrogates C1 and C2 binding activities and destroys transcriptional activity of the p41 promoter. C3 and C4 are present in uninfected cells only and were found to contain C/EBP factors. These findings indicate that in infected cells, transcriptional stimulation of the p41 promoter by the EA site requires C1 and C2 binding activities. These results further suggest that transcriptional activity may also depend upon the elimination of C3 and C4 binding activities.
Determination of the role for CD21 during Epstein-Barr virus infection of B-lymphoblastoid cells
Authors: Martin D R , Marlowe R L , Ahearn J M
Published: January 2020

Epstein-Barr virus (EBV), a herpesvirus with oncogenic potential, is camouflaged with glycoprotein 350/220, which mimics the human ligand C3dg and thereby binds to and exploits complement receptor type 2 (CR2; CD21), the EBV receptor. It has not been possible to determine the role of CR2 during postbinding events of viral infection because all B lymphocytes express endogenous CR2, precluding an informative study of receptor mutants. We have overcome this obstacle through creation of a novel experimental system based on molecular dissection of the ligand-binding domains of human CR2 and murine CR2. Our results demonstrate first, that two discontinuous amino acid substitutions within the ligand-binding domain of murine CR2 render it capable of mediating EBV infection of human B-lymphoblastoid cells, and second, that the specific role of CR2 during EBV infection is to capture virions at the cell surface, after which cofactors not associated with CR2 mediate postbinding events. These are the first studies to be described in which a cell that is normally susceptible to viral infection can be manipulated so as to direct entry of virions via recombinant or endogenous receptors.
Contrasting effects from a single major histocompatibility complex class II molecule (H-2E) in recovery from Friend virus leukemia
Authors: Perry L L , Miyazawa M , Hasenkrug K , Wehrly K , David C S , Chesebro B
Published: January 2020

Resistance to erythroleukemia induced by infection with the Friend virus complex (FV) has been mapped to several genes residing both within and outside the murine major histocompatibility complex (MHC). MHC genes located in the A, D, and Qa/Tla regions of the murine H-2 complex have been shown to affect disease resistance through their capacity to regulate various aspects of the host immune response to viral antigens. This study establishes H-2E as the fourth MHC locus controlling immunological resistance to FV. Our investigation into the role of H-2E molecules revealed two distinct and opposite effects on recovery from Friend disease. H-2b/b mice normally lack a functional E gene product and are resistant to high doses of FV. The expression of H-2E molecules in H-2 recombinant or transgenic mice of this genotype resulted in a significant decrease in spontaneous recovery from FV-induced leukemia. In contrast, H-2E expression also appeared to influence recovery from Friend disease in a positive manner, since blocking these molecules with anti-E antibodies in vivo significantly decreased recovery from Friend disease. The data indicate that the positive effects of H-2E molecules derive from their function as restriction elements for helper T-cell recognition of the viral envelope glycoprotein, and we postulate that the negative effects are due to H-2E-dependent deletion in the T-cell repertoire during development.
Inhibition of vesicular stomatitis virus RNA synthesis by protein hyperphosphorylation
Authors: Chang T L , Reiss C S , Huang A S
Published: January 2020

Vesicular stomatitis virus (VSV) RNA synthesis requires the template nucleocapsid, the polymerase (L) protein, and the cofactor phosphorylated (P/NS) protein. To determine whether the degree of phosphorylation regulated VSV RNA synthesis, infected Chinese hamster ovary cells were treated with okadaic acid (OKA), a serine/threonine phosphatase inhibitor. OKA reduced viral penetration and uncoating but had little or no effect on primary transcription or viral protein synthesis. However, approximately 80% of total viral RNA synthesis was inhibited when 2 microM or more OKA was added to infected cells after viral uncoating had taken place. Analysis of proteins and RNA species in infected cells labeled with 32P showed that OKA led to hyperphosphorylation of two viral phosphoproteins, the P/NS protein and matrix protein (M), resulting in inhibition of full-length RNA synthesis and subsequent secondary transcription. Pulse-chase experiments demonstrated that the hyperphosphorylated P/NS species was converted rapidly from the less phosphorylated form. Hyperphosphorylated P/NS as well as the less phosphorylated form, but not M, were found to be associated with nucleocapsids isolated from cytoplasmic extracts. These results suggest that phosphorylation played an important role in the regulation between viral transcription and viral RNA replication as well as the turning off of RNA replication. Thus, phosphatase inhibitors promise to be a valuable tool for dissecting the regulatory mechanisms involving phosphorylated viral proteins.
Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein
Authors: McCarty D M , Pereira D J , Zolotukhin I , Zhou X , Ryan J H , Muzyczka N
Published: January 2020

We have used baculovirus-expressed Rep68 that has been purified to homogeneity to reexamine the binding properties of the Rep protein. We find that Rep68 is capable of binding to a linear DNA sequence that is contained within a 25-bp sequence of the A stem of the adeno-associated virus (AAV) terminal repeat proximal to the B and C palindromes. This has been shown conclusively by demonstrating that Rep68 could specifically bind to a synthetic oligonucleotide containing the 25-bp region in the absence of the other sequences within the terminal repeat. Rep78 was also capable of binding the A stem recognition element, as demonstrated by the fact that a DNA affinity column containing the 25-bp sequence can be used to purify Rep78. The ability to recognize the linear DNA sequence within the A stem provides a mechanism by which the Rep protein can be oriented on the terminal repeat so that only the correct strand is cut at the terminal resolution site (trs site) during terminal resolution. In addition, computer analysis suggests that sequences similar to the A stem element are present within the three AAV promoter regions. Electrophoretic mobility shift experiments clearly demonstrate that the p5 promoter contains a Rep binding sequence. DNase protection experiments indicate that the Rep binding sequence within the p5 promoter is located between the YY1 initiator sequence and the TATA binding site. This position immediately suggests a mechanism by which the Rep protein could act as a repressor or a transactivator of p5 transcription by interacting with either YY1 or TBP. In addition, gel shift experiments suggest that the p19 promoter also contains a Rep binding site. The presence of Rep binding sites upstream of both promoters suggests that these sites may be involved in coordinate regulation of AAV transcription. In addition, we have identified a heterologous Rep binding sequence within pBR322 DNA. A comparison of the sequences within the A stem, p5, and pBR322 binding sites suggests that a repeating GAGC motif is at least part of the Rep recognition sequence. In the accompanying report (D. M. McCarty, J. H. Ryan, S. Zolutukhin, X. Zhou, and N. Muzyczka, J. Virol. 68:4998-5006, 1994), we examine the relative affinity of Rep to the A stem site and the complete terminal repeat. Finally, we also have reexamined the ability of Rep68 and Rep78 to cut at the trs site in substrates that do not contain the B and C palindromes or any apparent secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)
Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1
Authors: McElrath M J , Rabin M , Hoffman M , Klucking S , Garcia J V , Greenberg P D
Published: January 2020

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.
Differential Th1 and Th2 cell responses in male and female BALB/c mice infected with coxsackievirus group B type 3
Authors: Huber S A , Pfaeffle B
Published: January 2020

Male and female BALB/c mice differ dramatically in susceptibility to myocarditis subsequent to coxsackievirus B3 (CVB3) infection. CVB3 infection of male mice results in substantial inflammatory cell infiltration of the myocardium, and virus-immune lymphocytes from these animals give predominantly a Th1 cell phenotypic response, as determined by predominant immunoglobulin G2a isotypic antibody production and elevated numbers of gamma interferon and interleukin-2 (IL-2)-producing CD4+ T lymphocytes. Females infected with the same virus give predominantly a Th2 cell phenotypic response, as determined by preferential immunoglobulin G1 antibody isotypic responses and increased precursor frequencies of IL-4- and IL-5-producing CD4+ T cells. Treatment of females with testosterone or males with estradiol prior to infection alters subsequent Th subset differentiation, suggesting that the sex-associated hormones have either a direct or indirect effect on CD4+ lymphocyte responses in this model. Treatment of females with 0.1 mg of monoclonal antibody to IL-4 reduces precursor frequencies of IL-4-producing CD4+ T cells and increases frequencies of gamma interferon-producing cells. This treatment also enhances myocardial inflammation, indicating a correlation between Th1-like cell responses and pathogenicity in CVB3 infection. The Th2-like cell may regulate Th1 cell activation. Adoptive transfer of T lymphocytes from CVB3-infected female mice into male animals suppresses the development of myocarditis in the recipients. Treatment of the female donors with monoclonal antibodies to either CD3, CD4, or IL-4 molecules abrogates suppression.
Cervical antibodies in patients with oral herpes simplex virus type 1 (HSV-1) infection: local anamnestic responses after genital HSV-2 infection
Authors: Ashley R , Wald A , Corey L
Published: January 2020

Herpes simplex virus (HSV)-specific immunoglobulin A, immunoglobulin G, and secretory-component-containing immunoglobulins were identified in cervical and salivary secretions from six subjects with oral HSV type 1 (HSV-1) infections. Anamnestic cervical and salivary antibody responses were detected in two HSV-1-seropositive women with newly acquired genital HSV-2 infections. These data implicate the common mucosal immune system in antibody responses to HSV.
Specificity and sequence requirements for interactions between various retroviral Gag proteins
Authors: Franke E K , Yuan H E , Bossolt K L , Goff S P , Luban J
Published: January 2020

We previously established a genetic assay for retroviral Gag polyprotein multimerization (J. Luban, K. B. Alin, K. L. Bossolt, T. Humaran, and S. P. Goff, J. Virol. 66:5157-5160, 1992). Here we use this assay to demonstrate homomeric interactions between Gag polyproteins encoded by six different retroviruses. Of the Gag polyproteins tested, only those encoded by closely related retroviruses formed heteromultimers. To determine the primary sequence requirements for human immunodeficiency virus type 1 Gag polyprotein multimerization, we studied the effects on multimerization of deletion and linker insertion mutations. Sequences necessary for this process were located between the C-terminal one-third of the capsid domain and the C terminus of the nucleocapsid domain.
Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping reading frames
Authors: Barr J , Chambers P , Harriott P , Pringle C R , Easton A J
Published: January 2020

The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.
Identification and initial characterization of the IR6 protein of equine herpesvirus 1
Authors: O'Callaghan D J , Colle C F , Flowers C C , Smith R H , Benoit J N , Bigger C A
Published: January 2020

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.
Immune activation and viral burden in acute disease induced by simian immunodeficiency virus SIVsmmPBj14: correlation between in vitro and in vivo events
Authors: Schwiebert R , Fultz P N
Published: January 2020

The simian immunodeficiency virus SIVsmmPBj14 (SIV-PBj14) is an atypical lentivirus that causes acute disease and death in pig-tailed macaques and in vitro replicates efficiently in resting macaque lymphocytes and activates and induces proliferation of lymphocytes. The present study was conducted to test the hypothesis that production of large quantities of SIV-PBj14 induces widespread immune activation and elaboration of cytokines which lead directly to the death of infected pig-tailed macaques. Following intravenous inoculation of pig-tailed macaques with SIV-PBj14, acute disease developed and was characterized by high levels of plasma viremia, p27gag antigenemia, tumor necrosis factor alpha, and interleukin-6 (IL-6). All animals died within 10 days of infection, at which time some animals had as many as 100% CD4+ cells in the periphery and lymphoid tissues infected. During the last few days before death, titers of infectious virus in blood increased as much as 10(5)-fold. By using dual-label immunofluorescence assays for detection of cell surface activation markers, both CD4+ and CD8+ lymphocytes were shown to express the IL-2 and transferrin receptors following either in vivo or in vitro infection with SIV-PBj14. Furthermore, in vitro infection of quiescent macaque lymphocytes by SIV-PBj14 was accompanied by proliferation of both CD4+ and CD8+ lymphocyte subsets, as measured by incorporation of [3H]thymidine. Increases in numbers of activated lymphocytes and levels of proinflammatory cytokines in plasma coincided with increased amounts of detectable virus in vivo. Clinical signs of disease and pathologic findings were most consistent with death from a shock-like syndrome, in which acute-phase inflammatory cytokines are known to play a major role. Tumor necrosis factor alpha, IL-2, and IL-6 were detected in some cultures infected with SIV-PBj14, but this finding was not consistent. When cytokines were detected, their concentrations were essentially no different from those found in control cultures infected with SIVsmm9, a prototypic strain from which SIV-PBj14 was derived. The in vivo results suggest a synergistic cycle of activation of lymphocytes and monocytes, elaboration of cytokines, and virus production that accelerates uncontrolled and culminates in death. The observed correlations between in vivo and in vitro activation events following SIV-PBj14 infection validate the use of in vitro studies to clarify lentivirus-lymphocyte interactions that may contribute to the virulence of SIV-PBj14.
Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses
Authors: Duch M , Paludan K , Jørgensen P , Pedersen F S
Published: January 2020

Integrated retroviruses or retroviral vectors may be transcriptionally inactive although their promoter-enhancer regions are able to direct transcription in the host cell. We have used single-gene retroviral vectors with a long terminal repeat-directed neo marker gene to determine if the level of transcription relates to the susceptibility of a provirus to inactivation. We used two isogenic vectors, carrying long terminal repeats with a strong and a weak transcriptional enhancer derived from SL3-3 and Akv murine leukemia viruses, respectively. Nonselected cell clones of the murine lymphoid cell line L691 with single integrated vector proviruses exhibiting a 20-fold range of initial expression levels were studied. The basal expression level of a given cell clone with a single provirus did not show any pattern of correlation with the long-term stability of expression, as monitored for periods up to 150 days. Our results thus indicate that the inactivation mechanism operates independently of the initial transcriptional activity of the provirus.
Immunization with a soluble CD4-gp120 complex preferentially induces neutralizing anti-human immunodeficiency virus type 1 antibodies directed to conformation-dependent epitopes of gp120
Authors: Kang C Y , Hariharan K , Nara P L , Sodroski J , Moore J P
Published: January 2020

Preservation of the conformation of recombinant gp120 in an adjuvant, enabling it to elicit conformation-dependent, epitope-specific, broadly neutralizing antibodies, may be critical for the development of any gp120-based human immunodeficiency virus type 1 (HIV-1) vaccine. It was hypothesized that recombinant gp120 complexed with recombinant CD4 could stabilize the conformation-dependent neutralizing epitopes and effectively deliver them to the immune system. Therefore, a soluble CD4-gp120 complex in Syntex adjuvant formulation was tested with mice for its ability to induce neutralizing anti-gp120 antibody responses. Seventeen monoclonal antibodies (MAbs) were generated and characterized. Immunochemical studies, neutralization assays, and mapping studies with gp120 mutants indicated that the 17 MAbs fell into three groups. Four of them were directed to what is probably a conformational epitope involving the C1 domain and did not possess virus-neutralizing activities. Another four MAbs bound to V3 peptide 302-321 and exhibited cross-reactive gp120 binding and relatively weak virus-neutralizing activities. These MAbs were very sensitive to amino acid substitutions, not only in the V3 regions but also in the base of the V1/V2 loop, implying a conformational constraint on the epitope. The last group of nine MAbs recognized conformation-dependent epitopes near the CD4 binding site of gp120 and inhibited the gp120-soluble CD4 interaction. Four of these nine MAbs showed broadly neutralizing activities against multiple laboratory-adapted strains of HIV-1, three of them neutralized only HIVIIIB, and the two lower-affinity MAbs did not neutralize any strain tested. Collectively, the results from this study indicate that immunization with the CD4-gp120 complex can elicit antibodies to conformationally sensitive gp120 epitopes, with some of the antibodies having broadly neutralizing activities. We suggest that immunization with CD4-gp120 complexes may be worth evaluating further for the development of an AIDS vaccine.
In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop
Authors: Donaldson Y K , Bell J E , Holmes E C , Hughes E S , Brown H K , Simmonds P
Published: January 2020

The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences characteristic of NSI/macrophage-tropic isolates form the predominant population in a range of lymphoid and nonlymphoid tissues in vivo, even in patients with AIDS.
Expression of a herpes simplex virus 1 open reading frame antisense to the gamma(1)34.5 gene and transcribed by an RNA 3' coterminal with the unspliced latency-associated transcript
Authors: Lagunoff M , Roizman B
Published: January 2020

Sensory neurons harboring latent herpes simplex virus 1 express viral RNAs derived from one or more transcriptional units contained in the inverted repeats which flank the long unique sequence but which terminate in the inverted repeats flanking the small unique sequences of viral DNA. These transcripts are also made in productively infected cells. We have identified 16 potential open reading frames (ORFs) predicted to encode 50 or more codons within the domain of the largest reported unspliced transcript and examined 5 ORFs by in-frame insertion of a sequence encoding an epitope reacting with a monoclonal antibody against a human cytomegalovirus protein. One ORF (ORF P), coincident with but antisense to the gamma(1)34.5 gene, was expressed but only under conditions in which ICP4 was not functional. To ensure the authenticity of the expression, a second degenerate sequence encoding the same epitope was inserted into a distant site of the same ORF. The protein expressed by the ORF P with two insertions migrated more slowly than the one carrying one insertion only, indicating that ORF P is expressed.
A human immunodeficiency virus type 1 (HIV-1)-based retroviral vector system utilizing stable HIV-1 packaging cell lines
Authors: Carroll R , Lin J T , Dacquel E J , Mosca J D , Burke D S , St Louis D C
Published: January 2020

We have constructed stable human immunodeficiency virus (HIV) packaging cell lines that when transfected with an HIV-based retroviral vector produce packaged vectors capable of transducing susceptible CD4+ cells. This HIV-1-based retroviral vector system has the potential for providing targeted delivery and regulated expression of immunogens or antiviral agents in CD4+ cells.
Susceptibility of nonprimate cell lines to hepatitis A virus infection
Authors: Dotzauer A , Feinstone S M , Kaplan G
Published: January 2020

Hepatitis A virus (HAV) has been adapted to grow in primate cell cultures. We investigated replication of HAV in nonprimate cells by inoculating 20 cell lines from different species with the tissue culture-adapted HM175 strain. Slot blot hybridization and immunofluorescence analysis revealed that HAV replicated in GPE, SP 1K, and IB-RS-2 D10 cells of guinea pig, dolphin, and pig origin, respectively. Studies in IB-RS-2 D10 cells were discontinued because cultures were contaminated with classical swine fever virus. A growth curve showed that HAV grew poorly in GPE cells and intermediately in SP 1K cells compared with growth in FRhK-4 cells. Therefore, the cell surface receptor(s) and other host factor(s) required for HAV replication are present in nonprimate as well as primate cells.