bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutralizing Antibodies from the Sera of Human Immunodeficiency Virus Type 1-Infected Individuals Bind to Monomeric gp120 and Oligomeric gp140
Authors: Stamatos Nicholas M. , Mascola John R. , Kalyanaraman Vaniambadi S. , Louder Mark K. , Frampton Lynn M. , Birx Deborah L. , VanCott Thomas C.
Published: December 2019

Antibodies that neutralize primary isolates of human immunodeficiency virus type 1 (HIV-1) appear during HIV-1 infection but are difficult to elicit by immunization with current vaccine products comprised of monomeric forms of HIV-1 envelope glycoprotein gp120. The limited neutralizing antibody response generated by gp120 vaccine products could be due to the absence or inaccessibility of the relevant epitopes. To determine whether neutralizing antibodies from HIV-1-infected patients bind to epitopes accessible on monomeric gp120 and/or oligomeric gp140 (ogp140), purified total immunoglobulin from the sera of two HIV-1-infected patients as well as pooled HIV immune globulin were selectively depleted of antibodies which bound to immobilized gp120 or ogp140. After passage of each immunoglobulin preparation through the respective columns, antibody titers against gp120 and ogp140 were specifically reduced at least 128-fold. The gp120- and gp140-depleted antibody fraction from each serum displayed reduced neutralization activity against three primary and two T-cell line-adapted (TCLA) HIV-1 isolates. Significant residual neutralizing activity, however, persisted in the depleted sera, indicating additional neutralizing antibody specificities. gp120- and ogp140-specific antibodies eluted from each column neutralized both primary and TCLA viruses. These data demonstrate the presence and accessibility of epitopes on both monomeric gp120 and ogp140 that are specific for antibodies that are capable of neutralizing primary isolates of HIV-1. Thus, the difficulties associated with eliciting neutralizing antibodies by using current monomeric gp120 subunit vaccines may be related less to improper protein structure and more to ineffective immunogen formulation and/or presentation.
Activation of Antiviral Protein Kinase Leads to Immunoglobulin E Class Switching in Human B Cells
Authors: Rager Kelly J. , Langland Jeffrey O. , Jacobs Bertram L. , Proud David , Marsh David G. , Imani Farhad
Published: December 2019

An epidemiologic association between viral infections and the onset of asthma and allergy has been documented. Also, evidence from animal and human studies has suggested an increase in antigen-specific immunoglobulin E (IgE) production during viral infections, and elevated levels of IgE are characteristic of human asthma and allergy. Here, we provide molecular evidence for the roles of viral infection and of activation of the antiviral protein kinase (PKR) (double-stranded-RNA [dsRNA]-activated protein kinase) in the induction of IgE class switching. The presence of dsRNA, a known component of viral infection and an activator of PKR, induced IgE class switching as detected by the expression of germ line ɛ in the human Ramos B-cell line. Furthermore, dsRNA treatment of Ramos cells resulted in the activation of PKR and in vivo activation of the NF-κB complex. Interestingly, infection of Ramos cells with rhinovirus (common cold virus) serotypes 14 and 16 resulted in the induction of germ line ɛ expression. To further evaluate the role of PKR in the viral induction of IgE class switching, we infected Ramos cells with two different vaccinia virus (cowpox virus) strains. Infection with wild-type vaccinia virus failed to induce germ line ɛ expression; however, a deletion mutant of vaccinia virus (VP1080) lacking the PKR-inhibitory polypeptide E3L induced the expression of germ line ɛ. Collectively, the results of our study define a common molecular mechanism underlying the role of viral infections in IgE class switching and subsequent induction of IgE-mediated disorders such as allergy and asthma.
Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells
Authors: Paul Mousumi , Mazumder Suparna , Raja Nicholas , Jabbar M. Abdul
Published: December 2019

Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.
Differential Tropism and Chemokine Receptor Expression of Human Immunodeficiency Virus Type 1 in Neonatal Monocytes, Monocyte-Derived Macrophages, and Placental Macrophages
Authors: Fear Warwick R. , Kesson Alison M. , Naif Hassan , Lynch Garry W. , Cunningham Anthony L.
Published: December 2019

Laboratory-adapted (LA) macrophage-tropic (M-tropic) human immunodeficiency virus type 1 (HIV-1) isolates (e.g., HIV-1
Ba-L
) and low-passage primary (PR) isolates differed markedly in tropism for syngeneic neonatal monocytes, monocyte-derived macrophages (MDMs), and placental macrophages (PMs). Newly adherent neonatal monocytes and cultured PMs were highly refractory to infection with PR HIV-1 isolates yet were permissive for LA M-tropic isolates. Day 4 MDMs were also permissive for LA M-tropic isolates and additionally, were permissive for over half the PR isolates tested. Qualitative differences in PR HIV-1 infection of monocytes/MDMs could not be correlated with CD4 levels alone, and in all three cell types the block to PR HIV-1 strain replication preceded reverse transcription. Neonatal monocyte susceptibility to PR HIV-1 strains correlated with increasing CCR-5 expression during maturation. CCR-5 could not be detected on newly adherent (day 1) neonatal monocytes, in contrast to adult monocytes (H. Naif et al., J. Virol. 72:830–836, 1998), but was readily detectable after 4 to 7 days of culture. However, moderate CCR-5 mRNA levels were present in day 1 neonatal monocytes and remained constant during monocyte maturation. CCR-5 was not detectable on the surface of PMs, yet the receptor was present within permeabilized cells. Notably, two brain-derived PR HIV-1 isolates from a single patient, differing in their V3 loops, were discordant in their abilities to infect neonatal monocytes/MDMs and PMs, yet both isolates could infect newly adherent adult monocytes. Together these data strongly suggest that LA HIV-1 isolates are able to infect neonatal monocytes at earlier stages of maturation and lower-level expression of CCR-5 than PR isolates. The differences between neonatal and adult monocytes in susceptibility to PR isolates may also be related to the level of CCR-5 expression.
Herpesvirus Entry Mediator HVEM Mediates Cell-Cell Spread in BHK(TK
−
) Cell Clones
Authors: Roller Richard J. , Rauch Daniel
Published: December 2019

95-19 and U
S
11cl19.3 are BHK(TK
−
)-derived cell lines that are highly resistant to postattachment entry of herpes simplex virus type 1 (HSV-1) and HSV-2 but not to later steps in single-step replication. The resistance properties of these two cell types are not identical. U
S
11cl19.3 cells are fully susceptible to pseudorabies virus (PRV), as shown by single-step growth experiments, whereas 95-19 cells are resistant to entry of free PRV but not to entry by cell-cell spread. We have tested the ability of HVEM to overcome the block to infection in both cell lines following transient and stable transfection. HVEM was able to mediate entry of free HSV-1 into both cell lines, as shown by an increase in the number of β-galactosidase-expressing cells in cultures transiently transfected with an HVEM expression plasmid and infected with
lacZ
-expressing HSV-1. In stably transfected 95-19 cells, HVEM enhanced infection by free HSV-1, as shown by an increase in the number of infectious centers obtained following infection. In both cell types, HVEM strongly enhanced entry of HSV-1 and HSV-2 by cell-cell spread, suggesting that HVEM can function as an entry mediator both in entry of free virus and in entry by cell-cell spread.
Structural Localization of the E3 Glycoprotein in Attenuated Sindbis Virus Mutants
Authors: Paredes A. M. , Heidner H. , Thuman-Commike P. , Venkataram Prasad B. V. , Johnston R. E. , Chiu W.
Published: December 2019

We have determined the three-dimensional structures of the wild-type Sindbis virus and two of its mutants that retain the E3 sequence within PE2. Using difference imaging between these mutants and the wild-type virus, we have assigned a location for the 64-amino-acid sequence corresponding to E3 in the mutant spike complex. In the wild-type virus, the spike is composed of an E1-E2 heterotrimer. The E3 protein was found to protrude midway between the center of the spike complex and the tips. Based on these results and the work of others, we propose a distribution for the functional domains of the spike proteins within the structure of wild-type Sindbis virus. Within the structure of the virus, the E1 domains form the central portion of the spike complex, while the tips are formed by the E2 domains that flare out from the center of the complex. The structural similarity between these Sindbis virus mutants and Ross River virus suggests that E3 may also be present in the latter, which is also a member of the
Alphavirus
genus.
Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility
Authors: Kim Seon Hee , Yu Seung Shin , Park Jong Sang , Robbins Paul D. , An Chung Sun , Kim Sunyoung
Published: December 2019

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire
gag
coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.
Development of Improved Adenosine Deaminase Retroviral Vectors
Authors: Onodera Masafumi , Nelson David M. , Yachie Akihiro , Jagadeesh G. Jayashree , Bunnell Bruce A. , Morgan Richard A. , Blaese R. Michael
Published: December 2019

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA−) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA−severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.
Intercapsomeric Disulfide Bonds in Papillomavirus Assembly and Disassembly
Authors: Li Maolin , Beard Peter , Estes Patricia A. , Lyon Mary K. , Garcea Robert L.
Published: December 2019

In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in
Escherichia coli
and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.
An Endonuclease Switching Mechanism in the Virion RNA and cRNA Promoters of Thogoto Orthomyxovirus
Authors: Leahy Michael B. , Dessens Johannes T. , Pritlove David C. , Nuttall Patricia A.
Published: December 2019

An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3′ and 5′ termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5′ hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5′ hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.
The M2 Ectodomain Is Important for Its Incorporation into Influenza A Virions
Authors: Park Eun K. , Castrucci Maria R. , Portner Allen , Kawaoka Yoshihiro
Published: December 2019

M2 is an integral protein of influenza A virus that functions as an ion channel. The ratio of M2 to HA in influenza A virions differs from that found on the cell surface, suggesting selective incorporation of M2 and HA into influenza virions. To examine the sequences that are important for M2 incorporation into virions, we used an incorporation assay that involves expressing M2 from a plasmid, transfecting the plasmid into recipient cells, and then infecting those cells with influenza virus. To test the importance of the different regions of the protein (extracellular, transmembrane, and cytoplasmic) in determining M2 incorporation, we created chimeric mutants of M2 and Sendai virus F proteins, exchanging corresponding extracellular, transmembrane, and cytoplasmic domains. Of the six possible chimeric mutants, only three were expressed on the cell surface. Of these three chimeric proteins, only one mutant (with the extracellular domain from M2 and the rest from F) was incorporated into influenza virions. These results suggest that the extracellular domain of M2 is important for its incorporation into virions.
Adenovirus Preterminal Protein Binds to the CAD Enzyme at Active Sites of Viral DNA Replication on the Nuclear Matrix
Authors: Angeletti Peter C. , Engler Jeffrey A.
Published: December 2019

Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.
Extensive Diversification of Human Immunodeficiency Virus Type 1 Subtype B Strains during Dual Infection of a Chimpanzee That Progressed to AIDS
Authors: Wei Qing , Fultz Patricia N.
Published: December 2019

A chimpanzee (C-499) infected for more than 9 years with two subtype B isolates of human immunodeficiency virus type 1 (HIV-1), one (HIV-1SF2) that replicates poorly and one (HIV-1LAV-1b) that replicates efficiently in chimpanzees, died of AIDS 11 years after initial infection (F. J. Novembre et al., J. Virol. 71:4086–4091, 1997). Nucleotide sequence and phylogenetic analyses of the C2 to V5 region ofenv(C2-V5env) in proviral DNA from peripheral blood lymphocytes obtained 22 months before death revealed two distinct virus populations. One of these populations appeared to be a recombinant inenv, having the V3 loop from HIV-1SF2and the V4-V5 region from HIV-1LAV-1b; the other population had evolved from HIV-1LAV-1b. In addition to C2-V5env, the entire p17gagandnefgenes were sequenced; however, based on nucleotide sequences and phlyogeny, whether the progenitor of the p17gagandnefgenes was SF2 or LAV-1b could not be determined. Compared to the two original viruses, the divergence of all clones of C2-V5envranged from 9.37 to 20.2%, that of p17gagranged from 3.11 to 9.29%, and that ofnefranged from 4.02 to 7.9%. In contrast, compared to the maximum variation of 20.2% in C2-V5envfor C-499, the maximum diversities in C2-V5envin proviruses from two chimpanzees infected with HIV-1LAV-1bfor 9 and 10 years were 9.65 and 2.48%, respectively. These results demonstrate that (i) two distinct HIV-1 populations can coexist and undergo extensive diversification in chimpanzees with progressive HIV-1-induced disease and (ii) recombination between two subtype B strains occurred even though the second strain was inoculated 15 months after the first one. Furthermore, evaluation ofenvgenes from three chimpanzees infected with the same strain suggests that the magnitude of HIV-1 diversification could be related to higher viral burdens, manifestations of disease, and/or dual infection.
Type C Retrovirus Released from Porcine Primary Peripheral Blood Mononuclear Cells Infects Human Cells
Authors: Wilson Carolyn A. , Wong Susan , Muller Jacqueline , Davidson Cynthia E. , Rose Timothy M. , Burd Parris
Published: December 2019

As part of the evaluation of porcine cells, tissues, and organs intended for transplantation into humans, we investigated the conditions required to induce expression and release of porcine endogenous retrovirus (PoEV) from primary cells. Pigs contain endogenous retroviral sequences encoding infectious retrovirus, yet little is known about the conditions required to activate the expression and release of PoEV from primary cells. We show here that mitogenic activation of peripheral blood mononuclear cells (PBMC) isolated from the National Institutes of Health (NIH) miniature pig and the Yucatan pig resulted in the activation and release of an infectious type C retrovirus. Coculture of activated porcine PBMC with pig or human cell lines resulted in the transfer and expression of PoEV-specific sequences and the establishment of a productive infection. Sequence comparison of portions of the PoEV
pol
gene expressed in pig cell lines productively infected with virus derived from NIH miniature pig and Yucatan pig PBMC revealed marked similarity, suggesting that one or a few loci may be capable of being activated to yield an infectious virus. These findings demonstrate that the presence of endogenous viruses in source animals needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.
Adenovirus E1B 55K Represses p53 Activation In Vitro
Authors: Martin Michelle E. D. , Berk Arnold J.
Published: December 2019

Adenovirus E1B 55K protein cooperates with E1A gene products to induce cell transformation. E1B 55K mediates its effects by binding to and inhibiting the transcriptional activation and growth-suppression functions of the tumor suppressor p53. Previous studies in vivo have suggested that E1B 55K has an active role in repressing p53 transcriptional activation and that this repression function is directed to specific promoters through E1B 55K’s interaction with DNA-bound p53. Flag-tagged E1B 55K (e55K) was expressed with the baculovirus expression system and immunopurified. Gel filtration, velocity sedimentation centrifugation, and glutaraldehyde cross-linking indicated that e55K is a dimer with a nonglobular conformation. e55K bound directly to purified p53, causing an ∼10-fold increase in p53 affinity for tandem binding sites. Using in vitro transcription assays reconstituted with purified p53, e55K, and HeLa cell nuclear extracts, we found that e55K specifically repressed p53 activation. These results demonstrate that as postulated from earlier transient expression experiments, E1B 55K is a specific repressor of transcription from a promoter with bound p53. Since HeLa nuclear extracts contain little detectable histone protein, E1B 55K probably represses transcription through direct or indirect interactions with the RNA polymerase II transcription machinery.
B-Lymphocyte Proliferation during Bovine Leukemia VirusInduced Persistent Lymphocytosis Is Enhanced by T-Lymphocyte-Derived Interleukin-2
Authors: Trueblood Esther S. , Brown Wendy C. , Palmer Guy H. , Davis William C. , Stone Diana M. , McElwain Terry F.
Published: December 2019

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5+B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-γ) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.
Infection of Human B Lymphocytes with Lymphocryptoviruses Related to Epstein-Barr Virus
Authors: Moghaddam Amir , Koch Joachim , Annis Bethany , Wang Fred
Published: December 2019

Lymphocryptoviruses (LCVs) naturally infecting Old World nonhuman primates are closely related to the human LCV, Epstein-Barr virus (EBV), and share similar genome organization and sequences, biologic properties, epidemiology, and pathogenesis. LCVs can efficiently immortalize B lymphocytes from the autologous species, but the ability of a given LCV to immortalize B cells from other Old World primate species is variable. We found that LCV from rhesus monkeys did not immortalize human B cells, and EBV did not immortalize rhesus monkey B cells. In this study, baboon LCV could not immortalize human peripheral blood B cells but could readily immortalize rhesus monkey B cells. Thus, efficient LCV-induced B-cell immortalization across distant Old World primate species appears to be restricted by a species-specific block. To further characterize this species restriction, we first cloned the rhesus monkey LCV major membrane glycoprotein and discovered that the binding epitope for the EBV receptor, CD21, was highly conserved. Stable infections of human B cells with recombinant amplicons packaged in rhesus monkey or baboon LCV envelopes were also consistent with a species-restricted block occurring after virus binding and penetration. Transient infections of human B cells with simian LCV resulted in latent LCV EBNA-2 gene expression and activation of cell CD23 gene expression. EBV-immortalized human B cells could be coinfected with baboon LCV, and the simian virus persisted and replicated in human B cells. Thus, several lines of evidence indicate that the species restriction for efficient LCV-induced B-cell immortalization occurs beyond virus binding and penetration. This has important implications for the study of LCV infection in Old World primate models and for human xenotransplantation where simian LCVs may be inadvertently introduced into humans.
Molluscum Contagiosum Virus Topoisomerase: Purification, Activities, and Response to Inhibitors
Authors: Hwang Young , Wang Beibei , Bushman Frederic D.
Published: December 2019

Molluscum contagiosum virus (MCV), the only member of the
Molluscipoxvirus
genus, causes benign papules in healthy people but disfiguring lesions in immunocompromised patients. The sequence of MCV has been completed, revealing that MCV encodes a probable type I topoisomerase enzyme. All poxviruses sequenced to date also encode type I topoisomerases, and in the case of vaccinia virus the topoisomerase has been shown to be essential for replication. Thus, inhibitors of the MCV topoisomerase might be useful as antiviral agents. We have cloned the gene for MCV topoisomerase, overexpressed and purified the protein, and begun to characterize its activities in vitro. Like other eukaryotic type I topoisomerases, MCV topoisomerase can relax both positive and negative supercoils. An analysis of the cleavage of plasmid and oligonucleotide substrates indicates that cleavage by MCV topoisomerase is favored just 3′ of the sequence 5′ (T/C)CCTT 3′, resulting in formation of a covalent bond to the 3′ T residue, as with other poxvirus topoisomerases. We identified solution conditions favorable for activity and measured the rate of formation and decay of the covalent intermediate. MCV topoisomerase is sensitive to inhibition by coumermycin A1 (50% inhibitory concentration, 32 μM) but insensitive to five other previously reported topoisomerase inhibitors. This work provides the point of departure for studies of the mechanism of function of MCV topoisomerase and the development of medically useful inhibitors.
Elongation of the Cytoplasmic Tail Interferes with the Fusion Activity of Influenza Virus Hemagglutinin
Authors: Ohuchi Masanobu , Fischer Christian , Ohuchi Reiko , Herwig Astrid , Klenk Hans-Dieter
Published: December 2019

The hemagglutinin (HA) of fowl plague virus was lengthened and shortened by site-specific mutagenesis at the cytoplasmic tail, and the effects of these modifications on HA functions were analyzed after expression from a simian virus 40 vector. Elongation of the tail by the addition of one to six histidine (His) residues did not interfere with intracellular transport, glycosylation, proteolytic cleavage, acylation, cell surface expression, and hemadsorption. However, the ability to induce syncytia at a low pH decreased dramatically depending on the number of His residues added. Partial fusion (hemifusion), assayed by fluorescence transfer from octadecylrhodamine-labeled erythrocyte membranes, was also reduced, but even with the mutant carrying six His residues, significant transfer was observed. However, when the formation of fusion pores was examined with hydrophilic fluorescent calcein, transfer from erythrocytes to HA-expressing cells was not observed with the mutant carrying six histidine residues. The addition of different amino acids to the cytoplasmic tail of HA caused an inhibitory effect similar to that caused by the addition of His. On the other hand, a mutant lacking the cytoplasmic tail was still able to fuse at a reduced level. These results demonstrate that elongation of the cytoplasmic tail interferes with the formation and enlargement of fusion pores. Thus, the length of the cytoplasmic tail plays a critical role in the fusion process.
Grossly DefectivenefGene Sequences in a Human Immunodeficiency Virus Type 1-Seropositive Long-Term Nonprogressor
Authors: Salvi Roberto , Garbuglia Anna Rosa , Di Caro Antonino , Pulciani Simonetta , Montella Francesco , Benedetto Arrigo
Published: December 2019

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4+T-cell counts (>1,000 CD4 cells/μl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5′ long terminal repeat (5′LTR)/gagleader and thenefgene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms withnefdeletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of thenefgene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in thenef-alone and thenef/U3 overlapping regions. Length polymorphism analysis of the 5′LTR/gagleader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5′LTR. Evidence of the role ofnefgene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.
An Array of Novel Murine Spleen Focus-Forming Viruses That Activate the Erythropoietin Receptor
Authors: Gomez-Lucia Esperanza , Zhi Yu , Nabavi Melud , Zhang Weibin , Kabat David , Hoatlin Maureen E.
Published: December 2019

The Friend spleen focus-forming virus (SFFV)
env
gene encodes a 409-amino-acid glycoprotein with an apparent
M
r
of 55,000 (gp55) that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. We reported previously the in vivo selection during serial passages in mice of several evolutionary intermediates that culminated in the formation of a novel SFFV (M. E. Hoatlin, E. Gomez-Lucia, F. Lilly, J. H. Beckstead, and D. Kabat, J. Virol. 72:3602–3609, 1998). A mouse injected with a retroviral vector in the presence of a nonpathogenic helper virus developed long-latency erythroblastosis, and subsequent viral passages resulted in more pathogenic isolates. The viruses taken from these mice converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives. Western blot analysis of cell extracts with an antiserum that broadly reacts with murine retroviral envelope glycoproteins suggested that the spleen from the initial mouse with mild erythoblastosis contained an array of viral components that were capable of activating EpoR. DNA sequence analysis of the viral genomes cloned from different factor-independent cell clones revealed
env
genes with open reading frames encoding 644, 449, and 187 amino acids. All three
env
genes contained 3′ regions identical to that of SFFV, including a 6-bp duplication and a single-base insertion that have been shown previously to be critical for pathogenesis. However, the three
env
gene sequences did not contain any polytropic sequences and were divergent in their 5′ regions, suggesting that they had originated by recombination and partial deletions of endogenously inherited MuLV
env
sequences. These results suggest that the requirements for EpoR activation by SFFV-related viruses are dependent on sequences at the 3′ end of the
env
gene and not on the polytropic regions or on the 585-base deletions that are common among the classical strains of SFFV. Moreover, sequence analysis of the different recombinants and deletion mutants revealed that short direct and indirect repeat sequences frequently flanked the deletions that had occurred, suggesting a reverse transcriptase template jumping mechanism for this rapid retroviral diversification.
The Transmembrane Domains of Sindbis Virus Envelope Glycoproteins Induce Cell Death
Authors: Joe Andrew K. , Foo Hsin Hsin , Kleeman Linda , Levine Beth
Published: December 2019

Sindbis virus, the prototype alphavirus, kills cells by inducing apoptosis. To investigate potential mechanisms by which Sindbis virus induces apoptosis, we examined whether specific viral gene products were able to induce cell death. Genes encoding the three structural proteins—capsid, the precursor E1 (6K plus E1), and the precursor E2 (P62 or E3 plus E2)—were cotransfected with a β-galactosidase reporter plasmid in transient-transfection assays in rat prostate adenocarcinoma AT3 cells. Cell death, as determined by measuring the loss of blue cells, was observed in AT3 cells transfected with 6K plus E1 and with P62 but not in cells transfected with capsid. Deletion mutagenesis of P62 indicated that large regions of the cytoplasmic domain and extracellular domain were not essential for the induction of cell death. However, constructs containing the minimal E3 signal sequence fused to the E2 transmembrane domain and the minimal E3 signal sequence fused to the E1 transmembrane domain induced death as efficiently as full-length P62 and 6K plus E1, whereas no cell death was observed after transfection with a control construct containing the E3 signal sequence linked to the transmembrane domain of murine CD4. These data demonstrate that intracellular expression of the transmembrane domains of the Sindbis virus envelope glycoproteins can kill AT3 cells.
Two Types of Virus-Related Particles Are Found during Transmissible Gastroenteritis Virus Morphogenesis
Authors: Risco Cristina , Muntión María , Enjuanes Luis , Carrascosa José L.
Published: December 2019

The intracellular assembly of the transmissible gastroenteritis coronavirus (TGEV) was studied in infected swine testis (ST) cells at different postinfection times by using ultrathin sections of conventionally embedded infected cells, freeze-substitution, and methods for detecting viral proteins and RNA at the electron microscopy level. This ultrastructural analysis was focused on the identification of the different viral components that assemble in infected cells, in particular the spherical, potentially icosahedral internal core, a new structural element of the extracellular infectious coronavirus recently characterized by our group. Typical budding profiles and two types of virion-related particles were detected in TGEV-infected cells. While large virions with an electron-dense internal periphery and a clear central area are abundant at perinuclear regions, smaller viral particles, with the characteristic morphology of extracellular virions (exhibiting compact internal cores with polygonal contours) accumulate inside secretory vesicles that reach the plasma membrane. The two types of virions coexist in the Golgi complex of infected ST cells. In nocodazole-treated infected cells, the two types of virions coexist in altered Golgi stacks, while the large secretory vesicles filled with virions found in normal infections are not detected in this case. Treatment of infected cells with the Golgi complex-disrupting agent brefeldin A induced the accumulation of large virions in the cisternae that form by fusion of different membranous compartments. These data, together with the distribution of both types of virions in different cellular compartments, strongly suggest that the large virions are the precursors of the small viral particles and that their transport through a functional Golgi complex is necessary for viral maturation.
Characterization of a Human Foamy Virus 170-Kilodalton Env-Bet Fusion Protein Generated by Alternative Splicing
Authors: Lindemann Dirk , Rethwilm Axel
Published: December 2019

Primate foamy viruses (FVs) express, in addition to the 130-kDa envelope protein, a 170-kDa glycoprotein, which reacts with antisera specific for the envelope and Bel proteins. We determined the exact nature of this 170-kDa glycoprotein by using the molecularly cloned human FV (HFV). Radioimmunoprecipitation analysis of 293T cells transfected with appropriate expression constructs by using antisera specific for the HFV Env, Bel1, and Bel2 proteins, as well as reverse transcription-PCR analysis of HFV-infected cells, demonstrated that this protein is an Env-Bet fusion protein that is secreted into the supernatant. However, it is only loosely associated, or not associated, with viral particles. gp170 is generated by an alternatively spliced Env mRNA using a splice donor and splice acceptor pair localized within the
env
open reading frame (ORF), which is normally used to generate Bel1 and Bet transcripts derived from the internal promoter within the
env
ORF. gp170 is expressed at a level 30 to 50% of the Env precursor gp130. However, it alone does not confer infectivity to HFV particles, because capsids derived from proviruses expressing only the gp170 were not released into the supernatant. In contrast, viruses derived from proviral clones deficient in gp170 expression showed similar in vitro infectivity and replication kinetics to wild-type virus. Furthermore, both types of viruses were inactivated to a similar extent by neutralizing sera, indicating that shedding of gp170 probably does not affect the humoral immune response in the infected host.
Identification of a Novel Cellular TPR-Containing Protein, SGT, That Interacts with the Nonstructural Protein NS1 of Parvovirus H-1
Authors: Cziepluch Celina , Kordes Elisabeth , Poirey Rémy , Grewenig Annabel , Rommelaere Jean , Jauniaux Jean-Claude
Published: December 2019

The nonstructural protein NS1 of autonomous parvoviruses is essential for viral DNA amplification and gene expression and is also the major cytopathic effector of these viruses. NS1 acts as nickase, helicase, and ATPase and upregulates P38-driven transcription of the capsid genes. We report here the identification of a novel cellular protein that interacts with NS1 from parvovirus H-1 and which we termed SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein. The cDNA encoding full-length SGT was isolated through a two-hybrid screen with, as bait, the truncated NS1
dl
C69 polypeptide, which lacks the C-terminal transactivation domain of NS1. Full-length NS1 and SGT interacted in the two-hybrid system and in an in vitro interaction assay. Northern blot analysis revealed one major transcript of about 2 kb that was present in all rat tissues investigated. Rat
sgt
cDNA coded for 314 amino acids, and the protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 34 kDa. SGT could be detected in both the nucleus and the cytoplasm of rat cells, as determined by indirect immunofluorescence analysis and Western blotting of fractionated cellular extracts with an affinity-purified antiserum raised against recombinant SGT (AC1.1). In H-1 virus-infected rat and human cells, compared to mock-infected controls, differences in the migration of SGT polypeptides were revealed after Western blot analysis of total cellular extracts. Moreover, the transient expression of NS proteins was sufficient to induce SGT modification. These results show that cellular SGT, which we have identified as an NS1-interacting protein, is modified by parvovirus infection as well as NS expression.
Effects of Mutations in Residues near the Active Site of Human Immunodeficiency Virus Type 1 Integrase on Specific Enzyme-Substrate Interactions
Authors: Gerton Jennifer L. , Ohgi Sharron , Olsen Mari , DeRisi Joseph , Brown Patrick O.
Published: December 2019

The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5′ end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5′ dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.