bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro
Authors: McCarthy Michael P. , White Wendy I. , Palmer-Hill Frances , Koenig Scott , Suzich Joann A.
Published: December 2019

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.
The Murine CAR Homolog Is a Receptor for Coxsackie B Viruses and Adenoviruses
Authors: Bergelson Jeffrey M. , Krithivas Anita , Celi Leo , Droguett Gustavo , Horwitz Marshall S. , Wickham Thomas , Crowell Richard L. , Finberg Robert W.
Published: December 2019

Complementary DNA clones encoding the murine homolog (mCAR) of the human coxsackievirus and adenovirus receptor (CAR) were isolated. Nonpermissive CHO cells transfected with mCAR cDNA became susceptible to infection by coxsackieviruses B3 and B4 and showed increased susceptibility to adenovirus-mediated gene transfer. These results indicate that the same receptor is responsible for virus interactions with both murine and human cells. Analysis of receptor expression in human and murine tissues should be useful in defining factors governing virus tropism in vivo.
Analysis of the Temporal Relationship between Human Immunodeficiency Virus Type 1 Quasispecies in Sequential Blood Samples and Various Organs Obtained at Autopsy
Authors: van’t Wout Angélique B. , Ran Leonie J. , Kuiken Carla L. , Kootstra Neeltje A. , Pals Steven T. , Schuitemaker Hanneke
Published: December 2019

We studied the temporal relationship between human immunodeficiency type 1 (HIV-1) quasispecies in tissues and in peripheral blood mononuclear cells (PBMC) of infected individuals. Sequential PBMC and tissue samples from various organs obtained at autopsy from three patients who died of AIDS-related complications were available for analysis. Biological HIV-1 clones were isolated from PBMC samples, and cellular tropism and syncytium-inducing (SI) capacity were determined. Genomic DNA was isolated from 1 cm3of organ tissue, and proviral DNA was amplified by means of PCR and cloned with the PGEM-T vector system. A 185-bp region encompassing the third variable domain of the virus envelope, known to influence HIV-1 biological properties, was sequenced. HIV-1 could be amplified from all PBMC and organ samples, except from liver tissue for two patients. Both SI and non-syncytium-inducing (NSI) genotypes could be detected in the different tissues. Tissue-specific quasispecies were observed in brain, lung, and testis. Lymphoid tissues, such as bone marrow, lymph node, and spleen, harbored several different variants similar to those detected in blood in the last PBMC samples. In general, only tissues in which macrophages are likely to be the main target cell for HIV-1 harbored NSI HIV-1 sequences that clustered separately. Both SI and NSI sequences that clustered with sequences from late-stage PBMC were present in other tissues, which may indicate that the presence of HIV-1 in those tissues is secondary to lymphocyte infiltration rather than to tissue tropism of HIV-1 itself. These data suggest that the viral reservoir may be limited, which will have important implications for the success of HIV-1 eradication.
Role of Baculovirus IE2 and Its RING Finger in Cell Cycle Arrest
Authors: Prikhod’ko Elena A. , Miller Lois K.
Published: December 2019

The
ie2
gene of
Autographa californica
nuclear polyhedrosis virus (Ac
M
NPV) is known to transactivate transient expression from viral promoters in a host cell-specific manner. We report that transfection of
Spodoptera frugiperda
(SF-21) cells with
ie2
was sufficient to arrest the cell cycle, resulting in the accumulation of enlarged cells with abnormally high DNA contents. By 72 h posttransfection, more than 50% of
ie2
-transfected cells had DNA contents greater than 4N. There was no evidence of mitotic spindle formation in these cells, and expression of
ie2
appeared to block cell cycle progression in S phase. Several
ie2
mutants were analyzed to further define the region of IE2 responsible for arresting the cell cycle. Analysis of these mutants showed that deletion of the RING finger motif eliminated the ability of IE2 to arrest the cell cycle but did not affect its ability to transactivate the
ie1
promoter. Moreover, mutation of a single conserved cysteine (C251) of the RING finger motif abolished the ability of IE2 to block cell cycle progression but had no apparent effect on its
trans
-regulatory activity. In contrast, a mutant of IE2 containing a deletion of residues 94 to 173 was able to block cell division but lacked
trans
-regulatory activity. Thus, the ability of IE2 to arrest the cell cycle depended on the integrity of the RING finger motif and was distinct from and independent of its ability to
trans
-activate the
ie1
promoter. IE2 also arrested the division of cells derived from other insect species,
Trichoplusia ni
(TN-368 and BTI-TN-5B1-4) and
Helicoverpa zea
(Hz-AM1).
Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses
Authors: Ares X. , Calamante G. , Cabral S. , Lodge J. , Hemenway P. , Beachy R. N. , Mentaberry A.
Published: December 2019

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (
Nicotiana tabacum
Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.
Viral E6-E7 Transcription in the Basal Layer of Organotypic Cultures without Apparent p21cip1 Protein Precedes Immortalization of Human Papillomavirus Type 16- and 18-Transfected Human Keratinocytes
Authors: Steenbergen Renske D. M. , Parker Jacqueline N. , Isern Sharon , Snijders Peter J. F. , Walboomers Jan M. M. , Meijer Chris J. L. M. , Broker Thomas R. , Chow Louise T.
Published: December 2019

Organotypic cultures of human keratinocytes provide a useful model system to study human papillomavirus (HPV)-host cell interactions. In this study, we analyzed organotypic cultures of two HPV type 16 (HPV16) (FK16A and FK16B)- and two HPV18 (FK18A and FK18B)-transfected keratinocyte cell lines through the process of immortalization in vitro. For FK16A and FK18B cells, passages of both mortal cells in their extended life span and subsequent immortal stages were studied. Mortal cells of FK16A and FK18B showed a morphology reminiscent of mild to moderate dysplasia, whereas in their immortal descendants, severely dysplastic features were observed. Immortal FK18A cells were mildly to moderately dysplastic, while FK16B cells were severely dysplastic. The increasing degrees of dysplasia were associated with a decreasing expression of differentiation markers cytokeratin 10 and profilaggrin. All raft cultures expressed E6-E7 mRNAs in the basal layer, while the amount of viral transcripts in the suprabasal cells was in general proportional to the degree of dysplasia. In all cases, E6-E7 transcription and dysplastic features were highly correlated with cellular proliferation, as assessed by Ki-67 (MIB-1) antigen expression. Moreover, high levels of E6-E7 transcription and expression of p21cip1 protein in the basal layer seemed to be mutually exclusive. We conclude that expression of E6-E7 in the basal cells associated with increased proliferation in the absence of detectable p21cip1 protein is apparently necessary but not sufficient for immortalization, or for the loss of terminal differentiation, for which yet to be discovered additional events are required. The model system described in this study provides a valuable tool to analyze alterations in viral transcription regulation during HPV-mediated cell transformation.
An Arenavirus RING (Zinc-Binding) Protein Binds the Oncoprotein Promyelocyte Leukemia Protein (PML) and Relocates PML Nuclear Bodies to the Cytoplasm
Authors: Borden Katherine L. B. , Campbell Dwyer Elizabeth J. , Salvato Maria S.
Published: December 2019

The promyelocytic leukemia protein (PML) forms nuclear bodies which are altered in some disease conditions. We report that the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV) influences the distribution of PML bodies. In cells infected with LCMV, the Z protein and PML form large bodies primarily in the cytoplasm. Transient transfection studies indicate that Z alone is sufficient to redistribute PML to the cytoplasm and that PML and Z colocalize. Coimmunoprecipitation studies show specific interaction between PML and Z proteins. A similar result was observed with a Z protein from another arenavirus, Lassa virus, suggesting that this is a general feature of theArenaviridae. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML and does not need the PML RING or the nuclear localization signal to colocalize. The Z protein acts dominantly to overcome the diffuse phenotype observed in several PML mutants. The interaction between PML and Z may influence certain unique characteristics of arenavirus infection.
Mechanism of Borna Disease Virus Entry into Cells
Authors: Gonzalez-Dunia Daniel , Cubitt Beatrice , de la Torre Juan Carlos
Published: December 2019

We have investigated the entry pathway of Borna disease virus (BDV). Virus entry was assessed by detecting early viral replication and transcription. Lysosomotropic agents (ammonium chloride, chloroquine, and amantadine), as well as energy depletion, prevented BDV infection, indicating that BDV enters host cells by endocytosis and requires an acidic intracellular compartment to allow membrane fusion and initiate infection. Consistent with this hypothesis, we observed that BDV-infected cells form extensive syncytia upon low-pH treatment. Entry of enveloped viruses into animal cells usually requires the membrane-fusing activity of viral surface glycoproteins (GPs). BDV GP is expressed as two products of 84 and 43 kDa (GP-84 and GP-43, respectively). We show here that only GP-43 is present at the surface of BDV-infected cells and therefore is likely the viral polypeptide responsible for triggering fusion events. We also present evidence that GP-43, which corresponds to the C terminus of GP-84, is generated by cleavage of GP-84 by the cellular protease furin. Hence, we propose that BDV GP-84 is involved in attachment to the cell surface receptor whereas its furin-cleaved product, GP-43, is involved in pH-dependent fusion after internalization of the virion by endocytosis.
Characterization of Provirus Clones of Simian Foamy Virus Type 1
Authors: Mergia Ayalew , Wu Min
Published: December 2019

We have cloned proviral DNA of simian foamy virus type 1 (SFV-1) from linear unintegrated DNA (pSFV-1). Transfection of pSFV-1 induces cytopathology in several cell lines with supernatants from the transfected cell culture containing infectious viral particles. Electron microscopy of the transfected cells revealed foamy virus particles. Deletion analysis of pSFV-1 indicated that the transcriptional transactivator (
tas
) gene located between
env
and the long terminal repeat is critical for virus replication, whereas the second open reading frame (ORF-2) in this region is dispensable. Although the
tas
and ORF-2 regions of foamy viruses have significantly diverged, the results presented here suggested that the gene products have similar functions. Recombinant pSFV-1 containing the
cat
gene was able to transduce the heterologous gene, indicating the utility of SFV-1 as a vector. An infectious clone of SFV-1 which is distantly related to the human foamy virus will provide a means to understand the biology of this unique group of viruses.
CCR5 Expression Correlates with Susceptibility of Maturing Monocytes to Human Immunodeficiency Virus Type 1 Infection
Authors: Naif Hassan M. , Li Shan , Alali Mohammed , Sloane Andrew , Wu Lijun , Kelly Mark , Lynch Garry , Lloyd Andrew , Cunningham Anthony L.
Published: December 2019

The chemokine receptor CCR5 and to a lesser extent CCR3 and CCR2b have been shown to serve as coreceptors for human immunodeficiency virus type 1 (HIV-1) entry into blood- or tissue-derived macrophages. Therefore, we examined the expression of the chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 as RNAs or as membrane-expressed antigens in monocytes maturing into macrophages and correlated these results with the susceptibility of macrophages to HIV-1 infection, as measured by their concentrations of extracellular p24 antigen and levels of intracellular HIV DNA by quantitative PCR. There was little change in levels of CCR1, CCR2b, and CCR5 RNAs. CCR3 RNA and surface antigen were undetectable throughout maturation of adherent monocytes over 10 days. CXCR4 RNA and membrane antigen were strongly expressed in newly adherent monocytes, but their levels declined at day 7. The amounts of CCR5 RNA remained stable, but the amounts of CCR5 antigen increased from undetectable to peak levels at day 7 and then declined slightly at day 10. Levels of susceptibility to laboratory (HIV-1BaL) and clinical strains of HIV-1 showed parallel kinetics, peaking at day 7 and then decreasing at days 10 to 14. The concordance of levels of HIV DNA and p24 antigen suggested that the changes in susceptibility with monocyte maturation were at or immediately after entry and correlated well with CCR5 expression and inversely with CXCR4 expression.
Apoptotic Regulation of T Cells and Absence of Immune Deficiency in Virus-Infected Gamma Interferon Receptor Knockout Mice
Authors: Lohman Barbara L. , Welsh Raymond M.
Published: December 2019

Acute viral infections often induce a transient period of immune deficiency in which the host’s T cells fail to proliferate in response to T-cell mitogens and fail to make an antigen-specific memory recall response. This has been associated with the enhanced sensitivity of these highly activated T cells to undergo apoptosis, or activation-induced cell death (AICD), upon T-cell receptor ligation. Here we show that gamma interferon receptor-deficient (IFN-γ R−/−) mice mount a T-cell response to lymphocytic choriomeningitis virus (LCMV) infection but fail to undergo the transient immune deficiency. Instead, their T cells were hyperproliferative and relatively, but not completely, resistant to AICD. The immune response returned to homeostasis, but with delayed kinetics, in parallel with delayed clearance of the virus. Wild-type mice receiving high doses of disseminating LCMV Clone 13 are known to undergo clonal exhaustion of their virus-specific cytotoxic T lymphocytes (CTL). To determine whether this process was mediated by AICD associated with IFN-γ or with Fas-Fas ligand interactions, LCMV-specific precursor CTL frequencies were examined in LCMV Clone 13-infected IFN-γ R−/−orlpr(Fas-deficient) mice. In both instances, viral persistence was established and CTL precursors were greatly eliminated. This finding indicates that clonal exhaustion of CTL does not require IFN-γ or Fas, even though both molecules influence AICD and the transient immune deficiency seen in the LCMV infection.
An Envelope Modification That Renders a Primary, Neutralization-Resistant Clade B Human Immunodeficiency Virus Type 1 Isolate Highly Susceptible to Neutralization by Sera from Other Clades
Authors: Stamatatos Leonidas , Cheng-Mayer Cecilia
Published: December 2019

SF162 is a primary (PR), non-syncytium-inducing, macrophagetropic human immunodeficiency virus type 1 (HIV-1) clade B isolate which is resistant to antibody-mediated neutralization. Deletion of the first or second hypervariable envelope gp120 region (V1 or V2 loop, respectively) of this virus does not abrogate its ability to replicate in peripheral blood mononuclear cells and primary macrophages, nor does it alter its coreceptor usage profile. The mutant virus with the V1 loop deletion, SF162ΔV1, remains as resistant to antibody-mediated neutralization as the wild-type virus SF162. In contrast, the mutant virus with the V2 loop deletion, SF162ΔV2, exhibits enhanced susceptibility to neutralization by certain monoclonal antibodies whose epitopes are located within the CD4-binding site and conserved regions of gp120. More importantly, SF162ΔV2 is now up to 170-fold more susceptible to neutralization than SF162 by sera collected from patients infected with clade B HIV-1 isolates. In addition, it becomes susceptible to neutralization by sera collected from patients infected with clade A, C, D, E, and F HIV-1 isolates. These findings suggest that the V2, but not the V1, loop of SF162 shields an as yet unidentified region of the HIV envelope rich in neutralization epitopes and that the overall structure of this region appears to be conserved among clade B, C, D, E, and F HIV-1 PR isolates.
Env-Independent Protection Induced by Live, Attenuated Simian Immunodeficiency Virus Vaccines
Authors: Gundlach Björn R. , Reiprich Stefan , Sopper Sieghart , Means Robert E. , Dittmer Ulf , Mätz-Rensing Kerstin , Stahl-Hennig Christiane , Überla Klaus
Published: December 2019

Live attenuated simian immunodeficiency viruses (SIV), such as
nef
deletion mutants, are the most effective vaccines tested in the SIV-macaque model so far. To modulate the antiviral immune response induced by live attenuated SIV vaccines, we had previously infected rhesus monkeys with a
nef
deletion mutant of SIV expressing interleukin 2 (SIV-IL2) (B. R. Gundlach, H. Linhart, U. Dittmer, S. Sopper, S. Reiprich, D. Fuchs, B. Fleckenstein, G. Hunsmann, S. Stahl-Hennig, and K. Überla, J. Virol. 71:2225–2232, 1997). In the present study, SIV-IL2-infected macaques and macaques infected with the
nef
deletion mutant SIVΔNU were challenged with pathogenic SIV 9 to 11 months postvaccination. In contrast to the results with naive control monkeys, no challenge virus could be isolated from the SIV-IL2- and SIVΔNU-infected macaques. However, challenge virus sequences could be detected by nested PCR in some of the vaccinated macaques. To determine the role of immune responses directed against Env of SIV, four vaccinated macaques were rechallenged with an SIV-murine leukemia virus (MLV) hybrid in which the
env
gene of SIV had been functionally replaced by the
env
gene of amphotropic MLV. All vaccinated macaques were protected from productive infection with the SIV-MLV hybrid in the absence of measurable neutralizing antibodies, while two naive control monkeys were readily infected. Since the SIV-MLV hybrid uses the MLV Env receptor Pit2 and not CD4 and a coreceptor for virus entry, chemokine inhibition and receptor interference phenomena were not involved in protection. These results indicate that the protective responses induced by live attenuated SIV vaccines can be independent of host immune reactions directed against Env.
Role of the TRAF Binding Site and NF-κB Activation in Epstein-Barr Virus Latent Membrane Protein 1-Induced Cell Gene Expression
Authors: Devergne Odile , McFarland Ellen Cahir , Mosialos George , Izumi Kenneth M. , Ware Carl F. , Kieff Elliott
Published: December 2019

In this study, we investigated the induction of cellular gene expression by the Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1). Previously, LMP1 was shown to induce the expression of ICAM-1, LFA-3, CD40, and EBI3 in EBV-negative Burkitt lymphoma (BL) cells and of the epidermal growth factor receptor (EGF-R) in epithelial cells. We now show that LMP1 expression also increased Fas and tumor necrosis factor receptor-associated factor 1 (TRAF1) in BL cells. LMP1 mediates NF-κB activation via two independent domains located in its C-terminal cytoplasmic tail, a TRAF-interacting site that associates with TRAF1, -2, -3, and -5 through a PXQXT/S core motif and a TRADD-interacting site. In EBV-transformed B cells or transiently transfected BL cells, significant amounts of TRAF1, -2, -3, and -5 are associated with LMP1. In epithelial cells, very little TRAF1 is expressed, and only TRAF2, -3, and -5, are significantly complexed with LMP1. The importance of TRAF binding to the PXQXT/S motif in LMP1-mediated gene induction was studied by using an LMP1 mutant that contains alanine point mutations in this motif and fails to associate with TRAFs. This mutant, LMP1(P204A/Q206A), induced 60% of wild-type LMP1 NF-κB activation and had approximately 60% of wild-type LMP1 effect on Fas, ICAM-1, CD40, and LFA-3 induction. In contrast, LMP1(P204A/Q206A) was substantially more impaired in TRAF1, EBI3, and EGF-R induction. Thus, TRAF binding to the PXQXT/S motif has a nonessential role in up-regulating Fas, ICAM-1, CD40, and LFA-3 expression and a critical role in up-regulating TRAF1, EBI3, and EGF-R expression. Further, D1 LMP1, an LMP1 mutant that does not aggregate failed to induce TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 expression confirming the essential role for aggregation in LMP1 signaling. Overexpression of a dominant form of IκBα blocked LMP1-mediated TRAF1, EBI3, Fas, ICAM-1, CD40, and LFA-3 up-regulation, indicating that NF-κB is an important component of LMP1-mediated gene induction from both the TRAF- and TRADD-interacting sites.
The Coxsackievirus-Adenovirus Receptor Protein Can Function as a Cellular Attachment Protein for Adenovirus Serotypes from Subgroups A, C, D, E, and F
Authors: Roelvink Peter W. , Lizonova Alena , Lee Jennifer G. M. , Li Yuan , Bergelson Jeffrey M. , Finberg Robert W. , Brough Douglas E. , Kovesdi Imre , Wickham Thomas J.
Published: December 2019

Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other’s cellular binding. Recombinant fiber knobs and
3
H-labeled Ad virions from serotypes representing all six subgroups (A to F) were used to determine whether the knobs cross-blocked the binding of virions from different subgroups. With the exception of subgroup B, all subgroup representatives cross-competed, suggesting that they use CAR as a cellular fiber receptor as well. This result was confirmed by showing that CAR, produced in a soluble recombinant form (sCAR), bound to nitrocellulose-immobilized virions from the different subgroups except subgroup B. Similar results were found for blotted fiber knob proteins. The subgroup F virus Ad41 has both short and long fibers, but only the long fiber bound sCAR. The sCAR protein blocked the attachment of all virus serotypes that bound CAR. Moreover, CHO cells expressing human CAR, in contrast to untransformed CHO cells, all specifically bound the sCAR-binding serotypes. We conclude therefore that Ad serotypes from subgroups A, C, D, E, and F all use CAR as a cellular fiber receptor.
Inactivation of Human Immunodeficiency Virus Type 1 Infectivity with Preservation of Conformational and Functional Integrity of Virion Surface Proteins
Authors: Rossio J. L. , Esser M. T. , Suryanarayana K. , Schneider D. K. , Bess J. W. , Vasquez G. M. , Wiltrout T. A. , Chertova E. , Grimes M. K. , Sattentau Q. , Arthur L. O. , Henderson L. E. , Lifson J. D.
Published: December 2019

Whole inactivated viral particles have been successfully used as vaccines for some viruses, but procedures historically used for inactivation can denature virion proteins. Results have been inconsistent, with enhancement of disease rather than protection seen in some notable instances following vaccination. We used the compound 2,2′-dithiodipyridine (aldrithiol-2; AT-2) to covalently modify the essential zinc fingers in the nucleocapsid (NC) protein of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) virions, thereby inactivating infectivity. The inactivated virus was not detectably infectious in vitro (up to 5 log units of inactivation). However, in contrast to virions inactivated by conventional methods such as heat or formalin treatment, viral and host cell-derived proteins on virion surfaces retained conformational and functional integrity. Thus, immunoprecipitation of AT-2-treated virions was comparable to precipitation of matched untreated virus, even when using antibodies to conformational determinants on gp120. AT-2 inactivated virions bound to CD4+target cells and mediated virus-induced, CD4-dependent “fusion from without” comparably to native virions. However, viral entry assays demonstrated that the viral life cycle of AT-2-treated virions was arrested before initiation of reverse transcription. The major histocompatibility complex (MHC) class II molecules on the surface of AT-2-treated virions produced from MHC class II-expressing cells retained the ability to support class II-dependent, superantigen-triggered proliferative responses by resting T lymphocytes. These findings indicate that inactivation via this method results in elimination of infectivity with preservation of conformational and functional integrity of virion surface proteins, including both virally encoded determinants and proteins derived from the host cells in which the virus was produced. Such inactivated virions should provide a promising candidate vaccine antigen and a useful reagent for experimentally probing the postulated involvement of virion surface proteins in indirect mechanisms of HIV-1 pathogenesis.
Processing of Proteinase Precursors and Their Effect on Hepatitis A Virus Particle Formation
Authors: Probst Christian , Jecht Monika , Gauss-Müller Verena
Published: December 2019

Proteolytic processing of the picornaviral polyprotein mediated by the differential action of virus-encoded proteinase(s) is pivotal to both RNA genome replication and capsid formation. Possibly to enlarge the array of viral proteins, picornaviral polyprotein processing results in intermediate and mature products which apparently have distinct functions within the viral life cycle. For hepatitis A virus (HAV), we report here on the autoproteolysis of precursor polypeptides comprising the only viral proteinase, 3C
pro
, and on their role in viral particle formation. Following transient expression of a nested set of 3C
pro
-containing proteins (P3, 3ABC, 3BCD, 3CD, 3BC, and 3C) in eukaryotic cells, the extent of processing was determined by analyzing the cleavage products. The 3C/3D site was more efficiently cleaved than those at the 3A/3B and 3B/3C sites, leading to the accumulation of the intermediate product 3ABC. In the absence of 3A from the precursor, cleavage at the 3B/3C site was further reduced and a switch to an alternative 3C/3D site was observed. Coexpression of various parts of P3 with the precursor of the viral structural proteins P1-2A showed that all 3C-containing intermediates cleaved P1-2A with almost equal efficiency; however, viral particles carrying the neutralizing epitope form much more readily in the presence of the complete P3 domain than with parts of it. These data support the notion that efficient liberation of structural proteins from P1-2A is necessary but not sufficient for productive HAV capsid formation and suggest that the polypeptides flanking 3C
pro
promote the assembly of viral particles.
Comparison of the Neurovirulence of a Vaccine and a Wild-Type Mumps Virus Strain in the Developing Rat Brain
Authors: Rubin Steven A. , Pletnikov Mikhail , Carbone Kathryn M.
Published: December 2019

Prior to the adoption of widespread vaccination programs, mumps virus was the leading cause of virus-induced central nervous system (CNS) disease. Mumps virus-associated CNS complications in vaccinees continue to be reported; outside the United States, some of these complications have been attributed to vaccination with insufficiently attenuated neurovirulent vaccine strains. The development of potentially neurovirulent, live, attenuated mumps virus vaccines stems largely from the lack of an animal model that can reliably predict the neurovirulence of mumps virus vaccine candidates in humans. The lack of an effective safety test with which to measure mumps virus neurovirulence has also hindered analysis of the neuropathogenesis of mumps virus infection and the identification of molecular determinants of neurovirulence. In this report we show, for the first time, that mumps virus infection of the neonatal rat leads to developmental abnormalities in the cerebellum due to cerebellar granule cell migration defects. The incidence of the cerebellar abnormalities and other neuropathological and clinical outcomes of mumps virus infection of the neonatal rat brain demonstrated the ability of this model to distinguish neurovirulent (Kilham) from nonneurovirulent (Jeryl Lynn) mumps virus strains. Thus, this neonatal rat model may prove useful in evaluating the neurovirulence potential of new live, attenuated vaccine strains and may also be of value in elucidating the molecular basis of mumps virus neurovirulence.
Protection against Lethal Encephalomyocarditis Virus Infection in the Absence of Serum-Neutralizing Antibodies
Authors: Neal Zane C. , Splitter Gary A.
Published: December 2019

Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHAβ−/−) mice, which are unable to mediate CD4+T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHAβ−/−mice inoculated with 104PFU of attenuated Mengo virus (vMC24) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8+T lymphocytes prior to lethal EMCV challenge ablated protection in vMC24-immunized RHAβ−/−mice. The CD8+T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8+T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8+T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC24-immunized RHAβ−/−mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC24-immunized RHAβ−/−mice survived when rechallenged 90 days later. The present study demonstrating CD8+T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC24-specific CD4+T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.
Expression of Measles Virus V Protein Is Associated with Pathogenicity and Control of Viral RNA Synthesis
Authors: Tober Christiane , Seufert Marion , Schneider Henriette , Billeter Martin A. , Johnston Ian C. D. , Niewiesk Stefan , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: December 2019

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314–322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V−), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats (
Sigmodon hispidus
) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.
Measles Virus Fusion Protein Is Palmitoylated on Transmembrane-Intracytoplasmic Cysteine Residues Which Participate in Cell Fusion
Authors: Caballero Monserrat , Carabaña Juan , Ortego Javier , Fernández-Muñoz Rafael , Celma María L.
Published: December 2019

[
3
H]palmitic acid was metabolically incorporated into the viral fusion protein (F) of Edmonston or freshly isolated measles virus (MV) during infection of human lymphoid or Vero cells. The uncleaved precursor F
0
and the F
1
subunit from infected cells and extracellular virus were both labeled, indicating that palmitoylation can take place prior to F
0
cleavage and that palmitoylated F protein was incorporated into virus particles. [
3
H]palmitic acid was released from F protein upon hydroxylamine or dithiothreitol treatment, indicating a thioester linkage. In cells transfected with the cloned MV F gene, in which the cysteines located in the intracytoplasmic and transmembrane domains (Cys 506, 518, 519, 520, and 524) were replaced by serine, a major reduction of [
3
H]palmitic acid incorporation was observed for F mutated at Cys 506 and, to a lesser extent, at Cys 518 and Cys 524. We also observed incorporation of [
3
H]palmitic acid in the F
1
subunit of canine distemper virus F protein. Cell fusion induced by cotransfection of cells with MV F and H (hemagglutinin) genes was significantly reduced after replacement of Cys 506 or Cys 519 with serine in the MV F gene. Transfection with the F gene with a mutation for Cys 518 abolished cell fusion, although less mutant protein was detected on the cell surface. These results suggest that the F protein transmembrane domain cysteines 506 and 518 participate in structures involved in cell fusion, possibly mediated by palmitoylation.
Structure and Distribution of Endogenous Nonecotropic Murine Leukemia Viruses in Wild Mice
Authors: Tomonaga Keizo , Coffin John M.
Published: December 2019

Virtually all of our present understanding of endogenous murine leukemia viruses (MLVs) is based on studies with inbred mice. To develop a better understanding of the interaction between endogenous retroviruses and their hosts, we have carried out a systematic investigation of endogenous nonecotropic MLVs in wild mice. Species studied included four major subspecies of
Mus musculus
(
M. m. castaneus
,
M. m. musculus
,
M. m. molossinus
, and
M. m. domesticus
) as well as four common inbred laboratory strains (AKR/J, HRS/J, C3H/HeJ, and C57BL/6J). We determined the detailed distribution of nonecotropic proviruses in the mice by using both
env
- and long terminal repeat (LTR)-derived oligonucleotide probes specific for the three different groups of endogenous MLVs. The analysis indicated that proviruses that react with all of the specific probes are present in most wild mouse DNAs tested, in numbers varying from 1 or 2 to more than 50. Although in common inbred laboratory strains the linkage of group-specific sequences in
env
and the LTR of the proviruses is strict, proviruses which combine
env
and the LTR sequences from different groups were commonly observed in the wild-mouse subspecies. The “recombinant” nonecotropic proviruses in the mouse genomes were amplified by PCR, and their genetic and recombinant natures were determined. These proviruses showed extended genetic variation and provide a valuable probe for study of the evolutionary relationship between MLVs and the murine hosts.
Mx1-Based Resistance to Thogoto Virus in A2G Mice Is Bypassed in Tick-Mediated Virus Delivery
Authors: Dessens Johannes T. , Nuttall Patricia A.
Published: December 2019

The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruses, including Thogoto virus. Thus,
Mx1
+
A2G mice are apparently resistant to infection following needle- or tick-borne virus challenge. However, tick-borne challenge and, to a lesser degree, injection of virus mixed with tick salivary gland extract resulted in virus transmission to uninfected ticks feeding on the A2G mice. The data indicate that immunomodulatory components in tick saliva can overcome a natural antiviral mechanism.
The Major Open Reading Frame of the β2.7 Transcript of Human Cytomegalovirus: In Vitro Expression of a Protein Posttranscriptionally Regulated by the 5′ Region
Authors: Bergamini Giovanna , Reschke Marko , Battista Maria Concetta , Boccuni Maria Cristina , Campanini Fabio , Ripalti Alessandro , Landini Maria Paola
Published: December 2019

β2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5′ sequence of the transcript, which harbors two short upstream ORFs.
Optimal Induction of Hepatitis C Virus Envelope-Specific Immunity by Bicistronic Plasmid DNA Inoculation with the Granulocyte-Macrophage Colony-Stimulating Factor Gene
Authors: Lee Seung Woo , Cho Jae Ho , Sung Young Chul
Published: December 2019

In this study, we have constructed various DNA vaccine vectors that carried hepatitis C virus (HCV) envelope genes without and with the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in several different ways. In Buffalo rats that received plasmids carrying the HCV envelope genes, which encode envelope proteins E1 and E2, both antibody and lymphoproliferative responses against these proteins were induced. These responses were greatly enhanced by the codelivery of the GM-CSF gene. In particular, inoculation with a bicistronic plasmid that independently expressed the GM-CSF gene and the envelope genes in the same construct generated the highest antibody titers and significantly increased lymphoproliferative responses against these proteins. Moreover, strong antibody responses to homologous and heterologous hypervariable region 1 peptides were elicited in the immunized rats.
High-Mobility Group 1/2 Proteins Are Essential for Initiating Rolling-Circle-Type DNA Replication at a Parvovirus Hairpin Origin
Authors: Cotmore Susan F. , Tattersall Peter
Published: December 2019

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5′ end of the DNA at the nick and providing a 3′ hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5′) MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3′) viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.
Ribosomal S27a Coding Sequences Upstream of Ubiquitin Coding Sequences in the Genome of a Pestivirus
Authors: Becher Paul , Orlich Michaela , Thiel Heinz-Jürgen
Published: December 2019

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3′ part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5′ region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.
Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity
Authors: Yang Decheng , Hwang Dorothy , Qiu Zhiyong , Gillam Shirley
Published: December 2019

Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.