bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of Monoclonal Antibodies Directed against Hepatitis B Virus Polymerase Protein
Authors: zu Putlitz Jasper , Lanford Robert E. , Carlson Rolf I. , Notvall Lena , de la Monte Suzanne M. , Wands Jack R.
Published: December 2019

Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.
Genetic and Phenotypic Changes Accompanying the Emergence of Epizootic Subtype IC Venezuelan Equine Encephalitis Viruses from an Enzootic Subtype ID Progenitor
Authors: Wang Eryu , Barrera Roberto , Boshell Jorge , Ferro Cristina , Freier Jerome E. , Navarro Juan Carlos , Salas Rosalba , Vasquez Clovis , Weaver Scott C.
Published: December 2019

Recent studies have indicated that epizootic Venezuelan equine encephalitis (VEE) viruses can evolve from enzootic, subtype ID strains that circulate continuously in lowland tropical forests (A. M. Powers, M. S. Oberste, A. C. Brault, R. Rico-Hesse, S. M. Schmura, J. F. Smith, W. Kang, W. P. Sweeney, and S. C. Weaver, J. Virol. 71:6697–6705, 1997). To identify mutations associated with the phenotypic changes leading to epizootics, we sequenced the entire genomes of two subtype IC epizootic VEE virus strains isolated during a 1992–1993 Venezuelan outbreak and four sympatric, subtype ID enzootic strains closely related to the predicted epizootic progenitor. Analysis by maximum-parsimony phylogenetic methods revealed 25 nucleotide differences which were predicted to have accompanied the 1992 epizootic emergence; 7 of these encoded amino acid changes in the nsP1, nsP3, capsid, and E2 envelope glycoprotein, and 2 were mutations in the 3′ untranslated genome region. Comparisons with the genomic sequences of IAB and other IC epizootic VEE virus strains revealed that only one of the seven amino acid changes associated with the 1992 emergence, a threonine-to-methionine change at position 360 of the nsP3 protein, accompanied another VEE virus emergence event. Two changes in the E2 envelope glycoprotein region believed to include the major antigenic determinants, both involving replacement of uncharged residues with arginine, are also candidates for epizootic determinants.
Polyvalent Rev Decoys Act as Artificial Rev-Responsive Elements
Authors: Symensma Tonia L. , Baskerville Scott , Yan Amy , Ellington Andrew D.
Published: December 2019

Interactions between Rev and the Rev-responsive element (RRE) control the order, rate, and extent of gene expression in human immunodeficiency virus type 1. Rev decoys may therefore prove to be useful RNA therapeutics for the treatment of AIDS. To improve upon the current generation of Rev decoys that bind single Rev molecules, it would be useful to generate polyvalent Rev decoys that could bind multiple Rev molecules. J. Kjems and P. A. Sharp (J. Virol. 67:4769–4776, 1993) originally constructed functional polyvalent Rev decoys, but the structural context of these polyvalent decoys remains unclear, and it has been argued that the individual decoys were either structurally discrete (Kjems and Sharp, J. Virol. 67:4769–4776, 1993) or were part of an extended helix (R. W. Zemmel et al., Mol. Biol. 258:763–777, 1996). To resolve the differences between these models, we have designed and synthesized concatemers of Rev-binding elements (RBEs) that fold to form multiple, discrete, high-affinity Rev-binding sites. We find that the concatenated RBEs can facilitate the cytoplasmic transport of viral mRNAs and therefore likely bind multiple Rev molecules. These artificial RREs may simultaneously sequester Rev and hinder access to the cellular transport machinery.
Molecular Cloning and Phylogenetic Analysis of Human Immunodeficiency Virus Type 1 Subtype C: a Set of 23 Full-Length Clones from Botswana
Authors: Novitsky Vladimir A. , Montano Monty A. , McLane Mary F. , Renjifo Boris , Vannberg Fredrik , Foley Brian T. , Ndung’u Thumbi P. , Rahman Mafizur , Makhema Moeketsi J. , Marlink Richard , Essex Max
Published: December 2019

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9.1% versus 6.5 and 4.3%, respectively;
P 
< 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.
Effect of Bovine Papillomavirus E2 Protein-Specific Monoclonal Antibodies on Papillomavirus DNA Replication
Authors: Kurg Reet , Parik Jüri , Juronen Erkki , Sedman Tiina , Abroi Aare , Liiv Ingrid , Langel Ülo , Ustav Mart
Published: December 2019

The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and transactivation-defective but not the competent conformation of the transactivation domain of the E2 protein. MAb 5H4 prevented efficiently the formation of E2-DNA as well as E2-dependent E1-E2-origin complexes and also dissociated preformed complexes in a concentration-dependent manner. Cotransfection of several MAbs with the BPV-1 minimal origin plasmid pUCAlu into CHO4.15 cells resulted in a dose-dependent inhibition of replication. Inhibition of replication by MAb 5H4 and the Fab′ fragment of 5H4 correlated with their ability to dissociate the E2 protein from the DNA. MAb 3F12 and MAbs 1H10 and 1E4, directed against the hinge region, were also capable of inhibiting BPV-1 origin replication in CHO4.15 cells. However, the Fab′ fragments of 1H10 and 3F12 had no effect in the transient replication assay. These data suggest that MAbs directed against the hinge region sterically hinder the inter- or intramolecular interactions required for the replication activity of the E2 protein.
Trichostatin A Up-Regulates Human Papillomavirus Type 11 Upstream Regulatory Region-E6 Promoter Activity in Undifferentiated Primary Human Keratinocytes
Authors: Zhao Wei , Noya Francisco , Chen Wen Yong , Townes Tim M. , Chow Louise T. , Broker Thomas R.
Published: December 2019

Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using the
lacZ
reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the
lacZ
reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-
lacZ
and LTR-
lacZ
in murine retrovirus producer cell lines of fibroblast origin.
Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein
Authors: Zavorotinskaya Tatiana , Albritton Lorraine M.
Published: December 2019

Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.
In Vivo Replication of Recombinant Murine Cytomegalovirus Driven by the Paralogous Major Immediate-Early Promoter-Enhancer of Human Cytomegalovirus
Authors: Grzimek Natascha K. A. , Podlech Jürgen , Steffens Hans-Peter , Holtappels Rafaela , Schmalz Susanne , Reddehase Matthias J.
Published: December 2019

Transcription of the major immediate-early (MIE) genes of cytomegaloviruses (CMV) is driven by a strong promoter-enhancer (MIEPE) complex. Transactivator proteins encoded by these MIE genes are essential for productive infection. Accordingly, the MIEPE is a crucial control point, and its regulation by activators and repressors is pertinent to virus replication. Since the MIEPE contains multiple regulatory elements, it was reasonable to assume that specific sequence motifs are irreplaceable for specifying the cell-type tropism and replication pattern. Recent work on murine CMV infectivity (A. Angulo, M. Messerle, U. H. Koszinowski, and P. Ghazal, J. Virol. 72:8502–8509, 1998) has documented the proposed enhancing function of the enhancer in that its resection or its replacement by a nonregulatory stuffer sequence resulted in a significant reduction of infectivity, even though replication competence was maintained by a basal activity of the spared authentic MIE promoter. Notably, full capacity for productive in vitro infection of fibroblasts was restored in recombinant viruses by the human CMV enhancer. Using two-color in situ hybridization with MIEPE-specific polynucleotide probes, we demonstrated that a murine CMV recombinant in which the complete murine CMV MIEPE is replaced by the paralogous human CMV core promoter and enhancer (recombinant virus mCMVhMIEPE) retained the potential to replicate in vivo in all tissues relevant to CMV disease. Notably, mCMVhMIEPE was also found to replicate in the liver, a site at which transgenic hCMV MIEPE is silenced. We conclude that productive in vivo infection with murine CMV does not strictly depend on a MIEPE type-specific regulation.
Immunohistochemical Analysis, Human Papillomavirus DNA Detection, Hormonal Manipulation, and Exogenous Gene Expression of Normal and Dysplastic Human Cervical Epithelium in Severe Combined Immunodeficiency Mice
Authors: Taylor Jason A. , Tewari Krishnansu , Liao Shu Y. , Hughes Christopher C. W. , Villarreal Luis P.
Published: December 2019

The cervical squamocolumnar junction of normal and dysplastic human xenografts was maintained in SCID-
beige
mice. Dysplastic tissue maintained a dysplastic morphology, irregular pattern of keratin expression, elevated levels of cellular proliferation, and human papillomavirus type 16 and/or type 18 DNA. Hyperplastic changes of normal xenografts occurred via high-dose estrogen exposure, and through recombinant adenovirus infection, the introduction and stable expression of an exogenous gene was accomplished.
Characterization of Monoclonal Antibodies Raised against the Latent Nuclear Antigen of Human Herpesvirus 8
Authors: Kellam Paul , Bourboulia Dimitra , Dupin Nicolas , Shotton Chris , Fisher Cyril , Talbot Simon , Boshoff Chris , Weiss Robin A.
Published: December 2019

Human herpesvirus 8 (HHV-8; also designated Kaposi’s sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi’s sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA–glutathione
S
-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.
Role of the ATP-Binding Domain of the Human Papillomavirus Type 11 E1 Helicase in E2-Dependent Binding to the Origin
Authors: Titolo Steve , Pelletier Alex , Sauvé Frédéric , Brault Karine , Wardrop Elizabeth , White Peter W. , Amin Anthony , Cordingley Michael G. , Archambault Jacques
Published: December 2019

Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro. This region of E1 encompasses the ATP-binding domain. Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin. Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction. The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro. This stimulation was maximal at the physiological temperature (37°C) and did not require ATP hydrolysis. In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain. These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.
Genome Structure and Expression of the ev/J Family of Avian Endogenous Viruses
Authors: Ruis Brian L. , Benson Scott J. , Conklin Kathleen F.
Published: December 2019

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157–10164, 1998). Based on the fact that the endogenous subgroup J-related
env
genes were associated with long terminal repeats (LTRs), we concluded that these LTR-
env
sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the
pol
gene and various amounts of
gag
and
env
gene sequences. Sequencing of the
gag
genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J
gag
gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.
Effects of Soluble CD4 on Simian Immunodeficiency Virus Infection of CD4-Positive and CD4-Negative Cells
Authors: Schenten Dominik , Marcon Luisa , Karlsson Gunilla B. , Parolin Cristina , Kodama Toshiaki , Gerard Norma , Sodroski Joseph
Published: December 2019

A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by simian immunodeficiency virus (SIV) and human immunodeficiency virus. We investigated the basis for these varying effects by studying the entry of three SIV isolates into CD4-positive and CD4-negative cells expressing different chemokine receptors. Infection of CD4-negative cells depended upon the viral envelope glycoproteins and upon the chemokine receptor, with CCR5 and gpr15 being more efficient than STRL33. Likewise, enhancement of infection by sCD4 was observed when CCR5- and gpr15-expressing target cells were used but not when those expressing STRL33 were used. The sCD4-mediated enhancement of virus infection of CD4-negative, CCR5-positive cells was related to the sCD4-induced increase in binding of the viral gp120 envelope glycoprotein to CCR5. Inhibitory effects of sCD4 could largely be explained by competition for virus attachment to cellular CD4 rather than other detrimental effects on virus infectivity (e.g., disruption of the envelope glycoprotein spike). Consistent with this, the sCD4-activated SIV envelope glycoprotein intermediate on the virus was long-lived. Thus, the net effect of sCD4 on SIV infectivity appears to depend upon the degree of enhancement of chemokine receptor binding and upon the efficiency of competition for cellular CD4.
Isolation and Characterization of a Hantavirus from
Lemmus sibiricus
: Evidence for Host Switch during Hantavirus Evolution
Authors: Vapalahti Olli , Lundkvist Åke , Fedorov Vadim , Conroy Christopher J. , Hirvonen Sirpa , Plyusnina Angelina , Nemirov Kirill , Fredga Karl , Cook Joseph A. , Niemimaa Jukka , Kaikusalo Asko , Henttonen Heikki , Vaheri Antti , Plyusnin Alexander
Published: December 2019

A novel hantavirus, first detected in Siberian lemmings (
Lemmus sibiricus
) collected near the Topografov River in the Taymyr Peninsula, Siberia (A. Plyusnin et al., Lancet 347:1835–1836, 1996), was isolated in Vero E6 cells and in laboratory-bred Norwegian lemmings (
Lemmus lemmus
). The virus, named Topografov virus (TOP), was most closely related to Khabarovsk virus (KBR) and Puumala viruses (PUU). In a cross focus reduction neutralization test, anti-TOP
Lemmus
antisera showed titers at least fourfold higher with TOP than with other hantaviruses; however, a rabbit anti-KBR antiserum neutralized TOP and KBR at the same titer. The TOP M segment showed 77% nucleotide and 88% amino acid identity with KBR and 76% nucleotide and 82% amino acid identity with PUU. However, the homology between TOP and the KBR S segment was disproportionately higher: 88% at the nucleotide level and 96% at the amino acid level. The 3′ noncoding regions of KBR and the TOP S and M segments were alignable except for 113- and 58-nucleotide deletions in KBR. The phylogenetic relationships of TOP, KBR, and PUU and their respective rodent carriers suggest that an exceptional host switch took place during the evolution of these viruses; while TOP and KBR are monophyletic, the respective rodent host species are only distantly related.
La Autoantigen Specifically Recognizes a Predicted Stem-Loop in Hepatitis B Virus RNA
Authors: Heise Tilman , Guidotti Luca G. , Chisari Francis V.
Published: December 2019

We recently identified three nuclear proteins (p45, p39, and p26) that bind to a 91-nucleotide (nt) RNA element between nt 1243 and 1333 in hepatitis B virus (HBV) RNA, and we showed that these proteins and HBV RNA are regulated coordinately by gamma interferon and tumor necrosis factor alpha. Purification and sequence analysis of tryptic peptides obtained from p39 revealed sequence homology to the mouse La protein. Immunoprecipitation experiments showed that p45, p39, and p26 were recognized by anti-La-specific antiserum, indicating that p45 is the full-length La protein and that p39 and p26 are likely to be proteolytic La cleavage products. Furthermore, in competition experiments we found that all three La proteins bind, in a phosphorylation-dependent manner, to the same predicted stem-loop structure located between nt 1275 and 1291 of HBV, withKds of approximately 1.0 nM. Collectively, these results support the notion that the La protein may contribute to HBV RNA stability, constitutively and in response to inflammatory cytokines.
Discrimination between Sialic Acid-Containing Receptors and Pseudoreceptors Regulates Polyomavirus Spread in the Mouse
Authors: Bauer Paul H. , Cui Cunqi , Stehle Thilo , Harrison Stephen C. , DeCaprio James A. , Benjamin Thomas L.
Published: December 2019

Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal α-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.
Role of the Membrane-Proximal Domain in the Initial Stages of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein-Mediated Membrane Fusion
Authors: Muñoz-Barroso Isabel , Salzwedel Karl , Hunter Eric , Blumenthal Robert
Published: December 2019

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315–323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.
The GDVII Strain of Theiler’s Virus Spreads via Axonal Transport
Authors: Martinat Cécile , Jarousse Nadine , Prévost Marie-Christine , Brahic Michel
Published: December 2019

Following intracerebral inoculation, the DA strain of Theiler’s virus sequentially infects neurons in the gray matter and glial cells in the white matter of the spinal cord. It persists in the latter throughout the life of the animal. Several observations suggest that the virus spreads from the gray to the white matter by axonal transport. In contrast, the neurovirulent GDVII strain causes a fatal encephalitis with lytic infection of neurons. It does not infect the white matter of the spinal cord efficiently and does not persist in survivors. The inability of this virus to infect the white matter could be due to a defect in axonal transport. Using footpad inoculations, we showed that the GDVII strain is, in fact, transported in axons. Transport was prevented by sectioning the sciatic nerve. The kinetics of transport and experiments using colchicine suggested that the virus uses microtubule-associated fast axonal transport. Our results show that a cardiovirus can spread by fast axonal transport and suggest that the inability of the GDVII strain to infect the white matter is not due to a defect in axonal transport.
Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus
Authors: Chang Yu-Shiu , Liao Ching-Len , Tsao Chang-Huei , Chen Mei-Chieh , Liu Chiu-I , Chen Li-Kuang , Lin Yi-Ling
Published: December 2019

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or α-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducibleEscherichia colisystem, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or β-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.
Myxoma Virus Serp2 Is a Weak Inhibitor of Granzyme B and Interleukin-1β-Converting Enzyme In Vitro and Unlike CrmA Cannot Block Apoptosis in Cowpox Virus-Infected Cells
Authors: Turner Peter C. , Sancho M. Carmen , Thoennes S. R. , Caputo A. , Bleackley R. C. , Moyer Richard W.
Published: December 2019

The Serp2 protein encoded by the leporipoxvirus myxoma virus is essential for full virulence (F. Messud-Petit, J. Gelfi, M. Delverdier, M. F. Amardeilh, R. Py, G. Sutter, and S. Bertagnoli, J. Virol. 72:7830–7839, 1998) and, like crmA of cowpox virus (CPV), is reported to inhibit the interleukin-1β-converting enzyme (ICE, caspase-1) (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860–5866, 1996). Serp2 and CrmA both contain Asp at the P1 position within the serpin reactive site loop and yet are only 35% identical overall. Serp2 protein was cleaved by ICE but, unlike CrmA, did not form a stable complex with ICE that was detectable by native gel electrophoresis. Attempts to covalently cross-link ICE-serpin inhibitory complexes were successful with CrmA, but no complex between ICE and Serp2 was visible after cross-linking. Purified His
10
-tagged Serp2 protein was a relatively poor inhibitor of ICE, with a

K
i

of 80 nM compared to 4 pM for CrmA. Serp2 protein resembled CrmA in that a stable complex with the serine proteinase granzyme B was detectable after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, Serp2 was less effective at inhibiting granzyme B activity (

K
i

= 420 nM) than CrmA (

K
i

= 100 nM). Finally, Serp2 was tested for the ability to replace CrmA and inhibit apoptosis in LLC-PK1 cells infected with a CPV recombinant deleted for CrmA but expressing Serp2. Unlike wild-type-CPV-infected cells, apoptosis was readily observed in cells infected with the recombinant virus, as indicated by the induction of both nuclear fragmentation and caspase-mediated cleavage of DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl coumarin)]. These results indicate that Serp2 is unable to functionally substitute for CrmA within the context of CPV and that the inhibition spectra for Serp2 and CrmA are distinct.
Establishment of a Seronegative Human T-Cell Leukemia Virus Type 1 (HTLV-1) Carrier State in Rats Inoculated with a Syngeneic HTLV-1-Immortalized T-Cell Line Preferentially Expressing Tax
Authors: Koya Yoshihiro , Ohashi Takashi , Kato Hirotomo , Hanabuchi Shino , Tsukahara Tomonori , Takemura Fumiyo , Etoh Ken-ichiro , Matsuoka Masao , Fujii Masahiro , Kannagi Mari
Published: December 2019

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies in a small percentage of the population infected with the virus after a long carrier state. In the present study, we established a seronegative HTLV-1 carrier state in rats inoculated with a newly established HTLV-1-infected rat T cell line, FPM1. FPM1 originated from rat thymocytes cocultured with a human HTLV-1 producer, MT-2 cells, and expressed rat CD4, CD5, CD25, and HTLV-1 Tax. However, FPM1 scarcely expressed other major HTLV-1 structural proteins and failed to induce typical antibody responses against HTLV-1 in inoculated rats. In contrast, control rats inoculated with MT-2 cells generated significant levels of anti-HTLV-1 antibodies. HTLV-1 proviruses were detected in peripheral blood cells of syngeneic rats inoculated with FPM1 for more than 1 year. Analysis of the flanking region of HTLV-1 provirus integrated into host cells suggested that FPM1 cells remained in these animals over a relatively long period of time. However, a similar seronegative HTLV-1 carrier state was induced in the rats inoculated with mitomycin C-treated FPM1 cells and also in FPM1-inoculated allogeneic rats, suggesting that FPM1 could also transmit HTLV-1 into host cells in vivo. Our findings indicated that (i) HTLV-1-immortalized T cells which preferentially express HTLV-1 Tax persisted in vivo but failed to induce any diseases in immunocompetent syngeneic rats and that (ii) suboptimal levels of HTLV-1 for antibody responses allowed the establishment of persistent HTLV-1 infection.
A Putative Cell Surface Receptor for Anemia-Inducing Feline Leukemia Virus Subgroup C Is a Member of a Transporter Superfamily
Authors: Tailor Chetankumar S. , Willett Brian J. , Kabat David
Published: December 2019

Domestic cats infected with the horizontally transmitted feline leukemia virus subgroup A (FeLV-A) often produce mutants (termed FeLV-C) that bind to a distinct cell surface receptor and cause severe aplastic anemia in vivo and erythroblast destruction in bone marrow cultures. The major determinant for FeLV-C-induced anemia has been mapped to a small region of the surface envelope glycoprotein that is responsible for its receptor binding specificity. Thus, erythroblast destruction may directly or indirectly result from FeLV-C binding to its receptor. To address these issues, we functionally cloned a putative cell surface receptor for FeLV-C (FLVCR) by using a human T-lymphocyte cDNA library in a retroviral vector. Expression of the 2.0-kbp FLVCR cDNA in naturally resistant Swiss mouse fibroblasts and Chinese hamster ovary cells caused substantial susceptibility to FeLV-C but no change in susceptibilities to FeLV-B and other retroviruses. The predicted FLVCR protein contains 555 amino acids and 12 hydrophobic potential membrane-spanning sequences. Database searches indicated that FLVCR is a member of the major-facilitator superfamily of transporters and implied that it may transport an organic anion. RNA blot analyses showed that FLVCR mRNA is expressed in multiple hematopoietic lineages rather than specifically in erythroblasts. These results suggest that the targeted destruction of erythroblasts by FeLV-C may derive from their greater sensitivity to this virus rather than from a preferential susceptibility to infection.
Comparison of Genetic Variability at Multiple Loci across the Genomes of the Major Subtypes of Kaposi’s Sarcoma-Associated Herpesvirus Reveals Evidence for Recombination and for Two Distinct Types of Open Reading Frame K15 Alleles at the Right-Hand End
Authors: Poole Lynn J. , Zong Jian-Chao , Ciufo Dolores M. , Alcendor Donald J. , Cannon Jennifer S. , Ambinder Richard , Orenstein Jan M. , Reitz Marvin S. , Hayward Gary S.
Published: December 2019

Kaposi’s sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0.35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized “orphan” region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results suggest that an original recombination event with a related primate virus from an unknown source introduced exogenous right-hand side ORF-K15(M) sequences into an ancient M form of HHV8, followed by eventual acquisition into the subtype C lineage of the modern P-form of the HHV8 genome and subsequent additional, more recent transfers by homologous recombination events into several subtype A and B lineages as well.
The Baculovirus PE38 Protein Augments Apoptosis Induced by Transactivator IE1
Authors: Prikhod’ko Elena A. , Miller Lois K.
Published: December 2019

While studying apoptosis induced by baculovirus transactivator IE1 in SF-21 cells, we found that the levels of IE1-induced apoptosis were increased approximately twofold upon cotransfection with the baculovirus early
pe38
gene. However, no apoptotic activity was observed in cells transfected with
pe38
alone, even when placed under the control of a constitutive promoter. Thus,
pe38
was able to augment IE1-induced apoptosis but was unable to induce apoptosis when expressed in SF-21 cells alone. PE38, the full-length product of
pe38
, is a nuclear protein with RING finger and leucine zipper motifs. Deletion of the amino-terminal region, which contains a putative nuclear localization motif, resulted in cytoplasmic localization of the PE38 mutants. These N-terminal deletion mutants were unable to enhance IE1-induced apoptosis. Mutation of a single conserved leucine (L242) of the leucine zipper motif also eliminated the ability of PE38 to augment apoptosis induced by IE1. In contrast, PE38 mutants with alanine substitutions for conserved cysteine residues (C109 or C138) of the RING finger motif were able to increase IE1-induced apoptosis to levels equivalent to those of wild-type PE38. We propose that PE38 is one of at least two viral factors which collectively evoke a cellular apoptotic response during baculovirus infection.
The Murine Cytomegalovirus Chemokine Homolog, m131/129, Is a Determinant of Viral Pathogenicity
Authors: Fleming Peter , Davis-Poynter Nicholas , Degli-Esposti Mariapia , Densley Eloise , Papadimitriou John , Shellam Geoffrey , Farrell Helen
Published: December 2019

Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3′ end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Δm131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Δm131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Δm131/129 was reduced compared with that of wt MCMV. Furthermore, the Δm131/129 mutants failed to establish a high-titer infection in the salivary glands. These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Δm131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4+CD8+phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells.
Analysis of Cellular Mutants Resistant to Theiler’s Virus Infection: Differential Infection of L929 Cells by Persistent and Neurovirulent Strains
Authors: Jnaoui Karima , Michiels Thomas
Published: December 2019

Theiler’s murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse and belongs to the
Picornaviridae
family. TMEV strains are divided into two subgroups on the basis of their pathogenicity. The first group contains two neurovirulent strains, FA and GDVII, which cause a rapid fatal encephalitis. The second group includes persistent strains, like DA and BeAn, which produce a biphasic neurological disease in susceptible mice. Persistence of these viruses in the white matter of the spinal cord leads to chronic inflammatory demyelination. L929 cells, which are susceptible to TMEV infection, were subjected to physicochemical mutagenesis. Cellular clones that became resistant to TMEV infection were selected by viral infection. Three such mutants resistant to strain GDVII were characterized to determine the step of the virus cycle that was inhibited. The mutation present in one of these mutant cell lines inhibited, by more than 1,000-fold, the entry of strain GDVII but hardly decreased infection by strain DA. In the two other cellular mutants, replication of the viral genome was slowed down. Interestingly, one of these mutant cell lines resisted infection by both the persistent and neurovirulent strains while the second cell line resisted infection by strain GDVII but remained susceptible to the persistent virus. These results show that although they have 95% identity at the amino acid sequence level, neurovirulent and persistent viruses use partly distinct pathways for both entry into cells and genome replication.
RhoA Interacts with the Fusion Glycoprotein of Respiratory Syncytial Virus and Facilitates Virus-Induced Syncytium Formation
Authors: Pastey Manoj K. , Crowe James E. , Graham Barney S.
Published: December 2019

The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation.
Equine Herpesvirus 1 Gene 12 Can Substitute for vmw65 in the Growth of Herpes Simplex Virus (HSV) Type 1, Allowing the Generation of Optimized Cell Lines for the Propagation of HSV Vectors with Multiple Immediate-Early Gene Defects
Authors: Thomas S. K. , Lilley C. E. , Latchman D. S. , Coffin R. S.
Published: December 2019

Herpes simplex virus (HSV) has often been suggested for development as a vector, particularly for the nervous system. Considerable evidence has shown that for use of HSV as a vector, immediate-early (IE) gene expression must be minimized or abolished, otherwise such vectors are likely to be highly cytotoxic. Mutations of vmw65 which abolish IE promoter transactivating activity may also be included to reduce IE gene expression generally. However, when vmw65 mutations are combined with an IE gene deletion, such viruses are hard to propagate, even on cells which otherwise complement the IE gene deletion effectively. We have found that vmw65 mutants can be effectively grown on cell lines expressing equine herpesvirus 1 gene 12, a non-HSV homologue of vmw65 with little sequence similarity to its HSV counterpart. This prevents repair by homologous recombination of vmw65 mutations in the virus, which would occur if mutations were complemented by vmw65 itself. The gene 12 protein is not packaged into HSV virions, which is important if viruses grown on such cells are to be used as vectors. These results not only further strengthen the evidence for direct functional homology between and similar modes of action of the two proteins but have allowed the generation of gene 12-containing cell lines in which ICP4 and ICP27 expression is induced by virus infection (probably by ICP0) and which give efficient growth of viruses deficient in ICP27, ICP4, and vmw65 (the viruses also have ICP34.5/ORFP deleted). Efficient growth of such viruses has not previously been possible. As these viruses are highly deficient in IE gene expression generally, such virus-cell line combinations may provide an alternative to HSV vectors with deletions of all four of the regulatory IE genes which, for optimal growth, require cell lines containing all four IE genes but which are hard to generate due to the intrinsic cytotoxicity of each of the proteins.