bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic T Cells from Human Immunodeficiency Virus Type 2-Infected Patients Frequently Cross-React with Different Human Immunodeficiency Virus Type 1 Clades
Authors: Bertoletti Antonio , Cham Fatim , McAdam Stephen , Rostron Tim , Rowland-Jones Sarah , Sabally Sehu , Corrah Tumani , Ariyoshi Koya , Whittle Hilton
Published: December 2019

Knowledge of immune mechanisms responsible for the cross-protection between highly divergent viruses such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 may contribute to an understanding of whether virus variability may be overcome in the design of vaccine candidates which are broadly protective across the HIV subtypes. We demonstrate that despite the significant difference in virus amino acid sequence, the majority of HIV-2-infected individuals with different HLA molecules possess a dominant cytotoxic T-cell response which is able to recognize HIV-1 Gag protein. Furthermore, HLA-B5801-positive subjects show broad cross-recognition of HIV-1 subtypes since they mounted a T-cell response that tolerated extensive amino acid substitutions within HLA-B5801-restricted HIV-1 and HIV-2 epitopes. These results suggests that HLA-B5801-positive HIV-2-infected individuals have an enhanced ability to react with HIV-1 that could play a role in cross-protection.
The Herpes Simplex Virus Type 1 Cleavage/Packaging Protein, UL32, Is Involved in Efficient Localization of Capsids to Replication Compartments
Authors: Lamberti Carmela , Weller Sandra K.
Published: December 2019

Six genes, including UL32, have been implicated in the cleavage and packaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isolated temperature-sensitive herpes simplex virus type 1 mutant,
ts
N20 (P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virology 52:57–71, 1973). A polyclonal antibody which recognizes UL32 was previously used by Chang et al. (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938–3946, 1996) to demonstrate that UL32 accumulates predominantly in the cytoplasm of infected cells. In this report, a functional epitope-tagged version of UL32 showed that while UL32 is predominantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICP8, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific for the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results suggest that cleavage and packaging occur in replication compartments at least at 6 and 8 h postinfection. Cells infected with the UL32 mutant exhibit a hexon staining pattern which is more diffusely distributed throughout the nucleus and which is not restricted to replication compartments. We propose that UL32 may play a role in “bringing” preassembled capsids to the sites of DNA packaging and that the failure to localize to replication compartments may explain the cleavage/packaging defect exhibited by this mutant. These results suggest that the UL32 protein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correct localization of capsids within infected cells.
Lymantria dispar
Nucleopolyhedrovirus
hrf-1
Expands the Larval Host Range of
Autographa californica
Nucleopolyhedrovirus
Authors: Chen Chi-Ju , Quentin Martha E. , Brennan Lynn A. , Kukel Christine , Thiem Suzanne M.
Published: December 2019

The gypsy moth (
Lymantria dispar
) is nonpermissive for
Autographa californica
nucleopolyhedrovirus (AcNPV) infection. We previously isolated a gene, host range factor 1 (
hrf-1
), from
L. dispar
nucleopolyhedrovirus that promotes AcNPV replication in Ld652Y cells, a nonpermissive
L. dispar
cell line (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221–2229, 1996). In the present study, we investigated the ability of
hrf-1
to alter the larval host range of AcNPV. Bioassays using recombinant AcNPV bearing
hrf-1
were conducted with insect larvae by use of oral infection. AcNPV bearing
hrf-1
was infectious for neonate
L. dispar
larvae, with a 50% lethal concentration of 1.2 × 10
5
polyhedral inclusion bodies/ml of diet, which is similar to that of wild-type AcNPV for permissive hosts. AcNPV can kill neonate
L. dispar
larvae at high doses, but it does not kill third-instar larvae. However, electron microscopy studies of AcNPV-inoculated third-instar larvae revealed virus replication in the midgut cells. PCR analyses indicated that the virus was AcNPV. These results suggest that the block for AcNPV infection of
L. dispar
larvae is its inability to spread systematically from primary infection sites in the midgut epithelium and that this barrier is leaky in neonates.
hrf-1
allows AcNPV to overcome this barrier. AcNPV recombinants bearing
hrf-1
were also significantly more infectious for
Helicoverpa zea
, a resistant species, suggesting that the blocks for AcNPV infection of
L. dispar
and
H. zea
larvae may be similar.
Analysis of Hepatitis C Virus-Inoculated Chimpanzees Reveals Unexpected Clinical Profiles
Authors: Bassett Suzanne E. , Brasky Kathleen M. , Lanford Robert E.
Published: December 2019

The clinical course of hepatitis C virus (HCV) infections in a chimpanzee cohort was examined to better characterize the outcome of this valuable animal model. Results of a cross-sectional study revealed that a low percentage (39%) of HCV-inoculated chimpanzees were viremic based on reverse transcription (RT-PCR) analysis. A correlation was observed between viremia and the presence of anti-HCV antibodies. The pattern of antibodies was dissimilar among viremic chimpanzees and chimpanzees that cleared the virus. Viremic chimpanzees had a higher prevalence of antibody reactivity to NS3, NS4, and NS5. Since an unexpectedly low percentage of chimpanzees were persistently infected with HCV, a longitudinal analysis of the virological profile of a small panel of HCV-infected chimpanzees was performed to determine the kinetics of viral clearance and loss of antibody. This study also revealed that a low percentage (33%) of HCV-inoculated chimpanzees were persistently viremic. Analysis of serial bleeds from six HCV-infected animals revealed four different clinical profiles. Viral clearance with either gradual or rapid loss of anti-HCV antibody was observed in four animals within 5 months postinoculation. A chronic-carrier profile characterized by persistent HCV RNA and anti-HCV antibody was observed in two animals. One of these chimpanzees was RT-PCR positive, antibody negative for 5 years and thus represented a silent carrier. If extrapolated to the human population, these data would imply that a significant percentage of unrecognized HCV infections may occur and that silent carriers may represent potentially infectious blood donors.
A Replication-Competent Retrovirus Arising from a Split-Function Packaging Cell Line Was Generated by Recombination Events between the Vector, One of the Packaging Constructs, and Endogenous Retroviral Sequences
Authors: Chong Heung , Starkey William , Vile Richard G.
Published: December 2019

Previously we reported the presence of a replication-competent retrovirus in supernatant from a vector-producing line derived from a widely used split-function amphotropic packaging cell line. Rigorous routine screening of all retroviral stocks produced in our laboratory has not, previously or since, indicated the presence of such a virus. Replication-competent retroviruses have never previously been used in our laboratory, and stringent screening of all routinely used cell lines has not revealed the presence of any helper viruses. Therefore, it is highly unlikely that this virus represents an adventitious cross-contaminant or had been imported unknowingly with our cell line stocks. PCR studies with DNA from infected cell lines and Northern blot analysis and reverse transcriptase PCR with RNA from infected cells suggest that the helper virus arose by recombination events, at sites of partial homology, between sequences in the vector, one of the packaging constructs, and endogenous retroviral elements. These recombinations were not present in stocks of the packaging cell line or in an initial stock of the vector-producing line, indicating that these events occurred while the vector-producing line was being passaged for harvest of supernatant stocks.
Packaging of Endogenous Retroviral Sequences in Retroviral Vectors Produced by Murine and Human Packaging Cells
Authors: Patience Clive , Takeuchi Yasuhiro , Cosset Francois-Loic , Weiss Robin A.
Published: December 2019

Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in unwanted events, such as the formation of recombinant viruses and the mobilization of therapeutic vectors. Using sensitive reverse transcriptase PCR assays, we investigated human and murine gene therapy packaging cell lines for incorporation of endogenous retrovirus transcripts into murine leukemia virus (MLV) vector particles and, conversely, whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were efficiently packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived β-galactosidase (β-Gal) vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed several classes of HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Nonspecific packaging of the MLV Gag-Pol expression vector transcripts was detected in the FLY virions at a low level (1 in 17,000 sequences). These findings indicate that human packaging cells produce retrovirus particles far less contaminated by endogenous viral sequences than murine packaging cells. Human teratocarcinoma cells (GH cells), which produce HERV-K particles, were transduced with an MLV-derived β-Gal vector. Although both HERV-K and RTVL-H sequences were found in association with the particles, β-Gal transcripts were not detected, indicating that HERV Gag proteins do not efficiently package MLV-based vectors.
Temporal Usage of Multiple Promoters during the Life Cycle of Human Papillomavirus Type 31b
Authors: Ozbun Michelle A. , Meyers Craig
Published: December 2019

The life cycles of human papillomaviruses (HPVs) are dependent upon the differentiation of the epithelial cells they infect. HPV type 31b (HPV31b) virions can be purified following the growth of a latently HPV-infected cell line (CIN-612 9E) in the organotypic or raft system. Treatment of the CIN-612 9E raft tissues with protein kinase C (PKC) activators is required for upregulation of late gene expression and efficient production of virions. We employed the raft culture system to study the temporal usage of HPV31b promoters during the viral life cycle. We compared monolayer cultures of CIN-612 9E cells, untreated CIN-612 9E raft tissues, and PKC-induced CIN-612 9E raft tissues harvested at various time points during epithelial differentiation. We found that the HPV31b major early promoter precisely maps to nucleotide (nt) 99 (P
99
). A transcriptional start site for both early and late gene transcripts mapped upstream of P
99
at nt 77 (P
77
). The P
77
and P
99
promoters were used constitutively throughout the HPV31b life cycle; however, initiation from P
99
was much stronger than from P
77
. Mapping of the differentiation-induced P
742
promoter revealed multiple start sites. These start sites were difficult to detect in monolayer cultures, were induced in untreated rafts, and were greatest in PKC-induced raft tissues at 8 to 12 days. A constitutively active promoter, P
3320
, was also defined and is responsible for the transcription of unspliced and spliced RNAs containing E5a, E5b, L2, and L1 open reading frames.
Immature Dendritic Cells Selectively Replicate Macrophagetropic (M-Tropic) Human Immunodeficiency Virus Type 1, while Mature Cells Efficiently Transmit both M- and T-Tropic Virus to T Cells
Authors: Granelli-Piperno Angela , Delgado Elena , Finkel Victoria , Paxton William , Steinman Ralph M.
Published: December 2019

Dendritic cells (DCs) can develop from CD14
+
peripheral blood monocytes cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). By 6 days in culture, the cells have the characteristics of immature DCs and can be further induced to mature by inflammatory stimuli or by monocyte-conditioned medium. After infection with macrophagetropic (M-tropic) human immunodeficiency virus type 1 (HIV-1), monocytes and mature DCs show a block in reverse transcription and only form early transcripts that can be amplified with primers for the R/U5 region. In contrast, immature DCs cultured for 6 or 11 days in GM-CSF and IL-4 complete reverse transcription and show a strong signal when LTR/gag primers are used. Blood monocytes and mature DCs do not replicate HIV-1, whereas immature DCs can be productively infected, but only with M-tropic HIV-1. The virus produced by immature DCs readily infects activated T cells. Although mature DCs do not produce virus, these cells transmit both M- and T-tropic virus to T cells. In the cocultures, both DCs and T cells must express functional chemokine coreceptors for viral replication to occur. Therefore, the developmental stage of DCs can influence the interaction of these cells with HIV-1 and influence the extent to which M-tropic and T-tropic virus can replicate.
Characterization of Hepatitis G Virus (GB-C Virus) Particles: Evidence for a Nucleocapsid and Expression of Sequences Upstream of the E1 Protein
Authors: Xiang Jinhua , Klinzman Donna , McLinden James , Schmidt Warren N. , LaBrecque Douglas R. , Gish Robert , Stapleton Jack T.
Published: December 2019

Hepatitis G virus (HGV or GB-C virus) is a newly described virus that is closely related to hepatitis C virus (HCV). Based on sequence analysis and by evaluation of translational initiation codon preferences utilized during in vitro translation, HGV appears to have a truncated or absent core protein at the amino terminus of the HGV polyprotein. Consequently, the biophysical properties of HGV may be very different from those of HCV. To characterize HGV particle types, we evaluated plasma from chronically infected individuals with and without concomitant HCV infection by using sucrose gradient centrifugation, isopycnic banding in cesium chloride, and saline density flotation centrifugation. Similar to HCV, HGV particles included an extremely-low-density virion particle (1.07 to 1.09 g/ml) and a nucleocapsid of ∼1.18 g/ml. One major difference between the particle types was that HGV was consistently more stable in cesium chloride than HCV. Plasma samples from chronically HGV-infected individuals and controls were assessed by a synthetic peptide-based immunoassay to determine if they contained HGV antibody specific for a conserved region in the coding region upstream of the E1 protein. Chronically HGV-infected individuals contained antibody to the HGV core protein peptide, whereas no binding to a hepatitis A virus peptide control was observed. Competitive inhibition of binding to the HGV peptide confirmed the specificity of the assay. These data indicate that HGV has a nucleocapsid and that at least part of the putative core region of HGV is expressed in vivo.
Interferon Resistance of Hepatitis C Virus Genotype 1b: Relationship to Nonstructural 5A Gene Quasispecies Mutations
Authors: Pawlotsky Jean-Michel , Germanidis Georgios , Neumann Avidan U. , Pellerin Muriel , Frainais Pierre-Olivier , Dhumeaux Daniel
Published: December 2019

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-α) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224–230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77–81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-α sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-α and (ii) that HCV-1b resistance to IFN-α could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-α-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-α, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.
Expression of the pRb-Binding Regions of E1A Enables Efficient Transformation of Primary Epithelial Cells by v-
src
Authors: Fischer Robert S. , Quinlan Margaret P.
Published: December 2019

Primary cultures of rat embryo fibroblasts have been shown to be resistant to transformation by dominant oncogenes such as v-
src
. We sought to determine if similar resistance is displayed by primary epithelial cells, and, if so, whether an immortalizing oncogene such as E1A could enhance transformation of primary epithelial cells by v-
src
. Transformation of primary rat epithelial cells by v-
src
was synergistically enhanced when E1A expression plasmids were cotransfected with a v-
src
expression plasmid. Foci were more numerous and observed earlier (9 to 14 days) with E1A plus v-
src
than with v-
src
alone (18 to 28 days). This cotransformation ability was abrogated by deletions in CR1 or CR2 of E1A, which encode the binding regions for the pRb family and are responsible for E1A-mediated cell cycle activation. Mutations in the p300 binding site or the second exon, which abolish immortalization, did not affect v-
src
cooperation, in contrast to
ras
and adenovirus E1B. While kinase activation was required for growth in soft agar, differential activation of Src kinase did not correlate with transformation efficiency. Cell morphology and actin structures were not dramatically impacted by E1A expression; thus, hypertransformation, as previously described for
ras
cotransformation, was not observed with v-
src
and second-exon mutants of E1A. However, growth rates for cells expressing both E1A and v-Src were higher than those for cells expressing only v-Src. These results suggest that functions involved in cell cycle activation encoded by E1A first exon can enhance v-
src
transformation of primary epithelial cells.
Characterization of the Single-Stranded DNA Binding Protein Encoded by the Vaccinia Virus I3 Gene
Authors: Rochester S. Craig , Traktman Paula
Published: December 2019

The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in
Escherichia coli
and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.
Specific Methylation Patterns in Two Control Regions of Epstein-Barr Virus Latency: the LMP-1-Coding Upstream Regulatory Region and an Origin of DNA Replication (oriP)
Authors: Falk Kerstin I. , Szekely Laszlo , Aleman Anna , Ernberg Ingemar
Published: December 2019

The Epstein-Barr virus (EBV) can establish at least four different forms of latent infection. Previously, we have shown that the level of methylation of the EBV genome varies, depending on the form of latency. The methylation status of CpGs was analyzed by the bisulfite genomic sequencing technique in four different cell types representing different forms of latency. The dyad symmetry element of the origin of replication (oriP) region and the latent membrane protein 1 (LMP-1) regulatory sequence (LRS) were studied. The dyad symmetry element has four binding sites for EBNA-1. In a cell with type I latency, a region upstream of the dyad symmetry element was highly methylated, whereas the dyad symmetry element was unmethylated in the EBNA-1-binding region. The LRS was extensively methylated in the LMP-1-negative cell line Rael, in contrast to a LMP-1-expressing nasopharyngeal carcinoma tumor (NPC C15), which was almost completely unmethylated. The methylation pattern of LRS in type I and type III Burkitt lymphoma cells of similar parental origins confirmed that demethylation of some regions takes place upon phenotypic drift.
In Vitro Transcription of pe38/Polyhedrin Hybrid Promoters Reveals Sequences Essential for Recognition by the Baculovirus-Induced RNA Polymerase and for the Strength of Very Late Viral Promoters
Authors: Mans Ruud M. W. , Knebel-Mörsdorf Dagmar
Published: December 2019

In vitro transcription was used to analyze the promoter specificity of the α-amanitin-resistant RNA polymerase that is induced late during infection of
Autographa californica
multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5′ untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.
Bovine Herpesvirus 1 Glycoprotein M Forms a Disulfide-Linked Heterodimer with the U
L
49.5 Protein
Authors: Wu S. X. , Zhu X. P. , Letchworth G. J.
Published: December 2019

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U
L
10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione
S
-transferase fusion proteins in
Escherichia coli
. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U
L
10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U
L
49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448–1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U
L
49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.
Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of U
L
15-Encoded Proteins with B Capsids Requires at Least the U
L
6, U
L
17, and U
L
28 Genes
Authors: Salmon Brandy , Baines Joel D.
Published: December 2019

The U
L
15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U
L
15-encoded protein recognized three proteins with apparent
M
r
s of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-
M
r
protein was detected in type C capsids and comigrated with the product of a U
L
15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-
M
r
proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔU
L
15), a virus lacking an intact U
L
15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of U
L
15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent
M
r
s of 83,000, 80,000, and 79,000 are products of U
L
15 with identical C termini. The 79,000-, 80,000-, and 83,000-
M
r
proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U
L
15-encoded proteins are integral components of B capsids. Only the 83,000-
M
r
protein was detected in B capsids purified from cells infected with viruses lacking the U
L
6, U
L
17, or U
L
28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-
M
r
proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-
M
r
U
L
15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.
Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase
Authors: Sosnovtsev Stanislav V. , Sosnovtseva Svetlana A. , Green Kim Y.
Published: December 2019

Feline calicivirus (FCV), a member of the
Caliciviridae
, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates for
trans
cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.
Bombyx mori
Nucleopolyhedrovirus Encodes a DNA-Binding Protein Capable of Destabilizing Duplex DNA
Authors: Mikhailov Victor S. , Mikhailova Alla L. , Iwanaga Masashi , Gomi Sumiko , Maeda Susumu
Published: December 2019

A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from
Bombyx mori
) infected with the
B. mori
nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with
Achromobacter
protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no.
L33180
). This ORF (designated
dbp
) is a homolog of
Autographa californica
multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3′→5′ exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.
The Activity of Sendai Virus Genomic and Antigenomic Promoters Requires a Second Element Past the Leader Template Regions: a Motif (GNNNNN)
3
Is Essential for Replication
Authors: Tapparel Caroline , Maurice Diane , Roux Laurent
Published: December 2019

The paramyxovirus genome, a nonsegmented, negative-polarity, single-stranded RNA of ∼15 kb, contains six transcription units flanked at the 3′ and 5′ ends by a short (∼ 50- to 60-nucleotide) extracistronic sequence, dubbed the positive and negative leader regions. These leader template regions, present at the 3′ end of the genome and the antigenome, have been shown to contain essential signals governing RNA replication activity. Whether they are sufficient to promote replication is still open to question. By using a series of Sendai virus defective interfering RNAs carrying a nested set of deletions in the promoter regions, it is shown here that for both the genomic and antigenomic promoters, a 3′-end RNA sequence of 96 nucleotides is required to allow replication. Sequence comparison of active and inactive promoters led to the identification of a set of three nucleotide hexamers (nucleotides 79 to 84, 85 to 90, and 91 to 96) containing a repeated motif RXXYXX [shown as 5′-3′ positive-strand]. Sequential mutation of each hexamer into its complementary sequence confirmed their essential role. The three hexamers are required, and their relative positioning is important, since displacing them by 6 nucleotides destroyed promoter function. RNAs carrying degenerate nucleotides in the three hexamers were used as replication templates. They led to the selection of actively replicating RNA species exclusively carrying the basic motif (GNNNNN)
3
from nucleotides 79 to 96. These results clearly show that, apart from the region from nucleotides 1 to 31, previously identified as governing Sendai virus replication activity, a second element, spanning at the most nucleotides 79 to 96, appears essential. Thus, the paramyxovirus replication promoters are not confined to the leader template regions, as seems to be the case for the rhabdoviruses.
The Proteolytic Cleavage of Human Immunodeficiency Virus Type 1 Nef Does Not Correlate with Its Ability To Stimulate Virion Infectivity
Authors: Chen Yen-Liang , Trono Didier , Camaur Diana
Published: December 2019

The Nef protein of human immunodeficiency virus type 1 (HIV-1) promotes virion infectivity through mechanisms that are yet ill defined. Some Nef is incorporated into particles, where it is cleaved by the viral protease between amino acids 57 and 58. The functional significance of this event, which liberates the C-terminal core domain of the protein from its membrane-associated N terminus, is unknown. To address this question, we examined the modalities of Nef virion association and processing. We found that although significant levels of Nef were detected in HIV-1 virions partly in a cleaved form, cell-specific variations existed in the efficiency of Nef proteolytic processing. The virion association of Nef was strongly enhanced by myristoylation but did not require other HIV-1-specific proteins, since Nef was efficiently incorporated into and cleaved inside murine leukemia virus particles. Substituting alanine for tryptophan
57
decreased the efficiency of Nef processing, while mutating leucine
58
had little effect. In contrast, replacing both of these residues simultaneously almost completely prevented this process. However, when the resulting mutants were compared with a wild-type control in viral infectivity assays, no correlation was found between the levels of cleavage and the ability to stimulate virion infectivity. Furthermore, simian immunodeficiency virus Nef, which lacks the sequence recognized by the protease and as a consequence is not cleaved despite its incorporation into virions, could stimulate the infectivity of a
nef
-defective HIV-1 variant as efficiently as HIV-1 Nef. On these bases, we conclude that the proteolytic processing of Nef is not required for the ability of this protein to enhance virion infectivity.
In Vivo Replication Capacity Rather Than In Vitro Macrophage Tropism Predicts Efficiency of Vaginal Transmission of Simian Immunodeficiency Virus or Simian/Human Immunodeficiency Virus in Rhesus Macaques
Authors: Miller Christopher J. , Marthas Marta , Greenier Jennifer , Lu Ding , Dailey Peter J. , Lu Yichen
Published: December 2019

We used the rhesus macaque model of heterosexual human immunodeficiency virus (HIV) transmission to test the hypothesis that in vitro measures of macrophage tropism predict the ability of a primate lentivirus to initiate a systemic infection after intravaginal inoculation. A single atraumatic intravaginal inoculation with a T-cell-tropic molecular clone of simian immunodeficiency virus (SIV), SIVmac239, or a dualtropic recombinant molecular clone of SIV, SIVmac239/1A11/239, or uncloned dualtropic SIVmac251 or uncloned dualtropic simian/human immunodeficiency virus (SHIV) 89.6-PD produced systemic infection in all rhesus macaques tested. However, vaginal inoculation with a dualtropic molecular clone of SIV, SIVmac1A11, resulted in transient viremia in one of two rhesus macaques. It has previously been shown that 12 intravaginal inoculations with SIVmac1A11 resulted in infection of one of five rhesus macaques (M. L. Marthas, C. J. Miller, S. Sutjipto, J. Higgins, J. Torten, B. L. Lohman, R. E. Unger, H. Kiyono, J. R. McGhee, P. A. Marx, and N. C. Pedersen, J. Med. Primatol. 21:99–107, 1992). In addition, SHIV HXBc2, which replicates in monkey macrophages, does not infect rhesus macaques following multiple vaginal inoculations, while T-cell-tropic SHIV 89.6 does (Y. Lu, P. B. Brosio, M. Lafaile, J. Li, R. G. Collman, J. Sodroski, and C. J. Miller, J. Virol. 70:3045–3050, 1996). These results demonstrate that in vitro measures of macrophage tropism do not predict if a SIV or SHIV will produce systemic infection after intravaginal inoculation of rhesus macaques. However, we did find that the level to which these viruses replicate in vivo after intravenous inoculation predicts the outcome of intravaginal inoculation with each virus.
Enhancement of Aquareovirus Infectivity by Treatment with Proteases: Mechanism of Action
Authors: McPhillips Thomas H. , Dinan David , Subramanian Kothandaraman , Samal Siba K.
Published: December 2019

The effects of protease digestion on the polypeptide composition and on the infectivity of striped bass virus, an aquareovirus, were examined. Both trypsin and chymotrypsin enhanced the infectivity of the virus. Enhancement of infectivity was correlated with the digestion of the outer capsid protein, VP7. These studies support the assertion that VP7 is the outermost capsid protein and suggest that VP4 and VP5 are exposed on the outer surface of infectious particles. The possible role of VP7 in the variation in virulence observed among aquareovirus isolates is discussed.
T-Cell Lymphoma Caused by Herpesvirus Saimiri C488 Independently ofie14/vsag, a Viral Gene with Superantigen Homology
Authors: Knappe Andrea , Thurau Mathias , Niphuis Henk , Hiller Christian , Wittmann Sabine , Kuhn Eva-Maria , Rosenwirth Brigitte , Fleckenstein Bernhard , Heeney Jonathan , Fickenscher Helmut
Published: December 2019

The immediate-early geneie14/vsagof herpesvirus saimiri has homology with murine superantigens. We compared the pathogenesis of infection with eitherie14/vsagdeletion mutants or wild-type virus C488 in cottontop tamarin monkeys (Saguinus oedipus). Two weeks after infection, all animals developed acute T-cell lymphomas independently of the presence of the viralie14/vsaggene.
Induction of Mucosal B-Cell Memory by Intramuscular Inoculation of Mice with Rotavirus
Authors: Coffin Susan E. , Offit Paul A.
Published: December 2019

We investigated the capacity of intramuscular (i.m.) immunization with heterologous-host rotavirus (simian strain RRV) to induce mucosal virus-specific memory B cells in mice. We found that prior i.m. immunization enhanced the magnitude of mucosal virus-specific immunoglobulin A (IgA) production but did not alter the site and timing of induction of virus-specific IgA responses after challenge.
Identification and Characterization of a Porcine Torovirus
Authors: Kroneman A. , Cornelissen L. A. H. M. , Horzinek M. C. , de Groot R. J. , Egberink H. F.
Published: December 2019

A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genusTorovirus(familyCoronaviridae, orderNidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.
Neutralization of Human Immunodeficiency Virus Type 1 by Antibody to gp120 Is Determined Primarily by Occupancy of Sites on the Virion Irrespective of Epitope Specificity
Authors: Parren Paul W. H. I. , Mondor Isabelle , Naniche Denise , Ditzel Henrik J. , Klasse P. J. , Burton Dennis R. , Sattentau Quentin J.
Published: December 2019

We investigated the relative importance of binding site occupancy and epitope specificity in antibody neutralization of human immunodeficiency virus (HIV) type 1 (HIV-1). The neutralization of a T-cell-line-adapted HIV-1 isolate (MN) was analyzed with a number of monovalent recombinant Fab fragments (Fabs) and monoclonal antibodies with a range of specificities covering all confirmed gp120-specific neutralization epitopes. Binding of Fabs to recombinant monomeric gp120 was determined by surface plasmon resonance, and binding of Fabs and whole antibodies to functional oligomeric gp120 was determined by indirect immunofluorescence and flow cytometry on HIV-infected cells. An excellent correlation between neutralization and oligomeric gp120 binding was observed, and a lack of correlation with monomeric gp120 binding was confirmed. A similar degree of correlation was observed between oligomeric gp120 binding and neutralization with a T-cell-line-adapted HIV-1 molecular clone (Hx10). The ratios of oligomer binding/neutralization titer fell, in general, within a relatively narrow range for antibodies to different neutralization epitopes. These results suggest that the occupancy of binding sites on HIV-1 virions is the major factor in determining neutralization, irrespective of epitope specificity. Models to account for these observations are proposed.
Functional Role of Hepatitis C Virus Chimeric Glycoproteins in the Infectivity of Pseudotyped Virus
Authors: Lagging L. Martin , Meyer Keith , Owens Randall J. , Ray Ranjit
Published: December 2019

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5°C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.
Mutations in Rotavirus Nonstructural Glycoprotein NSP4 Are Associated with Altered Virus Virulence
Authors: Zhang Mingdong , Zeng Carl Q.-Y. , Dong Yanjie , Ball Judith M. , Saif Linda J. , Morris Andrew P. , Estes Mary K.
Published: December 2019

Rotaviruses are major pathogens causing life-threatening dehydrating gastroenteritis in children and animals. One of the nonstructural proteins, NSP4 (encoded by gene 10), is a transmembrane, endoplasmic reticulum-specific glycoprotein. Recently, our laboratory has shown that NSP4 causes diarrhea in 6- to 10-day-old mice by functioning as an enterotoxin. To confirm the role of NSP4 in rotavirus pathogenesis, we sequenced gene 10 from two pairs of virulent and attenuated porcine rotaviruses, the OSU and Gottfried strains. Comparisons of the NSP4 sequences from these two pairs of rotaviruses suggested that structural changes between amino acids (aa) 131 and 140 are important in pathogenesis. We next expressed the cloned gene 10 from the OSU virulent (OSU-v) and OSU attenuated (OSU-a) viruses by using the baculovirus expression system and compared the biological activities of the purified proteins. NSP4 from OSU-v virus increased intracellular calcium levels over 10-fold in intestinal cells when added exogenously and 6-fold in insect cells when expressed endogenously, whereas NSP4 from OSU-a virus had little effect. NSP4 from OSU-v caused diarrhea in 13 of 23 neonatal mice, while NSP4 from OSU-a caused disease in only 4 of 25 mice (
P
< 0.01). These results suggest that avirulence is associated with mutations in NSP4. Results from site-directed mutational analyses showed that mutated OSU-v NSP4 with deletion or substitutions in the region of aa 131 to 140 lost its ability to increase intracellular calcium levels and to induce diarrhea in neonatal mice, confirming the importance of amino acid changes from OSU-v NSP4 to OSU-a NSP4 in the alteration of virus virulence.