bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization of a Bovine Enteric Calicivirus: Relationship to the Norwalk-Like Viruses
Authors: Liu B. L. , Lambden P. R. , Günther H. , Otto P. , Elschner M. , Clarke I. N.
Published: December 2019

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5′ terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the
Caliciviridae
JV is most closely related to the group 1 NLVs.
The U
L
3 Protein of Herpes Simplex Virus 1 Is Translated Predominantly from the Second In-Frame Methionine Codon and Is Subject to at Least Two Posttranslational Modifications
Authors: Markovitz Nancy S. , Filatov Felix , Roizman Bernard
Published: December 2019

The U
L
3 open reading frame (ORF) has the coding capacity of 235 codons. The proteins reacting with the anti-U
L
3 antibody form in denaturing polyacrylamide gel bands with apparent
M
r
s of 34,000, 35,000, 38,000, 40,000, 41,000, and 42,000 and designated 1 to 6, respectively. Studies on their origins revealed the following. (i) The U
L
3 proteins forming all six bands were present in lysates of cells infected with wild-type virus and treated with tunicamycin or monensin or in cells infected with the mutant lacking the gene encoding the U
S
3 protein kinase. (ii) The proteins contained in the slower-migrating bands were absent from cells infected with the mutant lacking the U
L
13 protein kinase. Bands 1 and 3, however were phosphorylated in cells infected with this mutant. (iii) Band 2 protein was absent from cells transfected with a plasmid carrying a substitution of the predicted first methionine codon of the U
L
3 ORF and superinfected with the U
L
3
−
mutant. (iv) Band 1 and 3 proteins were absent from lysates of cells transfected with a plasmid carrying a substitution of the second (M12) methionine codon of the U
L
3 ORF and superinfected with the U
L
3
−
mutant. (v) Cells superinfected with mutants lacking both methionine codons did not accumulate any of the proteins contained in the six bands. (vi) In vitro transcription-translation studies indicated that the translation of band 1 protein was initiated from the second (M12) methionine codon and that band 3 protein represented a U
L
13-independent, posttranslationally processed form of these proteins. The results indicate that the U
L
3 protein of herpes simplex virus 1 is translated predominantly from the second in-frame methionine codon and is subject to at least two posttranslational modifications.
Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase
Authors: Goodarzi Goodarz , Pursley Michael , Felock Peter , Witmer Marc , Hazuda Daria , Brackmann Karl , Grandgenett Duane
Published: December 2019

Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.
Amino Acid Substitutions within the Leucine Zipper Domain of the Murine Coronavirus Spike Protein Cause Defects in Oligomerization and the Ability To Induce Cell-to-Cell Fusion
Authors: Luo Zongli , Matthews Avery M. , Weiss Susan R.
Published: December 2019

The murine coronavirus spike (S) protein contains a leucine zipper domain which is highly conserved among coronaviruses. To assess the role of this leucine zipper domain in S-induced cell-to-cell fusion, the six heptadic leucine and isoleucine residues were replaced with alanine by site-directed mutagenesis. The mutant S proteins were analyzed for cell-to-cell membrane fusion activity as well as for progress through the glycoprotein maturation process, including intracellular glycosylation, oligomerization, and cell surface expression. Single-alanine-substitution mutations had minimal, if any, effects on S-induced cell-to-cell fusion. Significant reduction in fusion activity was observed, however, when two of the four middle heptadic leucine or isoleucine residues were replaced with alanine. Double alanine substitutions that involved either of the two end heptadic leucine residues did not significantly affect fusion. All double-substitution mutant S proteins displayed levels of endoglycosidase H resistance and cell surface expression similar to those of the wild-type S. However, fusion-defective double-alanine-substitution mutants exhibited defects in S oligomerization. These results indicate that the leucine zipper domain plays a role in S-induced cell-to-cell fusion and that the ability of S to induce fusion may be dependent on the oligomeric structure of S.
Characterization of the Block in Replication of Nucleocapsid Protein Zinc Finger Mutants from Moloney Murine Leukemia Virus
Authors: Gorelick Robert J. , Fu William , Gagliardi Tracy D. , Bosche William J. , Rein Alan , Henderson Louis E. , Arthur Larry O.
Published: December 2019

Mutagenesis studies have shown that retroviral nucleocapsid (NC) protein Zn
2+
fingers (-Cys-X
2
-Cys-X
4
-His-X
4
-Cys- [CCHC]) perform multiple functions in the virus life cycle. Moloney murine leukemia virus mutants His 34→Cys (CCCC) and Cys 39→His (CCHH) were able to package their genomes normally but were replication defective. Thermal dissociation experiments showed that the CCHH mutant was not defective in genomic RNA dimer structure. Primer tRNA placement on the viral genome and the ability of the tRNA to function in reverse transcription initiation in vitro also appear normal. Some “full-length” DNA copies of the viral genome were synthesized in mutant virus-infected cells. The CCCC and CCHH mutants produced these DNA copies at greatly reduced levels. Circle junction fragments, amplified from two-long-terminal-repeat viral DNA (vDNA) by PCR, were cloned and characterized. Remarkably, it was discovered that vDNA isolated from cells infected with mutant virions had a wide variety of abnormalities at the site at which the two ends of the linear precursor had been ligated to form the circle (i.e., the junction between the 5′ end of U3 and the 3′ end of U5). In some molecules, bases were missing from regions corresponding to the U3 and U5 linear vDNA termini; in others, the viral sequences extended either beyond the U5 sequences into the primer-binding site and 5′ leader or beyond the U3 sequences into the polypurine tract into the
env
coding region. Still other molecules contained nonviral sequences between the linear vDNA termini. Such defective genomes would certainly be unsuitable substrates for integration. Thus, strict conservation of the CCHC structure in NC is required for infection events prior to and possibly including integration.
Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in
Escherichia coli
: a New Approach for Construction of HCMV Mutants
Authors: Borst Eva-Maria , Hahn Gabriele , Koszinowski Ulrich H. , Messerle Martin
Published: December 2019

We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in
Escherichia coli
(M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation in
E. coli
, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in
E. coli
. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.
Activation of cJUN N-Terminal Kinase by Herpes Simplex Virus Type 1 Enhances Viral Replication
Authors: McLean T. I. , Bachenheimer S. L.
Published: December 2019

Signal transduction pathways convey signals generated at the cell surface into the cell nucleus in order to initiate a program of gene expression that is characteristic for particular stimuli. Here we present evidence that infection by herpes simplex virus type 1 activated the two terminal kinases, cJUN N-terminal kinase (JNK) and p38, of stress-activated signal transduction kinase cascades. By using a solid-phase kinase assay, a phospho-specific antibody, and extracts prepared from a variety of infected cell types, we determined that activation of both kinases began 3 to 4 h postinfection (p.i.) and remained elevated out to 14 h p.i. Through the use of UV-irradiated or antibody-neutralized wild-type virus and the temperature-sensitive mutanttsB7, the high level of JNK activation was shown to be dependent on viral gene expression. Activation of JNK following infection by vi13, an ICP4 mutant virus that does not express early or late genes, suggested that only virus entry and immediate-early gene expression were necessary for JNK activation. The activation of JNK and p38 correlated with increased chloramphenicol acetyltransferase (CAT) activity in reporter assays dependent upon the activity of cJUN and ATF2trans-activation domains. Increased CAT activity dependent on TRE and CRE promoter sites was also observed in response to herpes simplex virus infection. The activities of ERK and ERK-dependent transcription factors were unchanged or depressed following infection, showing that activation of JNK and p38 was a specific event. Finally, the activation of JNK was important for the efficiency of viral replication. The yield of virus in NIH 3T3 cells stably expressing JIP-1, an inhibitor of JNK translocation to the nucleus, was reduced 70% compared to that of control cells, in single-step growth experiments.
Altering the Intracellular Environment Increases the Frequency of Tandem Repeat Deletion during Moloney Murine Leukemia Virus Reverse Transcription
Authors: Pfeiffer Julie K. , Topping Robert S. , Shin Nam-Hee , Telesnitsky Alice
Published: December 2019

During retroviral DNA synthesis reverse transcriptase frequently performs nonrequired template switches that can lead to genetic rearrangements or recombination. It has been postulated that template switching occurs after pauses in the action of reverse transcriptase. Hence factors which affect pausing, such as polymerization rate, may affect the frequency of template switching. To address the hypothesis that increasing the time required to complete reverse transcription increases the frequency of template switching, we established conditions that lengthened the time required to complete a single round of intracellular Moloney murine leukemia virus reverse transcription approximately threefold. Under these conditions, which resulted from intracellular nucleotide pool imbalances generated with hydroxyurea, we examined template switching frequency using a
lacZ
-based tandem repeat deletion assay. We observed that the frequency of deletion during reverse transcription in hydroxyurea-treated cells was approximately threefold higher than that in untreated control cells. These findings suggest that rates of retroviral recombination may vary when the intracellular environment is altered.
Decreased Processivity of Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) Containing Didanosine-Selected Mutation Leu74Val: a Comparative Analysis of RT Variants Leu74Val and Lamivudine-Selected Met184Val
Authors: Sharma Prem L. , Crumpacker Clyde S.
Published: December 2019

We previously showed that a didanosine-selected mutation in pNL4-3 background conferred a replication disadvantage on human immunodeficiency virus type 1, resulting in a loss of replication fitness. This work has been extended by showing that a recombinant virus with the HXBc2 backbone and reverse transcriptase (RT) fragments from pNL4-3 containing the Leu74Val mutation produce decreasing amounts of p24 antigen over a 3-week period. The HXBc2 recombinant containing the wild-type RT from pNL4-3 replicated efficiently. When the virion-associated RT containing the Leu74Val mutation was used in an RT processivity assay with homopolymer RNA template-primer, poly(A), and oligo(dT), the RT with altered Leu74Val mutation was less processive, generating fewer cDNA products in comparison to wild-type pNL4-3 RT. The replication kinetics and RT processivity of the mutant with the Leu74Val mutation were compared to those of a lamivudine-selected mutant Met184Val. In replication kinetics assays, mutant Leu74Val replicated slower than the mutant Met184Val. In a processivity assay, the mutant RTs from both viruses show comparable decreases in processivity. These observations provide biochemical evidence of decreased processivity to support the decrease in replication fitness observed with the Leu74Val or Met184Val mutations.
Genetic Analysis of the Role of Herpes Simplex Virus Type 1 Glycoprotein K in Infectious Virus Production and Egress
Authors: Foster Timothy P. , Kousoulas Konstantin G.
Published: December 2019

Herpes simplex virus type 1 (KOS)ΔgK is a mutant virus which lacks glycoprotein K (gK) and exhibits defects in virion egress (S. Jayachandra, A. Baghian, and K. G. Kousoulas, J. Virol. 69:5401–5413, 1997). To further understand the role of gK in virus egress, we constructed recombinant viruses, ΔgKhpd-1, -2, -3, and -4, that specified gK amino-terminal portions of 139, 239, 268, and 326 amino acids, respectively, corresponding to truncations immediately after each of the four putative membrane-spanning domains of gK. ΔgKhpd-1 and ΔgKhpd-2 viruses produced lower yields and smaller plaques than ΔgK. Numerous ΔgKhpd-1 capsids accumulated predominately within large double-membrane vesicles of which the inner membrane appeared to be derived from viral envelopes while the outer membrane appeared to originate from the outer nuclear membrane. The mutant virus ΔgKhpd-3 produced higher yields and larger plaques than the ΔgK virus. The mutant virus ΔgKhpd-4 produced yields and plaques similar to those of the wild-type virus strain KOS, indicating that deletion of the carboxy-terminal 12 amino acids did not adversely affect virus replication and egress. Comparisons of the gK primary sequences specified by alphaherpesviruses revealed the presence of a cysteine-rich motif (CXXCC), located within domain III in the lumen side of gK, and a tyrosine-based motif, YTKΦ (where Φ is any bulky hydrophobic amino acid), located between the second and third hydrophobic domains (domain II) in the cytoplasmic side of gK. The mutant virus gK/Y183S, which was constructed to specify gK with a single-amino-acid change (Y to S) within the YTKΦ motif, replicated less efficiently than the ΔgK virus. The mutant virus gK/C304S-C307S, which was constructed to specify two serine instead of cysteine residues within the cysteine-rich motif (CXXCC changed to SXXSC) of gK domain III, replicated more efficiently than the ΔgK virus. Our data suggests that gK contains domains in its amino-terminal portion that promote aberrant nucleocapsid envelopment and/or membrane fusion between different virion envelopes and contains domains within its domains II and III that function in virus replication and egress.
Secreted Respiratory Syncytial Virus G Glycoprotein Induces Interleukin-5 (IL-5), IL-13, and Eosinophilia by an IL-4-Independent Mechanism
Authors: Johnson Teresa R. , Graham Barney S.
Published: December 2019

The attachment glycoprotein G of respiratory syncytial virus (RSV) is produced as both membrane-anchored and secreted forms by infected cells. Immunization with secreted RSV G (Gs) or formalin-inactivated alumprecipitated RSV (FI-RSV) predisposes mice to immune responses involving a Th2 cell phenotype which results in more severe illness and pathology, decreased viral clearance, and increased pulmonary eosinophilia upon subsequent RSV challenge. These responses are associated with increased interleukin-4 (IL-4) production in FI-RSV-primed mice, and the responses are IL-4 dependent. RNase protection assays demonstrated that similar levels of IL-4 mRNA were induced after RSV challenge in mice primed with vaccinia virus expressing Gs (vvGs) or a construct expressing only membrane-anchored G (vvGr). However, upon RSV challenge, vvGs-primed mice produced significantly greater levels of IL-5 and IL-13 mRNA and protein than vvGr-primed mice. Administration of neutralizing anti-IL-4 antibody 11.B11 during vaccinia virus priming did not alter the levels of vvGs-induced IL-5, IL-13, pulmonary eosinophilia, illness, or RSV titers upon RSV challenge, although immunoglobulin G (IgG) isotype profiles revealed that more IgG2a was produced. vvGs-priming of IL-4-deficient mice demonstrated that G-induced airway eosinophilia was not dependent on IL-4. In contrast, airway eosinophilia induced by FI-RSV priming was significantly reduced in IL-4-deficient mice. Thus we conclude that, in contrast to FI-RSV, the secreted form of RSV G can directly induce IL-5 and IL-13, producing pulmonary eosinophilia and enhanced illness in RSV-challenged mice by an IL-4-independent mechanism.
The Capsid Gene of Feline Calicivirus Contains Linear B-Cell Epitopes in both Variable and Conserved Regions
Authors: Radford Alan D. , Willoughby Kim , Dawson Susan , McCracken Christina , Gaskell Rosalind M.
Published: December 2019

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5′ end of region E (amino acids [aa] 445 to 451 [antigenic site {ags} 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.
Anterograde Transport of Herpes Simplex Virus Proteins in Axons of Peripheral Human Fetal Neurons: an Immunoelectron Microscopy Study
Authors: Holland David J. , Miranda-Saksena Monica , Boadle Ross A. , Armati Patricia , Cunningham Anthony L.
Published: December 2019

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.
Activation of Herpesvirus Gene Expression by the Human Cytomegalovirus Protein pp71
Authors: Homer Elizabeth G. , Rinaldi Angela , Nicholl Mary Jane , Preston Chris M.
Published: December 2019

The activation of gene expression by the human cytomegalovirus (HCMV) particle was investigated. The HCMV major immediate-early (IE) promoter was cloned upstream of the
Escherichia coli lacZ
coding sequences, and the resulting cassette was introduced into the genome of a herpes simplex virus type 1 (HSV-1) mutant lacking functional VP16. Upon infection with the HSV-1 recombinant in the presence of cycloheximide, to block de novo protein synthesis, expression of
lacZ
-specific transcripts was increased by fivefold when HCMV was included in the inoculum. Accumulation of HSV-1 IE RNAs was also stimulated by coinfection with HCMV, as was expression of the adenovirus 5 VAI transcript when the VAI gene was cloned into the HSV-1 genome. Coinfection with HCMV did not alter mRNA stability or uncoating of the HSV-1 genome. The coding sequences for the HCMV phosphoprotein pp71, controlled by the HCMV IE promoter, were cloned into an HSV-1 recombinant impaired for the production of the three major transactivators (VP16, ICP0, and ICP4) to yield a recombinant (
in
1324) which expressed pp71 but did not cause significant cytotoxicity. Infection with
in
1324 resulted in stimulation of HCMV IE, HSV-1 IE, and VAI expression, demonstrating that pp71 is responsible for the effects we observed when using the entire HCMV particle. Therefore, HCMV pp71 exhibits novel properties in its ability to stimulate gene expression from a range of promoters present in a herpesvirus genome.
Latent Adeno-Associated Virus Infection Elicits Humoral but Not Cell-Mediated Immune Responses in a Nonhuman Primate Model
Authors: Hernandez Yosbani J. , Wang Jianming , Kearns William G. , Loiler Scott , Poirier Amy , Flotte Terence R.
Published: December 2019

Latent infection with wild-type (wt) adeno-associated virus (AAV) was studied in rhesus macaques, a species that is a natural host for AAV and that has some homology to humans with respect to the preferred locus for wt AAV integration. Each of eight animals was infected with an inoculum of 10
10
IU of wt AAV, administered by either the intranasal, intramuscular, or intravenous route. Two additional animals were infected intranasally with wt AAV and a helper adenovirus (Ad), while one additional animal was inoculated with saline intranasally as a control. There were no detectable clinical or histopathologic responses to wt AAV administration. Molecular analyses, including Southern blot, PCR, and fluorescence in situ hybridization, were performed 21 days after infection. These studies indicated that AAV DNA sequences persisted at the sites of administration, albeit at low copy number, and in peripheral blood mononuclear cells. Site-specific integration into the AAVS1-like locus was observed in a subset of animals. All animals, except those infected by the intranasal route with wt AAV alone, developed a humoral immune response to wt AAV capsid proteins, as evidenced by a ≥fourfold rise in anti-AAV neutralizing titers. However, only animals infected with both wt AAV and Ad developed cell-mediated immune responses to AAV capsid proteins. These findings provide some insights into the nature of anti-AAV immune responses that may be useful in interpreting results of future AAV-based gene transfer studies.
Prevalence of Varicella-Zoster Virus DNA in Dissociated Human Trigeminal Ganglion Neurons and Nonneuronal Cells
Authors: LaGuardia James J. , Cohrs Randall J. , Gilden Donald H.
Published: December 2019

Previous analyses using in situ hybridization alone or together with PCR have yielded conflicting results regarding the cell type in which latent varicella-zoster virus (VZV) resides. We separated human trigeminal ganglia (TG) into neuronal and nonneuronal fractions, followed by primary and nested PCR to quantitate VZV DNA at the single cell level. Both TG from each of eight cadavers were dissociated and separated into neuronal and nonneuronal cell suspensions by differential filtration. Analysis of the neuron fraction (5,000 neurons per sample) revealed VZV DNA in 9 of 16 samples, with copy numbers ranging from 1 to 12, whereas only 2 of 16 nonneuronal cell samples were positive for VZV DNA, with 1 copy each. Further analysis of 10 samples of 100 neurons and the corresponding nonneuronal cell fractions from each TG of a single subject revealed VZV DNA in 3 of 10 samples of the left TG (range, 2 to 5 copies) and in 1 of 10 samples of the right TG (2 copies) but in none of the 20 nonneuronal cell fractions. These data indicate that latent VZV DNA is present primarily, if not exclusively, in neurons, at a frequency of two to five copies per latently infected neuron.
Postinoculation PMPA Treatment, but Not Preinoculation Immunomodulatory Therapy, Protects against Development of Acute Disease Induced by the Unique Simian Immunodeficiency Virus SIVsmmPBj
Authors: Hodge Shekema , de Rosayro Juliette , Glenn Amanda , Ojukwu Ifeoma C. , Dewhurst Stephen , McClure Harold M. , Bischofberger Norbert , Anderson Daniel C. , Klumpp Sherry A. , Novembre Francis J.
Published: December 2019

The fatal disease induced by SIVsmmPBj4 clinically resembles endotoxic shock, with the development of severe gastrointestinal disease. While the exact mechanism of disease induction has not been fully elucidated, aspects of virus biology suggest that immune activation contributes to pathogenesis. These biological characteristics include induction of peripheral blood mononuclear cell (PBMC) proliferation, upregulation of activation markers and Fas ligand expression, and increased levels of apoptosis. To investigate the role of immune activation and viral replication on disease induction, animals infected with SIVsmmPBj14 were treated with one of two drugs: FK-506, a potent immunosuppressive agent, or PMPA, a potent antiretroviral agent. While PBMC proliferation was blocked in vitro with FK-506, pig-tailed macaques treated preinoculation with FK-506 were not protected from acutely lethal disease. However, these animals did show some evidence of modulation of immune activation, including reduced levels of CD25 antigen and FasL expression, as well as lower tissue viral loads. In contrast, macaques treated postinoculation with PMPA were completely protected from the development of acutely lethal disease. Treatment with PMPA beginning as late as 5 days postinfection was able to prevent the PBj syndrome. Plasma and cellular viral loads in PMPA-treated animals were significantly lower than those in untreated controls. Although PMPA-treated animals showed acute lymphopenia due to SIVsmmPBj14 infection, cell subset levels subsequently recovered and returned to normal. Based upon subsequent CD4
+
cell counts, the results suggest that very early treatment following retroviral infection can have a significant effect on modifying the subsequent course of disease. These results also suggest that viral replication is an important factor involved in PBJ-induced disease. These studies reinforce the idea that the SIVsmmPBj model system is useful for therapy and vaccine testing.
A Chimeric Type 2 Adenovirus Vector with a Type 17 Fiber Enhances Gene Transfer to Human Airway Epithelia
Authors: Zabner Joseph , Chillon Miguel , Grunst Teresa , Moninger Thomas O. , Davidson Beverly L. , Gregory Richard , Armentano Donna
Published: December 2019

In studies of the genetic disease cystic fibrosis, recombinant adenovirus type 2 (Ad2) and Ad5 are being investigated as vectors to transfer cystic fibrosis transmembrane conductance regulator cDNA to airway epithelia. However, earlier work has shown that human airway epithelia are resistant to infection by Ad2 and Ad5. Therefore, we examined the efficiency of other adenovirus serotypes at infecting airway epithelia. We found that several serotypes of adenoviruses, in particular, wild-type Ad17, infected a greater number of cells than wild-type Ad2. The increased efficiency of wild-type Ad17 could be explained by increased fiber-dependent binding to the epithelia. Therefore, we constructed a chimeric virus, Ad2(17f)/βGal-2, which is identical to Ad2/βGal-2 with the exception of having the fiber protein of Ad17 replace Ad2 fiber. This vector retained the increased binding and efficiency of gene transfer to well-differentiated human airway epithelia. These data suggest that inclusion of Ad17 fiber into adenovirus vectors may improve the outlook for gene delivery to human airway epithelia.
Replication of Aleutian Mink Disease Parvovirus In Vivo Is Influenced by Residues in the VP2 Protein
Authors: Fox James M. , McCrackin Stevenson Mary A. , Bloom Marshall E.
Published: December 2019

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288–296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.
Evidence of Recombination among Enteroviruses
Authors: Santti Juhana , Hyypiä Timo , Kinnunen Leena , Salminen Mika
Published: December 2019

Human enteroviruses consist of more than 60 serotypes, reflecting a wide range of evolutionary divergence. They have been genetically classified into four clusters on the basis of sequence homology in the coding region of the single-stranded RNA genome. To explore further the genetic relationships between human enteroviruses and to characterize the evolutionary mechanisms responsible for variation, previously sequenced genomes were subjected to detailed comparison. Bootstrap and genetic similarity analyses were used to systematically scan the alignments of complete genomic sequences. Bootstrap analysis provided evidence from an early recombination event at the junction of the 5′ noncoding and coding regions of the progenitors of the current clusters. Analysis within the genetic clusters indicated that enterovirus prototype strains include intraspecies recombinants. Recombination breakpoints were detected in all genomic regions except the capsid protein coding region. Our results suggest that recombination is a significant and relatively frequent mechanism in the evolution of enterovirus genomes.
The Severity of Murray Valley Encephalitis in Mice Is Linked to Neutrophil Infiltration and Inducible Nitric Oxide Synthase Activity in the Central Nervous System
Authors: Andrews D. M. , Matthews V. B. , Sammels L. M. , Carrello A. C. , McMinn P. C.
Published: December 2019

A study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414–423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.
Altered Virulence of Vaccine Strains of Measles Virus after Prolonged Replication in Human Tissue
Authors: Valsamakis Alexandra , Auwaerter Paul G. , Rima Bert K. , Kaneshima Hideto , Griffin Diane E.
Published: December 2019

To understand the molecular determinants of measles virus (MV) virulence, we have used the SCID-hu thymus/liver xenograft model (SCID-hu thy/liv) in which in vivo MV virulence phenotypes are faithfully duplicated. Stromal epithelial and monocytic cells are infected by MV in thymus implants, and virulent strains induce massive thymocyte apoptosis, although thymocytes are not infected. To determine whether passage of an avirulent vaccine strain in human tissue increases virulence, we studied a virus isolated from thymic tissue 90 days after infection with the vaccine strain Moraten (pMor-1) and a virus isolated from an immunodeficient child with progressive vaccine-induced disease (Hu2). These viruses were compared to a minimally passaged wild-type Edmonston strain (Ed-wt) and the vaccine strain Moraten. pMor-1, Hu2, and Ed-wt displayed virulent phenotypes in thymic implants, with high levels of virus being detected by 3 days after infection (10
5.2
, 10
2.8
, and 10
3.4
, respectively) and maximal levels being detected between 7 and 14 days after infection. In contrast, Moraten required over 14 days to grow to detectable levels. pMor-1 produced the highest levels of virus throughout infection, suggesting thymic adaptation of this strain. Similar to other virulent strains, Ed-wt, Hu2, and pMor-1 caused a decrease in the number of viable thymocytes as assessed by trypan blue exclusion and fluorescence-activated cell sorter analysis. Thymic architecture was also disrupted by these strains. Sequence analysis of the hemagglutinin (H) and matrix (M) genes showed no common changes in Hu2 and pMor-1. M sequences were identical in pMor-1 and Mor and varied in H at amino acid 469 (threonine to alanine), a position near the base of propeller 4 in the propeller blade/stem model of H structure. Further study will provide insights into the determinants of virulence.
Localization of a Passively Transferred Human Recombinant Monoclonal Antibody to Herpes Simplex Virus Glycoprotein D to Infected Nerve Fibers and Sensory Neurons In Vivo
Authors: Sanna Pietro Paolo , Deerinck Thomas J. , Ellisman Mark H.
Published: December 2019

A human recombinant monoclonal antibody to herpes simplex virus (HSV) glycoprotein D labeled with the fluorescent dye Cy5 was administered to mice infected in the cornea with HSV type 1 (HSV-1). The distribution of such antibody in the corneas and trigeminal ganglia of the mice was then investigated by confocal microscopy. The antibody was detected on HSV-infected nerve fibers in the cornea—identified by colocalization with HSV antigens and the neuritic markers neurofilament, GAP-43, synapsin-1, and CNPase—and on the perikarya of sensory neurons in the HSV-1-infected neurons in ipsilateral trigeminal ganglia. Antibodies have been shown to be effective against many neurotropic viruses, often in the absence of obvious cell damage. Observations from experimental HSV infections suggest that antibodies could act in part by interfering with virus expression in the ganglia and/or with axonal spread. The present results provide morphological evidence of the localization of antiviral antibodies at anatomical sites relevant to such putative antibody-mediated protective actions and suggest that viral glycoproteins are accessible to antibodies on infected nerve fibers and sensory neurons.
Determinants of Neutralization Resistance in the Envelope Glycoproteins of a Simian-Human Immunodeficiency Virus Passaged In Vivo
Authors: Etemad-Moghadam Bijan , Sun Ying , Nicholson Emma K. , Karlsson Gunilla B. , Schenten Dominik , Sodroski Joseph
Published: December 2019

In vivo passage of a simian-human immunodeficiency virus (SHIV-89.6) generated a virus, SHIV-89.6P, that exhibited increased resistance to some neutralizing antibodies (G. B. Karlsson et al., J. Exp. Med. 188:1159–1171, 1998). Here we examine the range of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies to which the passaged virus became resistant and identify envelope glycoprotein determinants of antibody resistance. Compared with the envelope glycoproteins derived from the parental SHIV-89.6, the envelope glycoproteins of the passaged virus were resistant to antibodies directed against the gp120 V3 variable loop and the CD4 binding site. By contrast, both viral envelope glycoproteins were equally sensitive to neutralization by two antibodies, 2G12 and 2F5, that recognize poorly immunogenic structures on gp120 and gp41, respectively. Changes in the V2 and V3 variable loops of gp120 were necessary and sufficient for full resistance to the IgG1b12 antibody, which is directed against the CD4 binding site. Changes in the V3 loop specified complete resistance to a V3 loop-directed antibody, while changes in the V1/V2 loops conferred partial resistance to this antibody. The epitopes of the neutralizing antibodies were not disrupted by the resistance-associated changes. These results indicate that in vivo selection occurs for HIV-1 envelope glycoproteins with variable loop conformations that restrict the access of antibodies to immunogenic neutralization epitopes.
Amino Acids of Conserved Kinase Motifs of Cytomegalovirus Protein UL97 Are Essential for Autophosphorylation
Authors: Michel Detlef , Kramer Silke , Höhn Simone , Schaarschmidt Peter , Wunderlich Kirsten , Mertens Thomas
Published: December 2019

Thirteen point mutations targeting predicted domains conserved in homologous protein kinases were introduced into the UL97 coding region of the human cytomegalovirus. All mutagenized proteins were expressed in cells infected with recombinant vaccinia viruses (rVV). Several mutations drastically reduced ganciclovir (GCV) phosphorylation. Mutations at amino acids G340, A442, L446, and F523 resulted in a complete loss of pUL97 phosphorylation, which was strictly associated with a loss of GCV phosphorylation. Our results confirm that in rVV-infected cells pUL97 phosphorylation is due to autophosphorylation and show that several amino acids conserved within domains of protein kinases are essential for this pUL97 phosphorylation. GCV phosphorylation is dependent on pUL97 phosphorylation.
African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages
Authors: Oliveros Mariano , García-Escudero Ramón , Alejo Alí , Viñuela Eladio , Salas María L. , Salas José
Published: December 2019

The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg
2+
, the preferred cation, and has an apparent

K
m

for dUTP of 1 μM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vΔE165R) has been obtained. Growth kinetics showed that vΔE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.
Nonreplicative RNA Recombination in Poliovirus
Authors: Gmyl Anatoly P. , Belousov Evgeny V. , Maslova Svetlana V. , Khitrina Elena V. , Chetverin Alexander B. , Agol Vadim I.
Published: December 2019

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these “hot spots” could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5′ partner, if its 3′ end was oxidized or phosphorylated prior to being mixed with the 3′ partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2′,3′-cyclic phosphate and 5′-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.
A Primary Determinant of Cap-Independent Translation Is Located in the 3′-Proximal Region of the Tomato Bushy Stunt Virus Genome
Authors: Wu Baodong , White K. Andrew
Published: December 2019

Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus
Tombusvirus
. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5′ cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3′ terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845–5851, 1998). In the present report, we provide evidence that a 3′-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5′ cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3′ proximal element, designated as the 3′ cap-independent translational enhancer (3′CITE), that is required for proficient cap-independent translation; (iv) the 3′CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3′CITE activity; and (vi) no significant 3′CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3′CITE from other characterized plant viral 3′-proximal cap-independent translational enhancers. Additionally, because the 3′CITE overlaps with
cis
-acting replication signals, it could potentially participate in regulating the initiation of genome replication.