bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of Infectious Pancreatic Necrosis Virus from Cloned cDNA
Authors: Yao Kun , Vakharia Vikram N.
Published: December 2019

We developed a reverse genetics system for infectious pancreatic necrosis virus (IPNV), a prototype virus of theBirnaviridae family, with the use of plus-stranded RNA transcripts derived from cloned cDNA. Full-length cDNA clones of the IPNV genome that contained the entire coding and noncoding regions of RNA segments A and B were constructed. Segment A encodes a 106-kDa precursor protein which is cleaved to yield mature VP2, nonstructural protease, and VP3 proteins, whereas segment B encodes the RNA-dependent RNA polymerase VP1. Plus-sense RNA transcripts of both segments were prepared by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of chinook salmon embryo (CHSE) cells with combined transcripts of segments A and B generated infectious IPNV particles 10 days posttransfection. Furthermore, a transfectant virus containing a genetically tagged sequence was generated to confirm the feasibility of this system. The presence and specificity of the recovered virus were ascertained by immunofluorescence staining of infected CHSE cells with rabbit anti-IPNV serum and by nucleotide sequence analysis. In addition, 3′-terminal sequence analysis of RNA from the recovered virus showed that extraneous nucleotides synthesized at the 3′ end during in vitro transcription were precisely trimmed or excluded during replication, and hence these were not incorporated into the genome. An attempt was made to determine if RNA-dependent RNA polymerase of IPNV and infectious bursal disease virus (IBDV), another birnavirus, can support virus rescue in heterologous combinations. Thus, CHSE cells were transfected with transcripts derived from IPNV segment A and IBDV segment B and Vero cells were transfected with transcripts derived from IBDV segment A and IPNV segment B. In either case, no infectious IPNV or IBDV particles were generated even after a third passage in cell culture, suggesting that viral RNA-dependent RNA polymerase is species specific. However, the reverse genetics system for IPNV that we developed will greatly facilitate studies of viral replication and pathogenesis and the design of a new generation of live attenuated vaccines.
Importance of Basic Residues in the Nucleocapsid Sequence for Retrovirus Gag Assembly and Complementation Rescue
Authors: Bowzard J. Bradford , Bennett Robert P. , Krishna Neel K. , Ernst Sandra M. , Rein Alan , Wills John W.
Published: December 2019

The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger motifs and function to provide the proper density to viral particles. There are two zinc fingers and at least two I domains within these Gag proteins. To more thoroughly characterize the important sequence features and properties of I domains, we analyzed Gag proteins that contain one or no zinc finger motifs. Chimeric proteins containing the amino-terminal half of RSV Gag and various portions of the carboxy terminus of murine leukemia virus (MLV) (containing one zinc finger) Gag had only one I domain, whereas similar chimeras with human foamy virus (HFV) (containing no zinc fingers) Gag had at least two. Mutational analysis of the MLV NC sequence and inspection of I domain sequences within the zinc-fingerless C terminus of HFV Gag suggested that clusters of basic residues, but not the zinc finger motif residues themselves, are required for the formation of particles of proper density. In support of this, a simple string of strongly basic residues was found to be able to substitute for the RSV I domains. We also explored the possibility that differences in I domains (e.g., their number) account for differences in the ability of Gag proteins to be rescued into particles when they are unable to bind to membranes. Previously published experiments have shown that such membrane-binding mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with RSV-MLV chimeras now map this difference to the NC sequence of MLV. Importantly, the same RSV-MLV chimeras could be rescued by complementation when the block to budding was after, rather than before, transport to the membrane. These results suggest that MLV Gag molecules begin to interact at a much later time after synthesis than those of RSV and HIV.
Persistent Baculovirus Infection Results from Deletion of the Apoptotic Suppressor Gene p35
Authors: Lee Jin-Ching , Chen Hong-Hwa , Chao Yu-Chan
Published: December 2019

Infection with the wild-type baculovirusAutographa californicamultiple nuclear polyhedrosis virus (AcMNPV) results in complete death ofSpodoptera frugiperda(Sf) cells. However, infection of Sf cells with AcMNPV carrying a mutation or deletion of the apoptotic suppressor genep35allowed the cloning of surviving Sf cells that harbored persistent viral genomes. Persistent infection established with the virus withp35mutated or deleted was blocked by stable transfection ofp35in the host genome or by insertion of the inhibitor of apoptosis (iap) gene into the viral genome. These artificially established persistently virus-infected cells became resistant to subsequent viral challenge, and some of the cell lines carried large quantities of viral DNA capable of early gene expression. Continuous release of viral progenies was evident in some of the persistently virus-infected cells, and transfection ofp35further stimulated viral activation of the persistent cells, including the reactivation of viruses in those cell lines without original continuous virus release. These results have demonstrated the successful establishment of persistent baculovirus infections under laboratory conditions and that their establishment may provide a novel continuous, nonlytic baculovirus expression system in the future.
Persistence of Herpes Simplex Virus Type 1 DNA in Chronic Conjunctival and Eyelid Lesions of Mice
Authors: Maggs David J. , Chang Ed , Nasisse Mark P. , Mitchell William J.
Published: December 2019

Herpes simplex virus type 1 (HSV-1) causes chronic blepharitis and conjunctivitis as well as keratitis in humans. The pathogenesis of these inflammatory ocular and dermal lesions is not well understood. We have examined the persistence of HSV-1 DNA and its relationship to inflammatory lesions in the conjunctiva and eyelid skin of mice which were inoculated with HSV-1 by the corneal route. Viral DNA was detected by in situ PCR in the conjunctiva and eyelid tissue of infected mice at 5, 11, 23, and 37 days postinfection (p.i.). This DNA was localized in the epithelial cells of the conjunctiva and hair follicles and in the epidermal cells of the eyelid skin. Viral proteins were not detected in the conjunctiva or the eyelid skin after 5 days p.i., even though histopathological lesions were found at 23 and 37 days p.i. in both tissues. The DNA-containing cells were adjacent to sites of inflammation in the chronic lesions in both the conjunctiva and the eyelid skin. A similar temporal and spatial relationship between HSV-1 DNA and inflammatory lesions has been previously reported for the cornea. Our data suggest that the lesions in the cornea, conjunctiva, and eyelid skin progress similarly. Further studies are required to determine whether the long-term presence of HSV-1 is involved in the mechanism by which these chronic inflammatory lesions develop. The presence of HSV-1 DNA in these extraocular tissues for extended periods may constitute persistent viral infection of nonneuronal cells.
The 5′ and 3′ TAR Elements of Human Immunodeficiency Virus Exert Effects at Several Points in the Virus Life Cycle
Authors: Das Atze T. , Klaver Bep , Berkhout Ben
Published: December 2019

The human immunodeficiency virus type 1 RNA genome contains a terminal repeat (R) sequence that encodes the TAR hairpin motif, which has been implicated in Tat-mediated activation of transcription. More recently, a variety of other functions have been proposed for this structured RNA element. To determine the replicative roles of the 5′ and 3′ TAR hairpins, we analyzed multiple steps in the life cycle of wild-type and mutant viruses. A structure-destabilizing mutation was introduced in either the 5′, the 3′, or both TAR motifs of the proviral genome. As expected, opening of the 5′ TAR hairpin caused a transcription defect. Because the level of protein expression was not similarly reduced, the translation of this mRNA was improved. No effect of the 3′ hairpin on transcription and translation was measured. Mutations of the 5′ and 3′ hairpin structures reduced the efficiency of RNA packaging to similar extents, and RNA packaging was further reduced in the 5′ and 3′ TAR double mutant. Upon infection of cells with these virions, a reduced amount of reverse transcription products was synthesized by the TAR mutant. However, no net reverse transcription defect was observed after correction for the reduced level of virion RNA. This result was confirmed in in vitro reverse transcription assays. These data indicate that the 5′ and 3′ TAR motifs play important roles in several steps of the replication cycle, but these structures have no significant effect on the mechanism of reverse transcription.
Subunit Rotavirus Vaccine Administered Parenterally to Rabbits Induces Active Protective Immunity
Authors: Ciarlet Max , Crawford Sue E. , Barone Christopher , Bertolotti-Ciarlet Andrea , Ramig Robert F. , Estes Mary K. , Conner Margaret E.
Published: December 2019

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund’s adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10350% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P< 0.001,r= 0.55; Pearson’s correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund’s adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund’s adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund’s adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.
The Bipartite Geminivirus Coat Protein Aids BR1 Function in Viral Movement by Affecting the Accumulation of Viral Single-Stranded DNA
Authors: Qin Shenwei , Ward Brian M. , Lazarowitz Sondra G.
Published: December 2019

The movement of bipartite geminiviruses such as squash leaf curl virus (SqLCV) requires the cooperative interaction of two essential virus-encoded movement proteins, BR1 and BL1. While the viral coat protein AR1 is not essential for systemic infection, genetic studies demonstrate that its presence masks the defective phenotype of certain BR1 missense mutants, thus suggesting that coat protein does interact with the viral movement pathway. To further examine the mechanism of this interaction, we have constructed alanine-scanning mutants of AR1 and studied them for the ability to mask the infectivity defects of appropriate BR1 mutants, for the ability to target to the nucleus and to bind viral single-stranded DNA (ssDNA) and multimerize, and for effects on the accumulation of replicated viral ssDNA. We identified a specific region of AR1 required for masking of appropriate BR1 mutants and showed that this same region of AR1 was also important for ssDNA binding and the accumulation of viral replicated ssDNA. This region of AR1 also overlapped that involved in multimerization of the coat protein. We also found that the accumulation in protoplasts of single-stranded forms of a recombinant plasmid that included the SqLCV replication origin but was too large to be encapsidated was dependent on the presence of AR1 but did not appear to require encapsidation. These findings extend our model for SqLCV movement, demonstrating that coat protein affects viral movement through its ability to induce the accumulation of replicated viral ssDNA genomes. They further suggested that encapsidation was not required for the AR1-dependent accumulation of viral ssDNA.
Chemokine Coreceptor Usage by Diverse Primary Isolates of Human Immunodeficiency Virus Type 1
Authors: Zhang Linqi , He Tian , Huang Yaoxing , Chen Zhiwei , Guo Young , Wu Sam , Kunstman Kevin J. , Brown R. Clark , Phair John P. , Neumann Avidan U. , Ho David D. , Wolinsky Steven M.
Published: December 2019

We tested chemokine receptor subset usage by diverse, well-characterized primary viruses isolated from peripheral blood by monitoring viral replication with CCR1, CCR2b, CCR3, CCR5, and CXCR4 U87MG.CD4 transformed cell lines and STRL33/BONZO/TYMSTR and GPR15/BOB HOS.CD4 transformed cell lines. Primary viruses were isolated from 79 men with confirmed human immunodeficiency virus type 1 (HIV-1) infection from the Chicago component of the Multicenter AIDS Cohort Study at interval time points. Thirty-five additional well-characterized primary viruses representing HIV-1 group M subtypes A, B, C, D, and E and group O and three primary simian immunodeficiency virus (SIV) isolates were also used for these studies. The restricted use of the CCR5 chemokine receptor for viral entry was associated with infection by a virus having a non-syncytium-inducing phenotype and correlated with a reduced rate of disease progression and a prolonged disease-free interval. Conversely, broadening chemokine receptor usage from CCR5 to both CCR5 and CXCR4 was associated with infection by a virus having a syncytium-inducing phenotype and correlated with a faster rate of CD4 T-cell decline and progression of disease. We also observed a greater tendency for infection with a virus having a syncytium-inducing phenotype in men heterozygous for the defectiveCCR5 Δ32 allele (25%) than in those men homozygous for the wild-type CCR5 allele (6%) (P = 0.03). The propensity for infection with a virus having a syncytium-inducing phenotype provides a partial explanation for the rapid disease progression among some men heterozygous for the defectiveCCR5 Δ32 allele. Furthermore, we did not identify any primary viruses that used CCR3 as an entry cofactor, despite this CC chemokine receptor being expressed on the cell surface at a level commensurate with or higher than that observed for primary peripheral blood mononuclear cells. Whereas isolates of primary viruses of SIV also used STRL33/BONZO/TYMSTR and GPR15/BOB, no primary isolates of HIV-1 used these particular chemokine receptor-like orphan molecules as entry cofactors, suggesting a limited contribution of these other chemokine receptors to viral evolution. Thus, despite the number of chemokine receptors implicated in viral entry, CCR5 and CXCR4 are likely to be the physiologically relevant chemokine receptors used as entry cofactors in vivo by diverse strains of primary viruses isolated from blood.
Level of ICAM-1 Surface Expression on Virus Producer Cells Influences both the Amount of Virion-Bound Host ICAM-1 and Human Immunodeficiency Virus Type 1 Infectivity
Authors: Paquette Jean-Sébastien , Fortin Jean-François , Blanchard Luc , Tremblay Michel J.
Published: December 2019

Using virions harvested from 293T cells stably expressing either low or high levels of surface ICAM-1, we determined that the number of virus-embedded host ICAM-1 proteins is positively influenced by the expression level of ICAM-1 on virus producer cells. Moreover, the increase in virion-bound host cell membrane ICAM-1 led to a concomitant enhancement of virus infectivity when a T-cell-tropic strain of human immunodeficiency virus type 1 (HIV-1) was used. The phenomenon was also seen when primary human cells were infected with virions pseudotyped with the envelope protein from a macrophage-tropic HIV-1 isolate, thus ruling out any envelope-specific effect. We also observed that target cells treated with NKI-L16, an anti-LFA-1 antibody known to increase the affinity of LFA-1 for ICAM-1, were markedly more susceptible to infection with HIV-1 particles bearing on their surfaces large numbers of host-derived ICAM-1 proteins. Given that cellular activation of leukocytes is known to modify the conformational state of LFA-1 and induce ICAM-1 surface expression, it is tempting to speculate that activation of virus-infected cells will lead to the production of HIV-1 particles bearing more host ICAM-1 on their surfaces and that such progeny virions will preferentially infect and replicate more efficiently in activated cells which are prevalent in lymphoid organs.
Roles of the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein in Annealing and Initiation versus Elongation in Reverse Transcription of Viral Negative-Strand Strong-Stop DNA
Authors: Rong Liwei , Liang Chen , Hsu Mayla , Kleiman Lawrence , Petitjean Patrice , de Rocquigny Hugues , Roques Bernard P. , Wainberg Mark A.
Published: December 2019

To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lysprimer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(−)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (−)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.
RNA-Binding Activity of the E1B 55-Kilodalton Protein from Human Adenovirus Type 5
Authors: Horridge Jackie J. , Leppard Keith N.
Published: December 2019

The human adenovirus 5 E1B 55-kDa protein is required for efficient nucleocytoplasmic transport of late viral mRNAs. This protein is shown to have RNA-binding activity which maps to a region of the protein with homology to a family of RNA-binding proteins and which has been shown previously to be essential for functionality of the protein in vivo.
Definition of Amino Acid Residues on the Epitope Responsible for Recognition by Influenza A Virus H1-Specific, H2-Specific, and H1- and H2-Cross-Reactive Murine Cytotoxic T-Lymphocyte Clones
Authors: Tamura Manabu , Kuwano Koichi , Kurane Ichiro , Ennis Francis A.
Published: December 2019

We defined the epitopes recognized by three influenza A virus-specific,H-2Kd-restricted CD8+cytotoxic T-lymphocyte (CTL) clones: H1-specific clone A-12, H2-specific clone F-4, and H1- and H2-cross-reactive clone B7-B7. The A-12 and B7-B7 clones recognized the same peptide, which comprises amino acids 533 to 541 (IYSTVASSL) of A/PR/8 hemagglutinin (HA). The F-4 and B7-B7 clones both recognized the peptide which comprise amino acids 529 to 537 (IYATVAGSL) of A/Jap HA. Amino acids 533 to 541 of A/PR/8 HA are compatible with amino acids 529 to 537 of A/Jap HA. Amino acid S at positions 3 and 7 was responsible for recognition by H1-specific clone A-12, while amino acid G at position 7 was responsible for recognition by H2-specific clone F-4. Two conserved amino acids, T at position 4 and A at position 6, were responsible for recognition by H1-, and H2-cross-reactive clone B7-B7. These results indicate that a single nine-amino-acid region is recognized by HA-specific CTL clones of three different subtype specificities and that the amino acids responsible for the recognition by the CTL clones are different.
Guanylyltransferase Activity of the LEF-4 Subunit of Baculovirus RNA Polymerase
Authors: Guarino Linda A. , Jin Jianping , Dong Wen
Published: December 2019

The baculovirus
Autographa californica
nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that transcribes viral late genes. This polymerase is composed of four equimolar subunits, LEF-4, LEF-8, LEF-9, and p47. Here we present data indicating that the LEF-4 subunit of RNA polymerase is a guanylyltransferase. Incubation of RNA polymerase in the presence of divalent cation and radiolabeled GTP resulted in the formation of a covalent enzyme-guanylate complex that comigrated with the LEF-4 subunit. The label transfer assay showed an absolute requirement for divalent cation which could be satisfied by either manganese or magnesium. The reaction was specific for guanine nucleotides, and GTP was more effective than dGTP in the formation of enzyme-guanylate complex. To demonstrate that LEF-4 was the guanylyltransferase, the single subunit was overexpressed in baculovirus-infected cells. The overexpressed protein was primarily cytosolic, indicating that other proteins in the RNA polymerase complex were responsible for nuclear targeting of LEF-4. LEF-4 alone was able to covalently bind GMP, although less efficiently than viral RNA polymerase.
Multigene Tracking of Hepatitis C Virus Quasispecies after Liver Transplantation: Correlation of Genetic Diversification in the Envelope Region with Asymptomatic or Mild Disease Patterns
Authors: Sullivan Daniel G. , Wilson Jeffrey J. , Carithers Robert L. , Perkins James D. , Gretch David R.
Published: December 2019

To investigate the role of hepatitis C virus (HCV) quasispecies mutation in the pathogenesis of HCV infection, we analyzed changes in the genetic diversity of HCV genomes in 22 patients before and after liver transplantation by using heteroduplex mobility assay (HMA) technology. All patients were infected with HCV genotype 1 and developed high-titer posttransplant viremia. Each patient was classified according to the severity of posttransplant hepatitis, as assessed by standard biochemical and histological criteria. HCV quasispecies were characterized by HMA analysis of eight separate subgenomic regions of HCV, which collectively comprise 44% of the entire genome. The glycoprotein genes E1 and E2, as well as the nonstructural protein genes NS2 and NS3, had the greatest genetic divergence after liver transplantation (the change in the heteroduplex mobility ratio [HMR] ranged from 2.5 to 7.0%). In contrast, genes encoding the core, NS4, and NS5b proteins had the least amount of genetic divergence after liver transplantation (range, 0.3 to 1.2%). The E1/E2 region showed the greatest change in genetic diversity after liver transplantation, and the change in HMRs was 2.5- to 3.3-fold greater in patients with asymptomatic or moderate disease than in those with severe disease. The E1-5′ region of HCV quasispecies isolated from patients in the asymptomatic group had a significantly greater degree of diversification after liver transplantation than the same regions of HCV quasispecies isolated from patients in the severe disease group (
P
= 0.05). While changes in the genetic diversity of some nonstructural genes were also greater in asymptomatic patients or in patients with mild disease than in patients with severe disease, the results were not significant. Data from this cohort demonstrate that greater rates of HCV quasispecies diversification are associated with mild or moderate liver disease activity in this immunosuppressed population.
Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage φ6
Authors: Paatero Anja O. , Mindich Leonard , Bamford Dennis H.
Published: December 2019

Bacteriophage φ6 is a complex enveloped double-stranded RNA virus with a segmented genome and replication strategy quite similar to that of the
Reoviridae
. An in vitro packaging and replication system using purified components is available. The positive-polarity genomic segments are translocated into a preformed polymerase complex (procapsid) particle. This particle is composed of four proteins: the shell-forming protein P1, the RNA polymerase P2, and two proteins active in packaging. Protein P7 is involved in stable packaging, and protein P4 is a homomultimeric potent nucleoside triphosphatase that provides the energy for the RNA translocation event. In this investigation, we used mutational analysis to study P4 multimerization and assembly. P4 is assembled onto a preformed particle containing proteins P2 and P7 in addition to P1. Only simultaneous production of P1 and P4 in the same cell leads to P4 assembly on P1 alone, whereas the P1 shell is incompetent for accepting P4 if produced separately. The C-terminal part of P4 is essential for particle assembly but not for multimerization or enzymatic activity. Altering the P4 nucleoside triphosphate binding site destroys the ability to form multimers.
The Herpes Simplex Virus Triplex Protein, VP23, Exists as a Molten Globule
Authors: Kirkitadze Marina D. , Barlow Paul N. , Price Nicholas C. , Kelly Sharon M. , Boutell Christopher J. , Rixon Frazer J. , McClelland David A.
Published: December 2019

Two proteins, VP19C (50,260 Da) and VP23 (34,268 Da), make up the triplexes which connect adjacent hexons and pentons in the herpes simplex virus type 1 capsid. VP23 was expressed in
Escherichia coli
and purified to homogeneity by Ni-agarose affinity chromatography. In vitro capsid assembly experiments demonstrated that the purified protein was functionally active. Its physical status was examined by differential scanning calorimetry, ultracentrifugation, size exclusion chromatography, circular dichroism, fluorescence spectroscopy, and 8-anilino-1-naphthalene sulfonate binding studies. These studies established that the bacterially expressed VP23 exhibits properties consistent with its being in a partially folded, molten globule state. We propose that the molten globule represents a functionally relevant intermediate which is necessary to allow VP23 to undergo interaction with VP19C in the process of capsid assembly.
Thymic Dendritic Cells Are Primary Targets for the Oncogenic Virus SL3-3
Authors: Uittenbogaart Christel H. , Law Wendy , Leenen Pieter J. M. , Bristol Gregory , van Ewijk Willem , Hays Esther F.
Published: December 2019

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70+cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70+cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70+cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70+dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.
Retrovirus Packaging Cells Expressing the
Mus dunni
Endogenous Virus Envelope Facilitate Transduction of CHO and Primary Hematopoietic Cells
Authors: Wolgamot Greg , Rasko John E. J. , Miller A. Dusty
Published: December 2019

Mus dunni
endogenous virus (MDEV) infects a wide variety of cell types from many different species. To take advantage of this broad host range, we have constructed packaging cells (PD223) that produce virions bearing the MDEV envelope. PD223 cells are able to package Moloney murine leukemia virus-based vectors at a titer of 4 × 10
5
infectious units per ml in the absence of contaminating replication-competent retrovirus. Vectors packaged by PD223 cells are able to transduce CHO cells, which are resistant to transduction by many retroviruses, at ≥25-fold higher efficiency than vectors having other pseudotypes. A vector packaged by PD223 was found to be among the most efficient for transducing primary baboon and human CD34
+
cells.
Vaccine Protection against a Heterologous, Non-Syncytium-Inducing, Primary Human Immunodeficiency Virus
Authors: Robert-Guroff Marjorie , Kaur Harvinder , Patterson L. Jean , Leno Michel , Conley Anthony J. , McKenna Philip M. , Markham Phillip D. , Richardson Ersell , Aldrich Kristine , Arora Kamalpreet , Murty Lalita , Carter Lucretia , Zolla-Pazner Susan , Sinangil Faruk
Published: December 2019

Vaccine-induced protection of chimpanzees against laboratory-adapted and syncytium-inducing, multiply passaged primary human immunodeficiency virus type 1 (HIV-1) isolates, but not against non-syncytium-inducing, minimally passaged ones, has been demonstrated. Following challenge with such an isolate, HIV-1
5016
, we obtained complete protection in one of three chimpanzees previously protected against low- and high-dose HIV-1
SF2
exposures after immunization with an adenovirus-HIV-1
MN
gp160 priming–HIV-1
SF2
gp120 boosting regimen. At challenge, the protected chimpanzee exhibited broad humoral immunity, including neutralizing antibody activity. These results demonstrate the potential of this combination vaccine strategy and suggest that vaccine protection against an HIV isolate relevant to infection of people is feasible.
The African Swine Fever Virus Thymidine Kinase Gene Is Required for Efficient Replication in Swine Macrophages and for Virulence in Swine
Authors: Moore D. M. , Zsak L. , Neilan J. G. , Lu Z. , Rock D. L.
Published: December 2019

African swine fever virus (ASFV) replicates in the cytoplasm of infected cells and contains genes encoding a number of enzymes needed for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant TK gene deletion viruses were produced by using two highly pathogenic isolates of ASFV through homologous recombination with an ASFV p72 promoter–β-glucuronidase indicator cassette (p72GUS) flanked by ASFV sequences targeting the TK region. Attempts to isolate double-crossover TK gene deletion mutants on swine macrophages failed, suggesting a growth deficiency of TK
−
ASFV on macrophages. Two pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted to Vero cells, were used successfully to construct TK deletion viruses on Vero cells. The selected viruses grew well on Vero cells, but both mutants exhibited a growth defect on swine macrophages at low multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type levels. At high MOI, the macrophage growth defect was not apparent. The Malawi TK deletion mutant showed reduced virulence for swine, producing transient fevers, lower viremia titers, and reduced mortality. In contrast, 100% mortality was observed for swine inoculated with the TK
+
revertant virus. Swine surviving TK
−
ASFV infection remained free of clinical signs of African swine fever following subsequent challenge with the parental pathogenic ASFV. The data indicate that the TK gene of ASFV is important for growth in swine macrophages in vitro and is a virus virulence factor in swine.
Human Lymphoblastoid CD4
+
T Cells Become Permissive to Macrophage-Tropic Strains of Human Immunodeficiency Virus Type 1 after Passage into Severe Combined Immunodeficient Mice through In Vivo Upregulation of CCR5: In Vivo Dynamics of CD4
+
T-Cell Differentiation in Pathogenesis of AIDS
Authors: Lapenta Caterina , Parlato Stefania , Spada Massimo , Santini Stefano M. , Rizza Paola , Logozzi Mariantonia , Proietti Enrico , Belardelli Filippo , Fais Stefano
Published: December 2019

In this article, we show that passage in SCID mice rendered a human CD4
+
T-cell line (CEM cells) highly susceptible to infection by macrophage-tropic (M-tropic) strains and primary clinical isolates of human immunodeficiency virus type 1 (HIV-1). This in vivo-acquired permissiveness of CEM cells was associated with the induction of a CD45RO
+
phenotype as well as of some β-chemokine receptors. Regulated upon activation, normal T-cell expressed and secreted chemokine entirely inhibited the ability of M-tropic HIV-1 strains to infect these cells. These findings may lead to new approaches in investigating in vivo the capacity of different HIV strains to exploit chemokine receptors in relation to the dynamics of the activation and/or differentiation state of human CD4
+
T cells.
Modulation of Immune System Function by Measles Virus Infection: Role of Soluble Factor and Direct Infection
Authors: Fujinami Robert S. , Sun Xinmin , Howell Joseph M. , Jenkin James C. , Burns James B.
Published: December 2019

Measles virus infection can result in a variety of immunologic defects. We have begun studies to determine the basis for the lack of immune responsiveness to antigen and mitogen following infection. Here we present data showing that Epstein-Barr virus-transformed B-cell lines infected with measles virus produce a soluble factor that can inhibit antigen-specific T-cell proliferation and inhibit the proliferation of uninfected B cells. The soluble factor was neither interleukin-10, transforming growth factor β, nor alpha/beta interferon. B cells infected with measles virus or treated with the soluble factor were unable to present antigen to T cells in a manner that supported antigen-specific proliferation. This could represent one mechanism of how measles virus limits T-cell expansion. However, we found that once CD4+or CD8+T cells were activated, their cytolytic activity was intact whether infected with measles virus or treated with soluble factor. Thus, while slow to be generated these cytoxic cells could participate in viral clearance.
Mouse Mammary Tumor Virus Sequences Responsible for Activating Cellular Oncogenes
Authors: Grimm Sandra L. , Nordeen Steven K.
Published: December 2019

Integration of mouse mammary tumor virus (MMTV) near the
int
genes results in the inappropriate expression of these proto-oncogenes and initiates events that lead to the formation of mammary adenocarcinomas. In most cases, the MMTV provirus integrates in a transcriptional orientation opposite that of the
int
genes. We have used a novel, vector-based system designed to recapitulate the integration of MMTV upstream of the
int-2
promoter. Compared to a cellular promoter or another retroviral promoter, the MMTV long terminal repeat (LTR) in this configuration is particularly efficacious at activating the
int-2
promoter. The sequences responsible for enhancing the activity of the
int-2
promoter map to two domains in the 5′ end of the MMTV LTR. One domain is a previously defined element; the second is an element delineated by these studies that acts synergistically with the first. Both of these elements display mammary cell-specific activity. Thus, even though the MMTV promoter itself is weak without hormonal stimulation, viral integration can position the 5′ LTR elements to efficiently activate transcription from cellular proto-oncogenes. Other functional elements in the LTR have little effect on the activation of the
int-2
promoter. Even stimulation of the MMTV promoter with steroid hormones only modestly activates transcription from the
int-2
promoter, suggesting that the 5′ elements of the LTR are the predominant determinants of the tissue- and orientation-specific activation of cellular promoters by MMTV.
Cytokine Response in Multiple Lymphoid Tissues during the Primary Phase of Feline Immunodeficiency Virus Infection
Authors: Dean Gregg A. , Pedersen Niels C.
Published: December 2019

Type 1 and 2 cytokine mRNA responses were measured at various time periods and in various lymphoid compartments during the acute stage (first 4 months) of feline immunodeficiency virus (FIV) infection in laboratory cats. Cytokine responses were correlated with virus replication. Virus was detected in plasma and tissue from day 14 postinfection (p.i.) onward, peaked at 56 to 70 days, and declined greatly by 70 days. Virus replication was highest in the thymus, followed by spleen, mesenteric lymph nodes, and cervical lymph nodes. Baseline cytokine levels were highest in the mesenteric lymph nodes and lowest in the cervical lymph nodes. Cytokine upregulation after FIV infection was most dramatic in the cervical lymph nodes, with the greatest increase in interleukin-10 (IL-10) and gamma interferon (IFN-γ). Cytokine transcription in the mesenteric lymph node increased above baseline by day 14 p.i. for IFN-γ, IL-12p40, IL-4, and IL-10, while elevations in the spleen were mainly for IFN-γ, IL-12p40 and IL-10. An increase in IFN-γ, IL-10, and IL-12p40 occurred in the thymus at day 56 p.i., concomitant with the onset of thymitis. In general, type 2 cytokines (IL-4 and IL-10) were increased greater than 1 log over baseline, while the elevations in type 1 cytokines were less than 1 log. In the tissues tested, CD4+cells were the primary source of IL-2, IL-4, and IL-10. Both CD4+and CD8+cells produced IFN-γ, while no cytokine mRNA was detected in B cells. These results demonstrate the presence of a heterogeneous cytokine response in lymphoid tissues during the primary stage of FIV infection. The nature and intensity of the response differed from one compartment to the other and, in the case of the thymus, also with inflammatory changes. Although limited in scope, the present study confirms the usefulness of the FIV infection model in studying early cytokine events that lead to the secondary subclinical carrier state typical of most lentivirus infections.
Control of Human Immunodeficiency Virus Type 1 RNA Metabolism: Role of Splice Sites and Intron Sequences in Unspliced Viral RNA Subcellular Distribution
Authors: Séguin Béatrice , Staffa Alfredo , Cochrane Alan
Published: December 2019

In the course of examining the various factors which affect the metabolism of human immunodeficiency virus type 1 (HIV-1) RNA, we examined the role of intron sequences and splice sites in determining the subcellular distribution of the RNA. Using in situ hybridization, we demonstrated that in the absence of Rev, unspliced RNA generated with an HIV-1
env
expression construct displayed discrete localization in the nucleus, coincident with the location of the gene and not associated with SC35-containing nuclear speckles. Expression of Rev resulted in a disperse signal for the unspliced RNA throughout both the nucleus and the cytoplasm. Subsequent fractionation of the nucleus revealed that the majority of unspliced viral RNA within the nucleus is associated with the nuclear matrix and that upon expression of Rev, a small proportion of the unspliced RNA is found within the nucleoplasm. Mutations which altered splice site utilization did not alter the sequestration of unspliced RNA into discrete nuclear regions. In contrast, a 2.2-kb deletion of intron sequence resulted in a shift from discrete regions within the nucleus to a disperse signal throughout the cell, indicating that intron sequences, and not just splice sites, are required for the observed nuclear sequestration of unspliced viral RNA.
Immunization with a Single Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Recognition Epitope of Herpes Simplex Virus Type 2 Confers Protective Immunity
Authors: Blaney Joseph E. , Nobusawa Eri , Brehm Michael A. , Bonneau Robert H. , Mylin Lawrence M. , Fu Tong-Ming , Kawaoka Yoshihiro , Tevethia Satvir S.
Published: December 2019

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8+cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8+T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8+CTL in HSV vaccine design.
A
cis
Repression Sequence Adjacent to the Transcription Start Site of the Human Cytomegalovirus US3 Gene Is Required To Down Regulate Gene Expression at Early and Late Times after Infection
Authors: Lashmit Philip E. , Stinski Mark F. , Murphy Eain A. , Bullock Grant C.
Published: December 2019

Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a
cis
repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MIE) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was determined by comparing the levels of transcription from these two enhancer-containing promoters in recombinant viruses with a wild-type or mutant CRS. Upstream of the CRS of the US3 promoter was either the endogenous enhancer (R
2
) or silencer (R
1
). The downstream US3 gene was replaced with the indicator gene chloramphenicol acetyltransferase (CAT). Infected permissive human fibroblast cells or nonpermissive, undifferentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the wild-type or mutant CRS. With the wild-type CRS, the maximum level of transcription in permissive cells was detected within 4 to 6 h after infection and then declined. With the mutant CRS and the R
2
enhancer upstream, expression from the US3 promoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS was mutated. Less expression was obtained when only the R
1
element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-containing US3 promoter appears to allow for a short burst of US3 gene expression followed by repression at early and late times after infection. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of the CRS and the viral IE86 protein in controlling enhancer-containing promoters is discussed.
Targeting of a Short Peptide Derived from the Cytoplasmic Tail of the G1 Membrane Glycoprotein of Uukuniemi Virus (
Bunyaviridae
) to the Golgi Complex
Authors: Andersson Agneta M. , Pettersson Ralf F.
Published: December 2019

Members of the
Bunyaviridae
family acquire an envelope by budding through the lipid bilayer of the Golgi complex. The budding compartment is thought to be determined by the accumulation of the two heterodimeric membrane glycoproteins G1 and G2 in the Golgi. We recently mapped the retention signal for Golgi localization in one
Bunyaviridae
member (Uukuniemi virus) to the cytoplasmic tail of G1. We now show that a myc-tagged 81-residue G1 tail peptide expressed in BHK21 cells is efficiently targeted to the Golgi complex and retained there during a 3-h chase. Green-fluorescence protein tagged at either end with this peptide or with a C-terminally truncated 60-residue G1 tail peptide was also efficiently targeted to the Golgi. The 81-residue peptide colocalized with mannosidase II (a medial Golgi marker) and partially with p58 (an intermediate compartment marker) and TGN38 (a
trans
-Golgi marker). In addition, the 81-residue tail peptide induced the formation of brefeldin A-resistant vacuoles that did not costain with markers for other membrane compartments. Removal of the first 10 N-terminal residues had no effect on the Golgi localization but abolished the vacuolar staining. The shortest peptide still able to become targeted to the Golgi encompassed residues 10 to 40. Subcellular fractionation showed that the 81-residue tail peptide was associated with microsomal membranes. Removal of the two palmitylation sites from the tail peptide did not affect Golgi localization and had only a minor effect on the association with microsomal membranes. Taken together, the results provide strong evidence that Golgi retention of the heterodimeric G1-G2 spike protein complex of Uukuniemi virus is mediated by a short region in the cytoplasmic tail of the G1 glycoprotein.