bluuebunny/crossref_metadata_2025 huggingface dataset:
Matrix Protein of Rabies Virus Is Responsible for the Assembly and Budding of Bullet-Shaped Particles and Interacts with the Transmembrane Spike Glycoprotein G
Authors: Mebatsion Teshome , Weiland Frank , Conzelmann Karl-Klaus
Published: December 2019

To elucidate the functions of rhabdovirus matrix (M) protein, we determined the localization of M in rabies virus (RV) and analyzed the properties of an M-deficient RV mutant. We provide evidence that M completely covers the ribonucleoprotein (RNP) coil and keeps it in a condensed form. As determined by cosedimentation experiments, not only the M-RNP complex but also M alone was found to interact specifically with the glycoprotein G. In contrast, an interaction of G with the nucleoprotein N or M-less RNP was not observed. In the absence of M, infectious particles were mainly cell associated and the yield of cell-free infectious virus was reduced by as much as 500,000-fold, demonstrating the crucial role of M in virus budding. Supernatants from cells infected with the M-deficient RV did not contain the typical bullet-shaped rhabdovirus particles but instead contained long, rod-shaped virions, demonstrating severe impairment of the virus formation process. Complementation with M protein expressed from plasmids rescued rhabdovirus formation. These results demonstrate the pivotal role of M protein in condensing and targeting the RNP to the plasma membrane as well as in incorporation of G protein into budding virions.
Generation of Bovine Respiratory Syncytial Virus (BRSV) from cDNA: BRSV NS2 Is Not Essential for Virus Replication in Tissue Culture, and the Human RSV Leader Region Acts as a Functional BRSV Genome Promoter
Authors: Buchholz Ursula J. , Finke Stefan , Conzelmann Karl-Klaus
Published: December 2019

In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all
cis
-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.
Two Nucleotides Immediately Upstream of the Essential A
6
G
3
Slippery Sequence Modulate the Pattern of G Insertions during Sendai Virus mRNA Editing
Authors: Hausmann Stéphane , Garcin Dominique , Morel Anne-Sophie , Kolakofsky Daniel
Published: December 2019

Editing of paramyxovirus P gene mRNAs occurs cotranscriptionally and functions to fuse an alternate downstream open reading frame to the N-terminal half of the P protein. G residues are inserted into a short G run contained within a larger purine run (A

n

G

n

) in this process, by a mechanism whereby the transcribing polymerase stutters (i.e., reads the same template cytosine more than once). Although Sendai virus (SeV) and bovine parainfluenza virus type 3 (bPIV3) are closely related, the G insertions in their P mRNAs are distributed differently. SeV predominantly inserts a single G residue within the G run of the sequence 5′ A
AC
AAAAAAGGG, whereas bPIV3 inserts one to six G’s at roughly equal frequency within the sequence 5′ A
UU
AAAAAAGGG
G
(differences are underlined). We have examined how the
cis
-acting editing sequence determines the number of G’s inserted, both in a transfected cell system using minigenome analogues and by generating recombinant viruses. We found that the presence of four rather than three G’s in the purine run did not affect the distribution of G insertions. However, when the underlined AC of the SeV sequence was replaced by the UU found in bPIV3, the editing phenotype from both the minigenome and the recombinant virus resembled that found in natural bPIV3 infections (i.e., a significant fraction of the mRNAs contained two to six G insertions). The two nucleotides located just upstream of the polypurine tract are thus key determinants of the editing phenotype of these viruses. Moreover, the minimum number of A residues that will promote SeV editing phenotype is six but can be reduced to five when the upstream AC is replaced by UU. A model for how the upstream dinucleotide controls the insertion phenotype is presented.
Diminished Production of Human Immunodeficiency Virus Type 1 in Astrocytes Results from Inefficient Translation ofgag,env, andnefmRNAs despite Efficient Expression of Tat and Rev
Authors: Gorry Paul R. , Howard Jane L. , Churchill Melissa J. , Anderson Jenny L. , Cunningham Anthony , Adrian Deborah , McPhee Dale A. , Purcell Damian F. J.
Published: December 2019

Astrocytes infected with human immunodeficiency virus type 1 (HIV-1) produce only minimal quantities of virus. The molecular events that limit acute-phase HIV-1 infection of astrocytes were examined after inducing acute-phase replication by transfection with the pNL4-3 proviral plasmid. The levels of HIV-1 mRNA were similarly high in both astrocytes and HeLa cells, but astrocytes produced approximately 50-fold less supernatant p24 than HeLa cells. We found that diminished HIV-1 production in astrocytes resulted from inefficient translation ofgag,env, andnefmRNAs that were efficiently transported to the cytoplasm. Tat- or Rev-dependent reporter constructs showed no defect in Tat or Rev function in astrocytes compared with HeLa cells. HIV-1 mRNAs were correctly spliced, but only Rev and Tat proteins were efficiently translated from their native mRNAs. Pulse-chase labelling and immunoblot experiments revealed no defect in protein processing, but levels of Gag, Env, or Nef protein expressed were dramatically reduced in astrocytes compared to HeLa cells. These results demonstrate that inefficient translation of HIV-1 structural proteins underlies the restricted infection of astrocytes. The efficient expression of functional Tat and Rev by astrocytes may contribute to HIV-1 neuropathogenesis.
Functional Coupling between Replication and Packaging of Poliovirus Replicon RNA
Authors: Nugent Constance I. , Johnson Kyle L. , Sarnow Peter , Kirkegaard Karla
Published: December 2019

Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687–1696, 1988), were encapsidated in
trans
by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its
trans
-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726–1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.
Hepatitis B Virus RNA-Binding Proteins Associated with Cytokine-Induced Clearance of Viral RNA from the Liver of Transgenic Mice
Authors: Heise Tilman , Guidotti Luca G. , Cavanaugh Victoria J. , Chisari Francis V.
Published: December 2019

Hepatitis B virus (HBV) gene expression is downregulated in the liver of HBV transgenic mice by a posttranscriptional mechanism that is triggered by the local production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) during intrahepatic inflammation (hepatitis). The molecular basis for this antiviral effect is unknown. In this study, we identified three HBV RNA-binding liver nuclear proteins (p45, p39, and p26) the relative abundance of which correlates with the abundance of HBV RNA in response to the induction of IFN-γ and TNF-α. All three proteins bind to a 91-bp element located at the 5′ end of a previously defined posttranscriptional regulatory element that is thought to mediate the nuclear export of HBV RNA. The presence of p45 correlates directly with the presence of HBV RNA, being detectable under baseline conditions when the viral RNA is abundant and undetectable when the viral RNA disappears in response to IFN-γ and TNF-α. In contrast, p26 is inversely related to HBV RNA, being detectable only when the viral RNA disappears following cytokine activation. Finally, p39 is constitutively expressed, and its abundance and mobility appear to be slightly increased by cytokine activation. These results suggest a model in which hepatocellular HBV RNA content might be controlled by the stabilizing and/or destabilizing influences of these RNA-binding proteins whose activity is regulated by cytokine-induced signaling pathways.
Herpes Simplex Virus Type 1 Vector-Mediated Expression of Nerve Growth Factor Protects Dorsal Root Ganglion Neurons from Peroxide Toxicity
Authors: Goins William F. , Lee Kevin A. , Cavalcoli James D. , O’Malley Mark E. , DeKosky Steven T. , Fink David J. , Glorioso Joseph C.
Published: December 2019

Nerve growth factor β subunit (β-NGF) transgene delivery and expression by herpes simplex virus type 1 (HSV-1) vectors was examined in a cell culture model of neuroprotection from hydrogen peroxide toxicity. Replication-competent (tk
−
K mutant background) and replication-defective (ICP4
−
;tk
−
S mutant background) vectors were engineered to contain the murine β-NGF cDNA under transcriptional control of either the human cytomegalovirus immediate-early gene promoter (HCMV IEp) (e.g., KHN and SHN) or the latency-active promoter 2 (LAP2) (e.g., KLN and SLN) within the viral thymidine kinase (tk) locus. Infection of rat B103 and mouse N2A neuronal cell lines, 9L rat glioma cells, and Vero cells with the KHN or SHN vectors resulted in the production of β-NGF-specific transcripts and β-NGF protein reaching a maximum at 3 days postinfection (p.i.). NGF protein was released into the culture media in amounts ranging from 10.83 to 352.86 ng/ml, with the highest levels being achieved in B103 cells, and was capable of inducing neurite sprouting of PC-12 cells. The same vectors produced high levels of NGF in primary dorsal root ganglion (DRG) cultures at 3 days. In contrast to HCMV IEp-mediated expression, the LAP2-NGF vectors showed robust expression in primary DRG neurons at 14 days. The neuroprotective effect of vector produced NGF was assessed by its ability to inhibit hydrogen peroxide-induced neuron toxicity in primary DRG cultures. Consistent with the kinetics of vector-mediated NGF expression, HCMV-NGF vectors were effective in abrogating the toxic effects of peroxide at 3 but not 14 days p.i. whereas LAP2-NGF vector transduction inhibited apoptosis in DRG neurons at 14 days p.i. but was ineffective at 3 days p.i. Similar kinetics of NGF expression were observed with the KHN and KLN vectors in latently infected mouse trigeminal ganglia, where high levels of β-NGF protein expression were detected at 4 wks p.i. only from the LAP2; HCMV-NGF-driven expression peaked at 3 days but could not be detected during HSV latency at 4 weeks. Together, these results indicate that (i) NGF vector-infected cells produce and secrete mature, biologically active β-NGF; (ii) vector-synthesized NGF was capable of blocking peroxide-induced apoptosis in primary DRG cultures; and (iii) the HCMV-IEp functioned to produce high levels of NGF for several days; but (iv) only the native LAP2 was capable of long-term expression of a therapeutic gene product in latently infected neurons in vivo.
Nuclear Targeting of the Cauliflower Mosaic Virus Coat Protein
Authors: Leclerc Denis , Chapdelaine Yvan , Hohn Thomas
Published: December 2019

The entry of the viral genomic DNA of cauliflower mosaic virus into the nucleus is a critical step of viral infection. We have shown by transient expression in plant protoplasts that the viral coat protein (CP), which is processed from the product of open reading frame IV, contains an N-terminal nuclear localization signal (NLS). The NLS is exposed on the surface of the virion and is thus available for interaction with a putative NLS receptor. Phosphorylation of the matured CP did not influence the nuclear localization of the protein but improved protein stability. Mutation of the NLS completely abolished viral infectivity, thus indicating its importance in the virus life cycle. The NLS seems to be regulated by the N terminus of the precapsid, which inhibits its nuclear targeting. This regulation could be important in allowing virus assembly in the cytoplasm.
Sin Nombre Virus Pathogenesis in
Peromyscus maniculatus
Authors: Netski Dale , Thran Brandolyn H. , St. Jeor Stephen C.
Published: December 2019

Sin Nombre virus (SNV), a member of the
Hantavirus
genus, causes acute viral pneumonia in humans and is thought to persistently infect mice. The deer mouse,
Peromyscus maniculatus
, has been identified as the primary reservoir host for SNV. To understand SNV infection of
P. maniculatus
, we examined wild deer mice for localization of viral antigens and nucleic acid. Morphologic examination consistently revealed septal edema within lung tissue and mononuclear cell infiltrates in portal areas of the liver. Immunohistochemical analysis of SNV-infected deer mice identified viral antigens within lung, liver, kidney, and spleen. The lungs consistently presented with the highest levels of viral antigen by immunohistochemistry and with the highest levels of nucleic acid by reverse transcriptase (RT) PCR. The mononuclear cell infiltrates surrounding liver portal triads were positive for SNV antigens in addition to resident macrophages in liver sinuses. Spleen tissue contained antigens in both the red pulp and the periartereolar region of the white pulp. The kidney presented with no gross pathology, although antigens could be localized to glomeruli. Virus antigen levels within the kidney were highest in deer mice that did not have antibodies to SNV but contained viral nucleic acid detectable by RT PCR. Since transmission is thought to occur via urine, our results suggest that virus transmission may be highest in the early stages of infection. In addition, these results indicate that SNV does cause some pathology within its reservoir host.
Herpes Simplex Virus Genome Isomerization: Origins of Adjacent Long Segments in Concatemeric Viral DNA
Authors: Slobedman B. , Zhang X. , Simmons A.
Published: December 2019

Herpes simplex virus type 1 DNA isomerization was studied by using a viral mutant, 5B8, lacking the unique
Spe
I site of its parent, SC16. In coinfected cells, SC16 genomic long segments flanked 5B8 genomes in all possible orientations with similar frequencies. Thus, recombination between progeny of different replication templates is sufficient to explain genomic isomerization.
Vpu Increases Susceptibility of Human Immunodeficiency Virus Type 1-Infected Cells to Fas Killing
Authors: Casella Carolyn R. , Rapaport Eric L. , Finkel Terri H.
Published: December 2019

The importance of the Fas death pathway in human immunodeficiency virus (HIV) infection has been the subject of many studies. Missing from these studies is direct measurement of infected cell susceptibility to Fas-induced death. To address this question, we investigated whether T cells infected with HIV are more susceptible to Fas-induced death. We found that Fas cross-linking caused a decrease in the number of HIV-infected Jurkat T cells and CD4+peripheral blood leukocytes (PBLs). We confirmed this finding by demonstrating that there were more apoptotic infected than uninfected cells after Fas ligation. The increase in sensitivity of HIV-infected cells to Fas killing mapped tovpu, whilenef,vif,vpr, and second exon oftatdid not appear to contribute. Furthermore, expression of Vpu in Jurkat T cells rendered them more susceptible to Fas-induced death. These results show that HIV-infected cells are more sensitive to Fas-induced death and that the Vpu protein of HIV contributes to this sensitivity. The increased sensitivity of HIV-infected cells to Fas-induced death might help explain why these cells have such a short in vivo half-life.
Infection of Chinese Hamster Ovary Cells by Pseudorabies Virus
Authors: Nixdorf Ralf , Schmidt Jerg , Karger Axel , Mettenleiter Thomas C.
Published: December 2019

Chinese hamster ovary (CHO) cells have recently been used for identification of receptors for several alphaherpesviruses, including pseudorabies virus (PrV) (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618–1620, 1998). The experiments were based on the fact that CHO cells are inefficient target cells for PrV. However, a detailed analysis of the interaction between PrV and CHO wild-type and recombinant PrV-receptor bearing cells has not been performed. We show here that PrV has a growth defect on CHO cells which leads to a ca. 100-fold reduction in plating efficiency, strongly delayed penetration kinetics, and a 10
4
-fold reduction in one-step growth. Entry of PrV into CHO cells is significantly delayed but is not affected by inhibitors of endocytosis, suggesting that the mechanism of penetration resembles that on permissive cells. The defects in plating efficiency and penetration could be corrected by expression of herpesvirus entry mediators B (HveB), HveC, or HveD, with HveC being the most effective. However, the defects in one-step growth and plaque formation were not corrected by expression of PrV receptors, indicating an additional restriction in viral replication after entry. Surprisingly, PrV infection of CHO cells was sensitive to neutralization by a gB-specific monoclonal antibody, which does not inhibit PrV infection of other host cells. Moreover, the same monoclonal antibody neutralized PrV infectivity on cells displaying the interference phenomenon by overexpression of gD and subsequent intracellular sequestration of gD receptors. Thus, absence of gD receptors on two different host cells leads to an increased sensitivity of PrV toward gB neutralization. We hypothesize that this is due to the increased requirement for interaction of gB with a cellular surface protein in the absence of the gD-gD receptor interaction. As expected, CHO cells are as susceptible as other host cells to infection by PrV gD
−
Pass, an infectious gD-negative PrV mutant. However, PrV gD
−
Pass was also not able to form plaques on CHO cells.
Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses
Authors: Basler Christopher F. , García-Sastre Adolfo , Palese Peter
Published: December 2019

The influenza virus neuraminidase (NA) is a tetrameric, virus surface glycoprotein possessing receptor-destroying activity. This enzyme facilitates viral release and is a target of anti-influenza virus drugs. The NA structure has been extensively studied, and the locations of disulfide bonds within the NA monomers have been identified. Because mutation of cysteine residues in other systems has resulted in temperature-sensitive (ts) proteins, we asked whether mutation of cysteine residues in the influenza virus NA would yield ts mutants. The ability to rationally design tight and stable ts mutations could facilitate the creation of efficient helper viruses for influenza virus reverse genetics experiments. We generated a series of cysteine-to-glycine mutants in the influenza A/WSN/33 virus NA. These were assayed for neuraminidase activity in a transient expression system, and active mutants were rescued into infectious virus by using established reverse genetics techniques. Mutation of two cysteines not involved in intrasubunit disulfide bonds, C49 and C146, had modest effects on enzymatic activity and on viral replication. Mutation of two cysteines, C303 and C320, which participate in a single disulfide bond located in the β5L0,1 loop, produced ts enzymes. Additionally, the C303G and C320G transfectant viruses were found to be attenuated and ts. Because both the C303G and C320G viruses exhibited stable ts phenotypes, they were tested as helper viruses in reverse genetics experiments. Efficiently rescued were an N1 neuraminidase from an avian H5N1 virus, an N2 neuraminidase from a human H3N2 virus, and an N7 neuraminidase from an H7N7 equine virus. Thus, these cysteine-to-glycine NA mutants allow the rescue of a variety of wild-type and mutant NAs into influenza virus.
The First Immunoglobulin-Like Domain of HveC Is Sufficient To Bind Herpes Simplex Virus gD with Full Affinity, While the Third Domain Is Involved in Oligomerization of HveC
Authors: Krummenacher Claude , Rux Ann H. , Whitbeck J. Charles , Ponce-de-Leon Manuel , Lou Huan , Baribaud Isabelle , Hou Wangfang , Zou Changhua , Geraghty Robert J. , Spear Patricia G. , Eisenberg Roselyn J. , Cohen Gary H.
Published: December 2019

The human herpesvirus entry mediator C (HveC/PRR1) is a member of the immunoglobulin family used as a cellular receptor by the alphaherpesviruses herpes simplex virus (HSV), pseudorabies virus, and bovine herpesvirus type 1. We previously demonstrated direct binding of the purified HveC ectodomain to purified HSV type 1 (HSV-1) and HSV-2 glycoprotein D (gD). Here, using a baculovirus expression system, we constructed and purified truncated forms of the receptor containing one [HveC(143t)], two [HveC(245t)], or all three immunoglobulin-like domains [HveC(346t)] of the extracellular region. All three constructs were equally able to compete with HveC(346t) for gD binding. The variable domain bound to virions and blocked HSV infection as well as HveC(346t). Thus, all of the binding to the receptor occurs within the first immunoglobulin-like domain, or V-domain, of HveC. These data confirm and extend those of Cocchi et al. (F. Cocchi, M. Lopez, L. Menotti, M. Aoubala, P. Dubreuil, and G. Campadelli-Fiume, Proc. Natl. Acad. Sci. USA 95:15700, 1998). Using biosensor analysis, we measured the affinity of binding of gD from HSV strains KOS and rid1 to two forms of HveC. Soluble gDs from the KOS strain of HSV-1 had the same affinity for HveC(346t) and HveC(143t). The mutant gD(rid1t) had an increased affinity for HveC(346t) and HveC(143t) due to a faster rate of complex formation. Interestingly, we found that HveC(346t) was a tetramer in solution, whereas HveC(143t) and HveC(245t) formed dimers, suggesting a role for the third immunoglobulin-like domain of HveC in oligomerization. In addition, the stoichiometry between gD and HveC appeared to be influenced by the level of HveC oligomerization.
Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins
Authors: Vincent Martin J. , Melsen Lawrence R. , Martin Annelet S. , Compans Richard W.
Published: December 2019

In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.
Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model
Authors: Kucerova Lucia , Altanerova Veronika , Altaner Cestmir , Boris-Lawrie Kathleen
Published: December 2019

Infection with a replication-competent bovine leukemia virus structural gene vector (BLV SGV) is an innovative vaccination approach to prevent disease by complex retroviruses. Previously we developed BLV SGV that constitutively expresses BLV
gag
,
pol
, and
env
and related
cis
-acting sequences but lacks
tax
,
rex
,
RIII
, and
GIV
and most of the BLV long terminal repeat sequences, including the
cis
-acting Tax and Rex response elements. The novel SGV virus is replication competent and replicates a selectable vector to a titer similar to that of the parental BLV in cell culture. The overall goal of this study was to test the hypothesis that infection with BLV SGV is nonpathogenic in rabbits. BLV infection of rabbits by inoculation of cell-free BLV or cell-associated BLV typically causes an immunodeficiency-like syndrome and death by 1 year postinfection. We sought to evaluate whether in vivo transfection of BLV provirus recapitulates pathogenic BLV infection and to compare BLV and BLV SGV with respect to infection, immunogenicity, and clinical outcome. Three groups of rabbits were subjected to in vivo transfection with BLV, BLV SGV, or negative control DNA. The results of our 20-month study indicate that in vivo transfection of rabbits with BLV recapitulates the fatal BLV infection produced by cell-free or cell-associated BLV. The BLV-infected rabbits exhibited sudden onset of clinical decline and immunodeficiency-like symptoms that culminated in death. BLV and BLV SGV infected peripheral blood mononuclear cells and induced similar levels of seroconversion to BLV structural proteins. However, BLV SGV exhibited a reduced proviral load and did not trigger the immunodeficiency-like syndrome. These results are consistent with the hypothesis that BLV SGV is infectious and immunogenic and lacks BLV pathogenicity in rabbits, and they support the use of this modified proviral vector delivery system for vaccines against complex retroviruses like BLV.
Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins
Authors: Wu Jianwen , Davis Michael D. , Owens Roland A.
Published: December 2019

The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in
Escherichia coli
cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.
Avirulent Avian Influenza Virus as a Vaccine Strain against a Potential Human Pandemic
Authors: Takada Ayato , Kuboki Noritaka , Okazaki Katsunori , Ninomiya Ai , Tanaka Hiroko , Ozaki Hiroichi , Itamura Shigeyuki , Nishimura Hidekazu , Enami Masayoshi , Tashiro Masato , Shortridge Kennedy F. , Kida Hiroshi
Published: December 2019

In the influenza H5N1 virus incident in Hong Kong in 1997, viruses that are closely related to H5N1 viruses initially isolated in a severe outbreak of avian influenza in chickens were isolated from humans, signaling the possibility of an incipient pandemic. However, it was not possible to prepare a vaccine against the virus in the conventional embryonated egg system because of the lethality of the virus for chicken embryos and the high level of biosafety therefore required for vaccine production. Alternative approaches, including an avirulent H5N4 virus isolated from a migratory duck as a surrogate virus, H5N1 virus as a reassortant with avian virus H3N1 and an avirulent recombinant H5N1 virus generated by reverse genetics, have been explored. All vaccines were formalin inactivated. Intraperitoneal immunization of mice with each of vaccines elicited the production of hemagglutination-inhibiting and virus-neutralizing antibodies, while intranasal vaccination without adjuvant induced both mucosal and systemic antibody responses that protected the mice from lethal H5N1 virus challenge. Surveillance of birds and animals, particularly aquatic birds, for viruses to provide vaccine strains, especially surrogate viruses, for a future pandemic is stressed.
Proteolytic Cleavage of the Amino Terminus of the U
L
15 Gene Product of Herpes Simplex Virus Type 1 Is Coupled with Maturation of Viral DNA into Unit-Length Genomes
Authors: Salmon Brandy , Nalwanga Dorothy , Fan Ying , Baines Joel D.
Published: December 2019

The U
L
15 gene of herpes simplex virus type 1 (HSV-1), like U
L
6, U
L
17, U
L
28, U
L
32, and U
L
33, is required for cleavage of concatameric DNA into genomic lengths and for packaging of cleaved genomes into preformed capsids. A previous study indicated that the U
L
15 gene encodes minor capsid proteins. In the present study, we have shown that the amino-terminal 509 amino acids of the U
L
15-encoded protein are sufficient to confer capsid association inasmuch as a carboxyl-terminally truncated form of the U
L
15-encoded protein with an
M
r
of approximately 55,000 readily associated with capsids. This and previous studies have shown that, whereas three U
L
15-encoded proteins with apparent
M
r
s of 83,000, 80,000, and 79,000 associated with wild-type B capsids, only the full-length 83,000-
M
r
protein associated with B capsids purified from cells infected with viruses lacking functional U
L
6, U
L
17, U
L
28, U
L
32, and U
L
33 genes (B. Salmon and J. D. Baines, J. Virol. 72:3045–3050, 1998). Thus, all viral mutants that fail to cleave viral DNA into genomic-length molecules also fail to produce capsid-associated U
L
15 80,000- and 79,000-
M
r
proteins. In contrast, the 80,000- and 79,000-
M
r
proteins were readily detected in capsids purified from cells infected with a U
L
25 null virus that cleaves, but does not package, DNA. The conclusion that the amino terminus of the 83,000-
M
r
protein is truncated to produce the 80,000- and/or 79,000-
M
r
protein was supported by the following observations. (i) Whereas the C termini of the 83,000-, 80,000-, and 79,000-
M
r
proteins are identical, immunoreactivity dependent on the first 35 amino acids of the U
L
15 83,000-
M
r
protein was absent from the 80,000- and 79,000-
M
r
proteins. (ii) The 79,000- and 80,000-
M
r
proteins were detected in capsids from cells infected with HSV-1(U
L
15M36V), an engineered virus encoding valine rather than methionine at codon 36. Thus, initiation at codon 36 is unlikely to account for production of the 80,000- and/or 79,000-
M
r
protein. Taken together, these data strongly suggest that capsid-associated U
L
15-encoded protein is proteolytically cleaved near the N terminus and indicate that this modification is tightly linked to maturation of genomic DNA.
A Phylogenetically Conserved Hairpin-Type 3′ Untranslated Region Pseudoknot Functions in Coronavirus RNA Replication
Authors: Williams Gwyn D. , Chang Ruey-Yi , Brian David A.
Published: December 2019

Secondary and tertiary structures in the 3′ untranslated region (UTR) of plus-strand RNA viruses have been postulated to function as control elements in RNA replication, transcription, and translation. Here we describe a 54-nucleotide (nt) hairpin-type pseudoknot within the 288-nt 3′ UTR of the bovine coronavirus genome and show by mutational analysis of both stems that the pseudoknotted structure is required for the replication of a defective interfering RNA genome. The pseudoknot is phylogenetically conserved among coronaviruses both in location and in shape but only partially in nucleotide sequence, and evolutionary covariation of bases to maintain G · U pairings indicates that it functions in the plus strand. RNase probing of synthetic transcripts provided additional evidence of its tertiary structure and also identified the possible existence of two conformational states. These results indicate that the 3′ UTR pseudoknot is involved in coronavirus RNA replication and lead us to postulate that it functions as a regulatory control element.
The Human Immunodeficiency Virus Type 1nefGene Can to a Large Extent Replace Simian Immunodeficiency VirusnefIn Vivo
Authors: Kirchhoff Frank , Münch Jan , Carl Silke , Stolte Nicole , Mätz-Rensing Kerstin , Fuchs Dietmar , Ten Haaft Peter , Heeney Jonathan L. , Swigut Tomek , Skowronski Jacek , Stahl-Hennig Christiane
Published: December 2019

Thenefgene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1)nefalleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of thenefalleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency.neflong terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6nefallele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1nefcan, to a large extent, functionally replace SIVmacnefin vivo.
Patchwork Pattern of Transcriptional Reactivation in the Lungs Indicates Sequential Checkpoints in the Transition from Murine Cytomegalovirus Latency to Recurrence
Authors: Kurz Sabine K. , Reddehase Matthias J.
Published: December 2019

The lungs are a significant organ site of murine cytomegalovirus (mCMV) latency. We have shown that activity of the major immediate-early promoter (MIEP), which drives the transcription from the
ie1-ie3
transcription unit, does not inevitably initiate the productive cycle (S. K. Kurz, M. Rapp, H.-P. Steffens, N. K. A. Grzimek, S. Schmalz, and M. J. Reddehase, J. Virol. 73:482–494, 1999). Thus, even though MIEP activity governed by the MIEP-enhancer is unquestionably the first condition for recurrence, regulation of the enhancer by transcription factors is not the only mechanism controlling latency. Specifically, during latency, focal and stochastic MIEP activity in lung tissue was found to selectively generate IE1 transcripts, while transactivator-specifying IE3 transcripts were missing. This suggested a control of mCMV latency that is effectual at IE1-IE3 precursor mRNA cotranscriptional processing. Here we have used this model for studying the kinetics of reactivation and recurrence in individual lung tissue pieces after hematoablative, genotoxic treatment. Notably, reactivation was triggered, but the number of transcriptionally active foci in the lungs did not increase over time. This result is not compatible with a model of spontaneous reactivations accumulating after withdrawal of immune control. Instead, the data support the idea that reactivation is an induced event. In some pieces, focal reactivation generated IE3 transcripts but not gB transcripts, while other pieces contained foci that had proceeded to gB transcription, and only a few foci actually reached the state of virus recurrence. This finding indicates the existence of several sequentially ordered control points in the transition from mCMV latency to recurrence.
Targeting Human Immunodeficiency Virus (HIV) Type 2 Integrase Protein into HIV Type 1
Authors: Liu Hongmei , Wu Xiaoyun , Xiao Hongling , Kappes John C.
Published: December 2019

Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell’s chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3′ processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN
2
) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN
2
into IN mutant HIV-1 virions by expression in
trans
as a Vpr-IN fusion protein.
Trans
-complementation with IN
2
markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous
trans
-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126–2135, 1999), cells infected with IN
2
-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous
trans
-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, the
trans
-IN
2
protein was shown to support integration up to a level of 55% compared with that of the homologous
trans
-IN protein. The delivery of heterologous IN protein into HIV-1 particles in
trans
offers a novel approach to understand IN protein function in vivo.
Development of Murine Leukemia Virus-Based Self-Activating Vectors That Efficiently Delete the Selectable Drug Resistance Gene during Reverse Transcription
Authors: Delviks Krista A. , Pathak Vinay K.
Published: December 2019

Expression of the selectable drug resistance gene in retroviral vectors used for gene therapy can lead to a decreased expression of the gene of interest and may induce a host immune response, resulting in a decreased efficiency of gene therapy. In this study, we demonstrate that high-frequency deletion of direct repeats, an inherent property of reverse transcriptases, can be used to efficiently excise the drug resistance gene during reverse transcription. One retroviral vector containing a direct repeat deleted the neomycin resistance expression cassette during a single replication cycle at >99% efficiency.
Walleye Dermal Sarcoma Virus: OrfA N-Terminal End Inhibits the Activity of a Reporter Gene Directed by Eukaryotic Promoters and Has a Negative Effect on the Growth of Fish and Mammalian Cells
Authors: Zhang Z. , Martineau D.
Published: December 2019

Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis. The WDSV genome contains three short open reading frames designated
orfA
,
orfB
, and
orfC
in addition to the viral structural genes,
gag
,
pol
, and
env
.
orfA
and
orfB
transcripts are detected in tumors by reverse transcription-PCR. Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain. We report that expression of the accessory gene
orfA
inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters. In addition, stable transfection with the wild-type
orfA
generated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector. An
orfA
mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects.
Divergent Requirements for the MAPKERKSignal Transduction Pathway during Initial Virus Infection of Quiescent Primary B Cells and Disruption of Epstein-Barr Virus Latency by Phorbol Esters
Authors: Fenton Mandy , Sinclair Alison J.
Published: December 2019

Quiescent primary B lymphocytes and Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines express components of the extracellular response kinase arm of the mitogen-activated protein kinase (MAPKERK) signal transduction pathway and transmit signals through the pathway when exposed to appropriate stimuli. Although the MAPKERKpathway is activated following infection with EBV, MAPK/ERK kinase (MEK1) activity is not required to drive the proliferation of infected cells. However, MEK1 contributes to EBV latency control.
Characterization of Intracellular Reverse Transcription Complexes of Moloney Murine Leukemia Virus
Authors: Fassati Ariberto , Goff Stephen P.
Published: December 2019

To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating reverse transcription. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete reverse transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of reverse transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during reverse transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further.
A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor
Authors: Trkola Alexandra , Matthews Jamie , Gordon Cynthia , Ketas Tom , Moore John P.
Published: December 2019

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies.