bluuebunny/crossref_metadata_2025 huggingface dataset:
Rescue and Autonomous Replication of Adeno-Associated Virus Type 2 Genomes Containing Rep-Binding Site Mutations in the Viral p5 Promoter
Authors: Wang Xu-Shan , Srivastava Arun
Published: December 2019

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.
Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding
Authors: Lutzke Ramon A. Puras , Plasterk Ronald H. A.
Published: December 2019

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN
220–270
) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN
220–270
, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN
220–270
, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN.
The Rep52 Gene Product of Adeno-Associated Virus Is a DNA Helicase with 3′-to-5′ Polarity
Authors: Smith Richard H. , Kotin Robert M.
Published: December 2019

The
rep
gene of adeno-associated virus type 2 encodes four overlapping proteins from two separate promoters, termed P
5
and P
19
. The P
5
-promoted Rep proteins, Rep78 and Rep68, are essential for viral DNA replication, and a wealth of data concerning the biochemical activities of these proteins has been reported. In contrast, data concerning the biochemical functions of the P
19
-promoted Rep proteins, Rep52 and Rep40, are lacking. Here, we describe enzymatic activities associated with a bacterially expressed maltose-binding protein (MBP)-Rep52 fusion protein. Purified MBP-Rep52 possesses 3′-to-5′ DNA helicase activity that is strictly dependent upon the presence of nucleoside triphosphate and divalent cation cofactors. In addition, MBP-Rep52 demonstrates a constitutive ATPase activity that is active in the absence of DNA effector molecules. An MBP-Rep52 chimera bearing a lysine-to-histidine substitution at position 116 (K116H) within a consensus helicase- and ATPase-associated motif (motif I or Walker A site) was deficient for both DNA helicase and ATPase activities. In contrast to a Rep78 A-site mutant protein bearing a corresponding amino acid substitution at position 340 (K340H), the MBP-Rep52 A-site mutant protein failed to exhibit a
trans
-dominant negative effect when it was mixed with wild-type MBP-Rep52 or MBP-Rep78 in vitro. This lack of
trans
dominance, coupled with the results of coimmunoprecipitation and gel filtration chromatography experiments reported here, suggests that the ability of Rep52 to engage in multimeric interactions may differ from that of Rep78 or -68.
Long-Term Evolution of the Hypervariable Region of Hepatitis C Virus in a Common-Source-Infected Cohort
Authors: McAllister Jane , Casino Carmela , Davidson Fiona , Power Joan , Lawlor Emer , Yap Peng Lee , Simmonds Peter , Smith Donald B.
Published: December 2019

The long-term evolution of the hepatitis C virus hypervariable region (HVR) and flanking regions of the E1 and E2 envelope proteins have been studied in a cohort of women infected from a common source of anti-D immunoglobulin. Whereas virus sequences in the infectious source were relatively homogeneous, distinct HVR variants were observed in each anti-D recipient, indicating that this region can evolve in multiple directions from the same point. Where HVR variants with dissimilar sequences were present in a single individual, the frequency of synonymous substitution in the flanking regions suggested that the lineages diverged more than a decade previously. Even where a single major HVR variant was present in an infected individual, this lineage was usually several years old. Multiple lineages can therefore coexist during long periods of chronic infection without replacement. The characteristics of amino acid substitution in the HVR were not consistent with the random accumulation of mutations and imply that amino acid replacement in the HVR was strongly constrained. Another variable region of E2 centered on codon 60 shows similar constraints, while HVR2 was relatively unconstrained. Several of these features are difficult to explain if a neutralizing immune response against the HVR is the only selective force operating on E2. The impact of PCR artifacts such as nucleotide misincorporation and the shuffling of dissimilar templates is discussed.
Permissive Cytomegalovirus Infection of Primary Villous Term and First Trimester Trophoblasts
Authors: Hemmings D. G. , Kilani R. , Nykiforuk C. , Preiksaitis J. , Guilbert L. J.
Published: December 2019

Forty percent of women with primary cytomegalovirus (CMV) infections during pregnancy infect their fetuses with complications for the baby varying from mild to severe. How CMV crosses the syncytiotrophoblast, the barrier between maternal blood and fetal tissue in the villous placenta, is unknown. Virus may cross by infection of maternal cells that pass through physical breaches in the syncytiotrophoblast or by direct infection of the syncytiotrophoblast, with subsequent transmission to underlying fetal placental cells. In this study, we show that pure (>99.99%), long-term and healthy (>3 weeks) cultures of syncytiotrophoblasts are permissively infected with CMV. Greater than 99% of infectious progeny virus remained cell associated throughout culture periods up to 3 weeks. Infection of term trophoblasts required a higher virus inoculum, was less efficient, and progressed more slowly than parallel infections of placental and human embryonic lung fibroblasts. Three laboratory strains (AD169, Towne, and Davis) and a clinical isolate from a congenitally infected infant all permissively infected trophoblasts, although infection efficiencies varied. The infection of first trimester syncytiotrophoblasts with strain AD169 occurred at higher frequency and progressed more rapidly than infection of term cells but less efficiently and rapidly than infection of fibroblasts. These results show that villous syncytiotrophoblasts can be permissively infected by CMV but that the infection requires high virus titers and proceeds slowly and that progeny virus remains predominantly cell associated.
Mapping of Homologous Interaction Sites in the Hepatitis B Virus Core Protein
Authors: König Sabine , Beterams Gertrud , Nassal Michael
Published: December 2019

Hepatitis B virus consists of an outer envelope and an inner capsid, or core, that wraps around the small genome plus the viral replication enzyme. The icosahedrally symmetric nucleocapsid is assembled from multiple dimeric subunits of a single 183-residue capsid protein, which must therefore contain interfaces for monomer dimerization and for dimer multimerization. The atomic structure of the protein is not known, but electron microscopy-based image reconstructions suggested a hammerhead shape for the dimer and, very recently, led to a tentative model for the main chain trace. Here we used a combination of interaction screening techniques and functional analyses of core protein variants to define, at the primary sequence level, the regions that mediate capsid assembly. Both the two-hybrid system and the pepscan technique identified a strongly interacting region I between amino acids (aa) 78 and 117 that probably forms part of the dimer interface. Surprisingly, mutations in this region, in the context of a C-terminally truncated but assembly-competent core protein variant, had no detectable effect on assembly. By contrast, mutations in a second region, bordered by aa 113 and 143, markedly influenced capsid stability, strongly suggesting that this region II is the main contributor to dimer multimerization. Based on the electron microscopic data, it must therefore be located at the basal tips of the dimer, experimentally supporting the proposed main chain trace.
Limited Transmission of Kaposi’s Sarcoma-Associated Herpesvirus in Cultured Cells
Authors: Renne Rolf , Blackbourn David , Whitby Denise , Levy Jay , Ganem Don
Published: December 2019

Kaposi’s sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) is a novel gammaherpesvirus strongly implicated in the pathogenesis of Kaposi’s sarcoma. Although virions can be produced in high yield from latently infected B-cell lines treated with phorbol esters, little is known about the infectivity of such virus, and efficient serial propagation of KSHV has been problematic. Here we report on the infectivity of KSHV produced from phorbol-induced BCBL-1 cells, employing an assay based on the detection of a spliced late mRNA by a sensitive reverse transcriptase PCR (RT-PCR) method. The results of this study confirm previous observations that 293 cells are susceptible to viral infection; however, infection with BCBL-1-derived virus is inefficient and the pattern of viral gene expression in infected cells may not fully reproduce that of authentic lytic infection. In keeping with this finding, serial propagation of BCBL-1-derived virus could not be demonstrated on 293 cells. Eleven of 38 other cell lines tested also supported KSHV infection, as judged by this RT-PCR assay, including cells of B-cell, endothelial, epithelial, and fibroblastic origin; however, in all cases, infection proceeded at or below the levels observed in 293 cells.
Relative Rates of Retroviral Reverse Transcriptase Template Switching during RNA- and DNA-Dependent DNA Synthesis
Authors: Bowman Robert R. , Hu Wei-Shau , Pathak Vinay K.
Published: December 2019

Retroviral reverse transcriptases (RTs) frequently switch templates during DNA synthesis, which can result in mutations and recombination. The relative rates of in vivo RT template switching during RNA- and DNA-dependent DNA synthesis are unknown. To determine the relative rates of RT template switching during copying of RNA and DNA templates, we constructed spleen necrosis virus-based retroviral vectors containing a 400-bp direct repeat. The directly repeated sequences were upstream of the polypurine tract (PPT) in the RB-LLP vector; the same direct repeats flanked the PPT and attachment site (
att
) in the RB-LPL vector. RT template switching events could occur during either RNA- or DNA-dependent DNA synthesis and delete one copy of the direct repeat plus the intervening sequences. RB-LLP vectors that underwent direct repeat deletions during RNA- and DNA-dependent DNA synthesis generated viral DNA that could integrate into the host genome. However, any deletion of the direct repeats in the RB-LPL vector that occurred during RNA-dependent DNA synthesis resulted in deletion of the essential PPT and
att
site and generated a dead-end viral DNA product. Thus, only RB-LPL vectors that underwent direct repeat deletions during DNA-dependent DNA synthesis could integrate to form proviruses. The RB-LLP and RB-LPL vectors were permitted to undergo a single replication cycle, and the frequencies of direct repeat deletions were determined by PCR and Southern analysis of the resulting proviruses. A comparison of the frequency of direct repeat deletions in the RB-LLP and RB-LPL vectors indicated that the in vivo rates of RT template switching during RNA- and DNA-dependent DNA synthesis are nearly identical.
Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes
Authors: Ponnazhagan Selvarangan , Weigel Kirsten A. , Raikwar Sudhanshu P. , Mukherjee Pinku , Yoder Mervin C. , Srivastava Arun
Published: December 2019

A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.
Incorporation of Fowl Plague Virus Hemagglutinin into Murine Leukemia Virus Particles and Analysis of the Infectivity of the Pseudotyped Retroviruses
Authors: Hatziioannou Theodora , Valsesia-Wittmann Sandrine , Russell Stephen J. , Cosset François-Loïc
Published: December 2019

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a
lacZ
retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.
Threshold Number of Provirus Copies Required per Cell for Efficient Virus Production and Interference in Moloney Murine Leukemia Virus-Infected NIH 3T3 Cells
Authors: Odawara Takashi , Oshima Masamichi , Doi Kent , Iwamoto Aikichi , Yoshikura Hiroshi
Published: December 2019

The
gag-pol
readthrough mutant of Moloney murine leukemia virus, MLV-B(CAG) (T. Odawara, H. Yoshikura, M. Oshima, T. Tanaka, D. S. Jones, F. Nemoto, Y. Kuchino, and A. Iwamoto, J. Virol. 65:6376–6379, 1991), was poorly complemented by a mutant encoding only Gag. This is because with all the genetic elements necessary for
env
expression present in MLV-B(CAG), insufficient Env protein was produced by the cells expressing MLV-B(CAG) for efficient virus production. Since the
env
mRNA expression per provirus in the MLV-B(CAG)- and wild-type-MLV-producing cells were the same and since the cells expressing the former contained eightfold fewer proviral copies, the insufficient Env expression by the former was found to be due to insufficient proviral copies in the cells. Examination of the cell clones having various proviral copies of Δwt MLV (M. Oshima, T. Odawara, T. Matano, H. Sakahira, Y. Kuchino, A. Iwamoto, and H. Yoshikura, J. Virol. 70:2286–2295, 1996) showed that mRNA level was proportional to the number of proviral copies while interference and virus production followed a sigmoid curve with a sharp rise at the threshold number of proviral copies of around four per cell. Multicycle infection probably continues until the threshold level of proviral copies is attained in natural infection too.
Requirement for Cellular Cyclin-Dependent Kinases in Herpes Simplex Virus Replication and Transcription
Authors: Schang Luis M. , Phillips Joanna , Schaffer Priscilla A.
Published: December 2019

Several observations indicate that late-G
1
/S-phase-specific cellular functions may be required for herpes simplex virus (HSV) replication: (i) certain mutant HSV strains are replication impaired during infection of cells in the G
0
/G
1
but not in the G
1
/S phase of the cell cycle, (ii) several late-G
1
/S-phase-specific cellular proteins and functions are induced during infection, and (iii) the activity of a cellular protein essential for expression of viral immediate-early (IE) genes, HCF, is normally required during the late G
1
/S phase of the cell cycle. To test the hypothesis that late-G
1
/S-phase-specific cellular functions are necessary for HSV replication, HEL or Vero cells were infected in the presence of the cell cycle inhibitors roscovitine (Rosco) and olomoucine (Olo). Both drugs inhibit cyclin-dependent kinase 1 (cdk-1) and cdk-2 (required for cell cycle progression into the late G
1
/S phase) and cdk-5 (inactive in cycling cells) but not cdk-4 or cdk-6 (active at early G
1
). We found that HSV replication was inhibited by Rosco and Olo but not by lovastatin (a cell cycle inhibitor that does not inhibit cdk activity), staurosporine (a broad-spectrum protein serine-threonine kinase inhibitor), PD98059 (an inhibitor specific for erk-1 and -2) or iso-Olo (a structural isomer of Olo that does not inhibit cdk activity). The concentrations of Rosco and Olo required to inhibit cell cycle progression and viral replication in both HEL and Vero cells were similar. Inhibition of viral replication was found not to be mediated by drug-induced cytotoxicity. Efforts to isolate Rosco- or Olo-resistant HSV mutants were unsuccessful, indicating that these drugs do not act by inhibiting a single viral target. Viral DNA replication and accumulation of IE and early viral RNAs were inhibited in the presence of cell cycle-inhibitory concentrations of Rosco or Olo. We therefore conclude that one or more cdks active from late G
1
onward or inactive in nonneuronal cells are required for accumulation of HSV transcripts, viral DNA replication, and production of infectious virus.
The Role of Mouse Adenovirus Type 1 Early Region 1A in Acute and Persistent Infections in Mice
Authors: Smith Kimberley , Brown Corrie C. , Spindler Katherine R.
Published: December 2019

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) viral mutants were used to determine the importance of this region in pathogenesis and establishment of a persistent infection in the natural host. Lethal dose analysis with adult male Swiss outbred mice revealed a significant reduction in virulence for all of the E1A mutants. During acute infections with 105PFU of virus, an E1A null mutant,pmE109, was found in the same organs (brain, spleen, and spinal cord) and the same cell types (endothelial cells and mononuclear cells in lymphoid tissue) as wild-type virus. Another null mutant,pmE112, was detected in the same organs but in lower numbers. However, when mice were given a lower dose, 1 PFU,pmE109 andpmE112 reached none of the target organs analyzed by 14 days postinfection (p.i.). The absence of E1A did not hinder the ability of MAV-1 to establish a persistent infection. Viral nucleic acid was detected by PCR amplification or in situ hybridization in the kidneys, brains, spleens, and prefemoral lymph nodes of mice infected with wild-type or mutant virus up to 55 weeks p.i. The brain, spleen, and lymph node are recognized sites of acute viral infection but are previously unrecognized sites for MAV-1 persistence. Evidence for the potential reactivation of persistent MAV-1 infections is also presented.
Protective Immunity Induced by Oral Immunization with a Rotavirus DNA Vaccine Encapsulated in Microparticles
Authors: Chen Shing C. , Jones David H. , Fynan Ellen F. , Farrar Graham H. , Clegg J. Christopher S. , Greenberg Harry B. , Herrmann John E.
Published: December 2019

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.
DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles
Authors: Cassetti Maria Cristina , Merchlinsky Michael , Wolffe Elizabeth J. , Weisberg Andrea S. , Moss Bernard
Published: December 2019

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-β-
d
-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.
Uncoupled Expression of p33 and p92 Permits Amplification of Tomato Bushy Stunt Virus RNAs
Authors: Oster Sara K. , Wu Baodong , White K. Andrew
Published: December 2019

Tomato bushy stunt virus (TBSV) is a plus-sense RNA virus which encodes a 33-kDa protein in its 5′-most open reading frame (ORF). Readthrough of the amber stop codon of the p33 ORF results in the production of a 92-kDa fusion protein. Both of these products are expressed directly from the viral genome and are suspected to be involved in viral RNA replication. We have investigated further the roles of these proteins in the amplification of viral RNAs by using a complementation system in which p33 and p92 are expressed from different viral RNAs. Our results indicate that (i) both of these proteins are necessary for viral RNA amplification; (ii) translation of these proteins can be uncoupled while maintaining amplification of viral RNAs; (iii) if compatibility requirements exist between p33 and p92, they are not exceptionally strict; and (iv) the C-terminal ∼6% of p33 is necessary for its functional activity. Interestingly, no complementation was observed when a p33-encoding replicon containing a deletion of a 3′-located segment, region 3.5, was tested. However, when 5′-capped transcripts of the same replicon were analyzed, complementation allowing for RNA amplification was observed. This ability to compensate functionally for the absence of region 3.5 by the addition of a 5′ cap suggests that this RNA segment may act as a translational enhancer for the expression of virally encoded products.
Adeno-Associated Virus Vector-Mediated Transgene Integration into Neurons and Other Nondividing Cell Targets
Authors: Wu Ping , Phillips M. Ian , Bui John , Terwilliger Ernest F.
Published: December 2019

The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.
Human Herpesvirus 6 Open Reading Frame U12 Encodes a Functional β-Chemokine Receptor
Authors: Isegawa Yuji , Ping Zou , Nakano Kazushi , Sugimoto Nakaba , Yamanishi Koichi
Published: December 2019

Human herpesvirus 6 (HHV- 6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. HHV- 6 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors (GCR), while three other betaherpesviruses, human cytomegalovirus, murine cytomegalovirus, and human herpesvirus 7, have three, one, and two GCR-homologous genes, respectively. The U12 gene is expressed late in infection from a spliced mRNA. The U12 gene was cloned, and the protein was expressed in cells and analyzed for its biological characteristics. U12 functionally encoded a calcium-mobilizing receptor for β-chemokines such as regulated upon activation, normal T expressed and secreted (RANTES), macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and monocyte chemoattractant protein 1 but not for the α-chemokine interleukin-8, suggesting that the chemokine selectivity of the U12 product was distinct from that of the known mammalian chemokine receptors. These findings suggested that the product of U12 may play an important role in the pathogenesis of HHV- 6 through transmembrane signaling by binding with β-chemokines.
Human Herpesvirus 8 in the Prostate Glands of Men with Kaposi’s Sarcoma
Authors: Diamond Catherine , Brodie Scott J. , Krieger John N. , Huang Meei-Li , Koelle David M. , Diem Kurt , Muthui David , Corey Lawrence
Published: December 2019

The epidemiology of human immunodeficiency virus (HIV)-associated Kaposi’s sarcoma (KS) resembles that of a sexually transmitted pathogen. However, human herpesvirus 8 (HHV-8), the proposed cause of KS, is found in semen only infrequently and at low titers. To determine whether HHV-8 was present in the urogenital tract, transrectal ultrasound-guided prostate biopsies were obtained from six men with KS (five with concurrent HIV infection) and four without KS (three with concurrent HIV) and assayed for HHV-8 by PCR. Nine of the 10 men were seropositive for HHV-8. Five of nine HHV-8-seropositive men had HHV-8 DNA detected in prostate tissue by solution-based PCR. All five currently had KS or had it previously. In two subjects, prostate tissue was the only identified source of HHV-8. In situ PCR on serial sections of prostate indicated that HHV-8 infection was localized to discrete areas of the prostate. When detected, HHV-8 DNA was present in the nuclei of >90% of the glandular epithelial cells. In situ hybridization for HHV-8 mRNA revealed that between 1 and 5% of cells harboring HHV-8 DNA expressed viral transcripts associated with HHV-8 replication (T1.1 transcript), while >90% expressed gene products associated with viral latency (T0.7 transcript). Intermittent replication of HHV-8 in the prostate and subsequent shedding of virus in semen may be crucial factors for determining whether HHV-8 can be transmitted through sexual activity.
Molecular Mechanisms of Serum Resistance of Human Influenza H3N2 Virus and Their Involvement in Virus Adaptation in a New Host
Authors: Matrosovich Mikhail , Gao Peng , Kawaoka Yoshihiro
Published: December 2019

H3N2 human influenza viruses that are resistant to horse, pig, or rabbit serum possess unique amino acid mutations in their hemagglutinin (HA) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by measuring their affinity for total serum protein inhibitors and for soluble receptor analogs. Pig serum-resistant variants displayed a markedly decreased affinity for total pig serum sialylglycoproteins (which contain predominantly 2-6 linkage between sialic acid and galactose residues) and for the sialyloligosaccharide 6′-sialyl(
N
-acetyllactosamine). These properties correlated with the substitution 186S→I in HA1. The major inhibitory activity in rabbit serum was found to be a β inhibitor with characteristics of mannose-binding lectins. Rabbit serum-resistant variants exhibited decreased sensitivity to this inhibitor due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6′-sialyl(
N
-acetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties of equine α
2
-macroglobulin because of the mutation 145N→K/D in their HA1. These results indicate that influenza viruses become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we compared the receptor-binding properties of H3N8 avian and equine viruses, including two strains isolated during the 1989 to 1990 equine influenza outbreak, which was caused by an avian virus in China. Avian strains bound 4-O-acetylated sialic acid residues of equine α
2
-macroglobulin, whereas equine strains did not. The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, however, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant sialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.
Biochemical Analysis of the Secreted and Virion Glycoproteins of Ebola Virus
Authors: Sanchez Anthony , Yang Zhi-Yong , Xu Ling , Nabel Gary J. , Crews Tamara , Peters Clarence J.
Published: December 2019

The glycoproteins expressed by a Zaire species of Ebola virus were analyzed for cleavage, oligomerization, and other structural properties to better define their functions. The 50- to 70-kDa secreted and 150-kDa virion/structural glycoproteins (SGP and GP, respectively), which share the 295 N-terminal residues, are cleaved near the N terminus by signalase. A second cleavage event, occurring in GP at a multibasic site (RRTRR↓) that is likely mediated by furin, results in two glycoproteins (GP1 and GP2) linked by disulfide bonding. This furin cleavage site is present in the same position in the GPs of all Ebola viruses (R[R/K]X[R/K]R↓), and one is predicted for Marburg viruses (R[R/K]KR↓), although in a different location. Based on the results of cross-linking studies, we were able to determine that Ebola virion peplomers are composed of trimers of GP1-GP2 heterodimers and that aspects of their structure are similar to those of retroviruses, paramyxoviruses, and influenza viruses. We also determined that SGP is secreted from infected cells almost exclusively in the form of a homodimer that is joined by disulfide bonding.
In Vivo Pathogenesis of a Human Immunodeficiency Virus Type 1 Reporter Virus
Authors: Jamieson Beth D. , Zack Jerome A.
Published: December 2019

Our understanding of human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis is hampered by the inability to detect HIV-1 gene expression in infected viable cells. In this report, we describe two HIV-1 reporter constructs that are replication competent and cytopathic in vivo. These constructs contain DNA regions of two different lengths that bear the cDNA for the murine heat-stable antigen in thevprregion of a CXCR4-tropic virus. We used the SCID-hu mouse model and these reporter viruses to perform detailed kinetic studies of HIV-1 infection of human thymocytes in vivo. We document that the CD4+/CD8+thymocytes are the first to express virus and that this subset demonstrates the most rapid and extensive HIV-1-induced cell depletion. Following depletion of this subset, subsequent virus expression occurs predominantly in phenotypically CD4−cells, suggesting that CD4 down-regulation occurs in HIV-1-infected thymocytes in vivo. These results demonstrate the utility of these HIV-1 reporter constructs to monitor HIV pathogenesis in vitro and in vivo.
Identification of a Cytotoxic T-Lymphocyte Response to the Novel BARF0 Protein of Epstein-Barr Virus: a Critical Role for Antigen Expression
Authors: Kienzle Norbert , Sculley Tom B. , Poulsen Leith , Buck Marion , Cross Simone , Raab-Traub Nancy , Khanna Rajiv
Published: December 2019

The Epstein-Barr virus (EBV)-encoded BARF0 open reading frame gene products are consistently expressed in EBV-positive Burkitt’s lymphoma (BL) cell lines, nasopharyngeal carcinoma cell lines, and lymphoblastoid cell lines (LCLs). Here we show that the BARF0 sequence includes an HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitope. By using theoretically predicted HLA A2 binding motifs and peptide-loaded antigen presentation-deficient T2 cells, polyclonal BARF0-specific CD8+CTLs were isolated from four different healthy EBV-seropositive donors but not from two seronegative donors. These CTL lines recognized the peptide epitope LLWAARPRL, which was found to be conserved in 33 of 34 virus strains originating from Caucasian, African, and Asian individuals. The BARF0-specific CTL lines could lyse EBV-negative BL cells stably transfected with the BARF0 gene but did not kill HLA A2-matched EBV-positive BL cells and LCLs in a standard51Cr release assay. Reverse transcriptase PCR analysis demonstrated that these EBV-positive cell lines expressed significantly lower levels of BARF0 mRNA than transfected cells. This data indicated that the BARF0 epitope could be endogenously processed; however, antigen levels in the target cell were a limiting factor for the effective interaction between BARF0-expressing cells and CTLs. The limited expression of BARF0 antigen in EBV-infected BL cells and LCLs might contribute to the escape of immune recognition from virus-specific CTLs present in the host.
Dendritic Cells Transmit Human Immunodeficiency Virus Type 1 to Monocytes and Monocyte-Derived Macrophages
Authors: Kacani Laco , Frank Ines , Spruth Martin , Schwendinger Michael G. , Müllauer Brigitte , Sprinzl Georg M. , Steindl Franz , Dierich Manfred P.
Published: December 2019

Previous studies have shown that human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DC) to replicate and spread among CD4
+
T cells. To explain the predominance of non-syncytium-inducing (NSI) over syncytium-inducing (SI) strains during the initial viremia of HIV, we investigated the ability of blood monocyte (Mo)-derived DC to transmit HIV-1 to CD4
+
cells of the monocytoid lineage. First, we demonstrate that in our system, DC are able to transmit NSI strains, but not SI strains, of HIV-1 to fresh blood Mo and to Mo-derived macrophages (MDM). To establish a productive infection, a 10-fold-lower amount of virus was necessary for DC-mediated transmission of HIV-1 to Mo than in case of cell-free infection. Second, immature CD83
−
DC (imDC) transmit virus to Mo and MDM with higher efficacy compared to mature CD83
+
DC (maDC); this finding is in contrast to data previously obtained with CD4
+
T cells. Third, maturation from imDC to maDC efficiently silenced expression of β
2
-integrins CD11b, CD11c, and CD18 by maDC. Moreover, monoclonal antibody against CD18 inhibited transmission of HIV-1 from imDC to Mo. We propose that the adhesion molecules of the CD11/CD18 family, involved in cell-cell interactions of DC with the microenvironment, may play a major role in imDC-mediated HIV-1 infection of Mo and MDM.
The Immunogenic Glycoprotein gp35-37 of Human Herpesvirus 8 Is Encoded by Open Reading Frame K8.1
Authors: Raab Marc-Steffen , Albrecht Jens-Christian , Birkmann Alexander , Yağuboğlu Svenja , Lang Dieter , Fleckenstein Bernhard , Neipel Frank
Published: December 2019

Human herpesvirus 8 (HHV-8) is likely to be involved in the pathogenesis of Kaposi’s sarcoma (KS) and body cavity-based lymphomas (BCBLs). The HHV-8 genome is primarily in a latent state in BCBL-derived cell lines like BCBL-1, but lytic replication can be induced by phorbol esters (R. Renne, W. Zhang, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. E. Ganem, Nat. Med. 2:342–346, 1996). A 35- to 37-kDa glycoprotein (gp35-37) is the polypeptide most frequently and intensively recognized by KS patient sera on Western blots with induced BCBL-1 cells. Its apparent molecular mass is reduced to 30 kDa by digestion with peptide-
N
-glycosidase F. By searching the known HHV-8 genomic sequence for open reading frames (ORF) with the potential to encode such a glycoprotein, an additional, HHV-8-specific reading frame was identified adjacently to ORF K8. This ORF, termed K8.1, was found to be transcribed primarily into a spliced mRNA encoding a glycoprotein of 228 amino acids. Recombinant K8.1 was regularly recognized by KS patient sera in Western blots, and immunoaffinity-purified antibodies to recombinant K8.1 reacted with gp35-37. This shows that the immunogenic gp35-37 is encoded by HHV-8 reading frame K8.1, which will be a useful tool for studies of HHV-8 epidemiology and pathogenesis.
Naturally Occurring Mutations Define a Novel Function of the Hepatitis B Virus Core Promoter in Core Protein Expression
Authors: Baumert Thomas F. , Marrone Aldo , Vergalla John , Liang T. Jake
Published: December 2019

Functional analysis of naturally occurring hepatitis B virus (HBV) mutations is crucial in understanding their impact on disease. We have recently identified two mutations in the HBV core promoter of an HBV strain associated with fulminant hepatitis leading to highly (15-fold) enhanced replication as a result of increased viral encapsidation of pregenomic RNA into the core particles (T. F. Baumert et al., J. Clin. Invest. 98:2268–2276, 1996). Functional studies in an encapsidation assay had demonstrated that the increase in encapsidation was largely independent of pregenomic RNA transcription. In this study, we define the molecular mechanism whereby the two core promoter mutations (C to T at nucleotide [nt] 1768 and T to A at nt 1770) result in enhanced viral encapsidation and replication. The effect of these mutations leading to increased encapsidation is mediated through enhanced core protein synthesis (15-fold) by the mutant virus. The marked increase in core protein synthesis is largely a result of posttranscriptional or translational effect of the mutations because the mutations resulted in only a twofold increase in pregenomic RNA transcription. In addition, this effect appears to be selective for core expression since reverse transcriptase-polymerase expression was increased only twofold.
trans
-complementation analyses of HBV replication demonstrated that enhanced replication occurred only when the mutations were provided together with the core protein in
trans
, confirming the functional association of the core promoter mutations and core protein expression. In addition, the effect of the mutations appears to be quantitatively dependent on the strain background to which the mutations were introduced. Our study suggests that the HBV core promoter regulates core protein expression at both transcriptional and posttranscriptional levels.
CXCR4 Is Required by a Nonprimate Lentivirus: Heterologous Expression of Feline Immunodeficiency Virus in Human, Rodent, and Feline Cells
Authors: Poeschla Eric M. , Looney David J.
Published: December 2019

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV
env
specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.
The Probability of In Vivo Reactivation of Herpes Simplex Virus Type 1 Increases with the Number of Latently Infected Neurons in the Ganglia
Authors: Sawtell N. M.
Published: December 2019

The purpose of this study was to define the relationship between herpes simplex virus (HSV) latency and in vivo ganglionic reactivation. Groups of mice with numbers of latently infected neurons ranging from 1.9 to 24% were generated by varying the input titer of wild-type HSV type 1 strain 17syn+. Reactivation of the virus in mice from each group was induced by hyperthermic stress. The number of animals that exhibited virus reactivation was positively correlated with the number of latently infected neurons in the ganglia over the entire range examined (
r
= 0.9852,
P
< 0.0001 [Pearson correlation]).
Antigenic Structure of Human Respiratory Syncytial Virus Fusion Glycoprotein
Authors: López Juan A. , Bustos Regla , Örvell Claes , Berois Mabel , Arbiza Juan , García-Barreno Blanca , Melero José A.
Published: December 2019

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.