bluuebunny/crossref_metadata_2025 huggingface dataset:
Coxsackievirus and Adenovirus Receptor Cytoplasmic and Transmembrane Domains Are Not Essential for Coxsackievirus and Adenovirus Infection
Authors: Wang Xianghong , Bergelson Jeffrey M.
Published: December 2019

Coxsackievirus and adenovirus receptor (CAR) from which the cytoplasmic domain had been deleted and glycosylphosphatidylinositol (GPI)-anchored CAR lacking both transmembrane and cytoplasmic domains were both capable of facilitating adenovirus 5-mediated gene delivery and infection by coxsackievirus B3. These results indicate that the CAR extracellular domain is sufficient to permit virus attachment and entry and that the presence of a GPI anchor does not prevent infection.
Foamy Virus Capsids Require the Cognate Envelope Protein for Particle Export
Authors: Pietschmann Thomas , Heinkelein Martin , Heldmann Martina , Zentgraf Hanswalter , Rethwilm Axel , Lindemann Dirk
Published: December 2019

Unlike other subclasses of the
Retroviridae
the
Spumavirinae
, its prototype member being the so-called human foamy virus (HFV), require the expression of the envelope (Env) glycoprotein for viral particle egress. Both the murine leukemia virus (MuLV) Env and the vesicular stomatitis virus G protein, which efficiently pseudotype other retrovirus capsids, were not able to support export of HFV particles. Analysis of deletion and point mutants of the HFV Env protein revealed that the HFV Env cytoplasmic domain (CyD) is dispensable for HFV particle envelopment, release, and infectivity, whereas deletion of the membrane-spanning-domain (MSD) led to an accumulation of naked capsids in the cytoplasm. Neither alternative membrane association of HFV Env deletion mutants lacking the MSD and CyD via phosphoglycolipid anchor nor domain swapping mutants, with the MSD or CyD of MuLV Env and VSV-G exchanged against the corresponding HFV domains, could restore particle envelopment and the release defect of pseudotypes. However, replacement of the HFV MSD with that of MuLV led to budding of HFV capsids at the intracellular membranes. These virions were of apparently wild-type morphology but were not naturally released into the supernatant and they were noninfectious.
Bacterial Lipopolysaccharide Inhibits Dengue Virus Infection of Primary Human Monocytes/Macrophages by Blockade of Virus Entry via a CD14-Dependent Mechanism
Authors: Chen Yun-Chi , Wang Sheng-Yuan , King Chwan-Chuen
Published: December 2019

Monocytes/macrophages (MO/Mφ) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mφ when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1β (IL-1β), IL-6, IL-8, IL-12, alpha interferon, MIP-1α, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mφ that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the “binding” of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mφ via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.
Sufficient Length of a Poly(A) Tail for the Formation of a Potential Pseudoknot Is Required for Efficient Replication of Bamboo Mosaic Potexvirus RNA
Authors: Tsai Ching-Hsiu , Cheng Chi-Ping , Peng Chih-Weng , Lin Biing-Yuan , Lin Na-Sheng , Hsu Yau-Heiu
Published: December 2019

RNAs transcribed from a full-length infectious cDNA clone of the bamboo mosaic potexvirus (strain O) genome, pBaMV-O, were infectious to
Nicotiana benthamiana
plants. Mutant genomes in which the poly(A) tail is absent or replaced by a 3′ tRNA-like structure from turnip yellow mosaic virus RNA failed to amplify detectably in
N. benthamiana
protoplasts. No amplification was detected in protoplasts inoculated with transcripts containing 4, 7, or 10 adenylate residues at the 3′ end, whereas transcript inocula with 15 adenylate residues resulted in coat protein accumulation to a level 26% of that resulting from inoculation with transcripts with 25 adenylate residues (designated as wild type). Coat protein accumulation levels of 69 and 98% relative to wild type were observed after inoculation of protoplasts with transcripts bearing poly(A) tails 18 and 22 nucleotides long, respectively. The presence of a putative 3′ pseudoknot structure including at least 13 adenylate residues of the 3′-terminal poly(A) tail was supported by enzymatic and chemical structural analysis. The functional relevance of this putative pseudoknot was tested by mutations that affected basepairing within the pseudoknot. These results support the existence of functional 3′ pseudoknot that includes part of the 3′ poly(A) tail.
Effect of the Attenuating Deletion and of Sequence Alterations Evolving In Vivo on Simian Immunodeficiency Virus C8-Nef Function
Authors: Carl Silke , Iafrate A. John , Skowronski Jacek , Stahl-Hennig Christiane , Kirchhoff Frank
Published: December 2019

The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8nef-long terminal repeat region encompasses only 12 bp and is “repaired” in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmacnefvariants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in thenefgene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.
A Single Amino Acid in the Adenovirus Type 37 Fiber Confers Binding to Human Conjunctival Cells
Authors: Huang Shuang , Reddy Vijay , Dasgupta Nabarun , Nemerow Glen R.
Published: December 2019

A 46-kDa receptor, coxsackievirus-adenovirus (Ad) receptor (CAR), mediates cell attachment of a number of different Ad serotypes; however, not all Ad serotypes utilize this receptor for infection. Moreover, the precise amino acid sequences in the Ad fiber protein that mediate cell attachment have yet to be identified. We investigated the interaction of subgroup D Ads with human ocular cells. Ad serotype 37 (Ad37), a virus associated with epidemic keratoconjunctivitis, but not a closely related virus serotype, Ad19p, exhibited preferential binding to and infection of human conjunctival cells. A single amino acid substitution in the Ad19p fiber distal domain (knob), Glu
240
to Lys, conferred binding to conjunctival cells, while the reverse substitution in the Ad37 fiber abrogated cell binding. These findings provide new information on the fiber sequences that regulate Ad host cell tropism.
Effects of Human Cytomegalovirus Major Immediate-Early Proteins in Controlling the Cell Cycle and Inhibiting Apoptosis: Studies with
ts
13 Cells
Authors: Lukac David M. , Alwine James C.
Published: December 2019

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) encodes several MIE proteins (MIEPs) produced as a result of alternative splicing and polyadenylation of the primary transcript. Previously we demonstrated that the HCMV MIEPs expressed from the entire MIE gene could rescue the temperature-sensitive (
ts
) transcriptional defect in the
ts
13 cell line. This defect is caused by a
ts
mutation in TAF
II
250, the 250-kDa TATA binding protein-associated factor (TAF). These and other data suggested that the MIEPs perform a TAF-like function in complex with the basal transcription factor TFIID. In addition to the transcriptional defect, the
ts
mutation in
ts
13 cells results in a defect in cell cycle progression which ultimately leads to apoptosis. Since all of these defects can be rescued by wild-type TAF
II
250, we asked whether the MIEPs could rescue the cell cycle defect and/or affect the progression to apoptosis. We have found that the MIEPs, expressed from the entire MIE gene, do not rescue the cell cycle block in
ts
13 cells grown at the nonpermissive temperature. However, despite the maintenance of the cell cycle block, the
ts
13 cells which express the MIEPs are resistant to apoptosis. MIEP mutants, which have previously been shown to be defective in rescuing the
ts
transcriptional defect, maintained the ability to inhibit apoptosis. Hence, the MIEP functions which affect transcription appear to be separable from the functions which inhibit apoptosis. We discuss these data in the light of the HCMV life cycle and the possibility that the MIEPs promote cellular transformation by a “hit-and-run” mechanism.
Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors
Authors: Zufferey Romain , Donello John E. , Trono Didier , Hope Thomas J.
Published: December 2019

The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3′ untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegalovirus or the herpesvirus thymidine kinase promoter and with both human immunodeficiency virus- and murine leukemia virus-based vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of retroviral vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.
Acute Hepatitis C Virus Structural Gene Sequences as Predictors of Persistent Viremia: Hypervariable Region 1 as a Decoy
Authors: Ray Stuart C. , Wang Yu-Ming , Laeyendecker Oliver , Ticehurst John R. , Villano Stephen A. , Thomas David L.
Published: December 2019

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5′ half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (

d
N
/d
S

) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1

d
N
/d
S

ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1

d
N
/d
S

ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.
The Retinoblastoma Protein Alters the Phosphorylation State of Polyomavirus Large T Antigen in Murine Cell Extracts and Inhibits Polyomavirus Origin DNA Replication
Authors: Reynisdóttir Inga , Bhattacharyya Subarna , Zhang Dong , Prives Carol
Published: December 2019

The retinoblastoma tumor suppressor protein (pRb) can associate with the transforming proteins of several DNA tumor viruses, including the large T antigen encoded by polyomavirus (Py T Ag). Although pRb function is critical for regulating progression from G
1
to S phase, a role for pRb in S phase has not been demonstrated or excluded. To identify a potential effect of pRb on DNA replication, pRb protein was added to reaction mixtures containing Py T Ag, Py origin-containing DNA (Py ori-DNA), and murine FM3A cell extracts. We found that pRb strongly represses Py ori-DNA replication in vitro. Unexpectedly, however, this inhibition only partially depends on the interaction of pRb with Py T Ag, since a mutant Py T Ag (
dl
141) lacking the pRb interaction region was also significantly inhibited by pRb. This result suggests that pRb interferes with or alters one or more components of the murine cell replication extract. Furthermore, the ability of Py T Ag to be phosphorylated in such extracts is markedly reduced in the presence of pRb. Since cyclin-dependent kinase (CDK) phosphorylation of Py T Ag is required for its replication function, we hypothesize that pRb interferes with this phosphorylation event. Indeed, the S-phase CDK complex (cyclin A-CDK2), which phosphorylates both pRb and Py T Ag, alleviates inhibition caused by pRb. Moreover, hyperphosphorylated pRb is incapable of inhibiting replication of Py ori-DNA in vitro. We propose a new requirement for maintaining pRb phosphorylation in S phase, namely, to prevent deleterious effects on the cellular replication machinery.
T Cells Contribute to Disease Severity during Coxsackievirus B4 Infection
Authors: Ramsingh Arlene I. , Lee William T. , Collins Doris N. , Armstrong Lori E.
Published: December 2019

By using a model of coxsackievirus B4-induced disease, the question of whether tissue damage is due to the virus or to immune-mediated mechanisms was addressed. Both viral replication and T-cell function were implicated in contributing to the severity of disease. Three stages (I to III) of disease, which correspond to periods of high viral titers, low viral titers, and no infectious virus, have been identified. Stage I disease is considered to be primarily the result of viral replication. Immunopathological mechanisms appear to contribute to the severity of stage II and III disease. To investigate the role of T cells in contributing to the severity of disease, viral infection in CD8 knockout (ko) mice and CD4 ko mice was analyzed. CD8 T-cell responses appear to be beneficial during early, viral disease but detrimental in later disease when viral titers are diminishing. CD4 ko mice, unlike the parental strain, survived infection. Viral replication was lower in the CD4 ko mice. Was survival due to decreased viral replication or to the lack of T-helper-cell function? To investigate further the role of T helper cells in contributing to tissue damage, viral infection in two additional ko strains (interleukin-4 [IL-4] ko and gamma interferon ko strains) was examined. A clear correlation between viral replication and the outcome of infection was not observed. The absence of IL-4, which may influence T-helper-cell subset development, was advantageous during early viral disease but deleterious in later disease. The results suggest that T-cell-mediated immunity is both beneficial and detrimental during coxsackievirus B4 infection.
Sendai Virus and Simian Virus 5 Block Activation of Interferon-Responsive Genes: Importance for Virus Pathogenesis
Authors: Didcock L. , Young D. F. , Goodbourn S. , Randall R. E.
Published: December 2019

Sendai virus (SeV) is highly pathogenic for mice. In contrast, mice (including SCID mice) infected with simian virus 5 (SV5) showed no overt signs of disease. Evidence is presented that a major factor which prevented SV5 from productively infecting mice was its inability to circumvent the interferon (IFN) response in mice. Thus, in murine cells that produce and respond to IFN, SV5 protein synthesis was rapidly switched off. In marked contrast, once SeV protein synthesis began, it continued, even if the culture medium was supplemented with alpha/beta IFN (IFN-α/β). However, in human cells, IFN-α/β did not inhibit the replication of either SV5 or SeV once virus protein synthesis was established. To begin to address the molecular basis for these observations, the effects of SeV and SV5 infections on the activation of an IFN-α/β-responsive promoter and on that of the IFN-β promoter were examined in transient transfection experiments. The results demonstrated that (i) SeV, but not SV5, inhibited an IFN-α/β-responsive promoter in murine cells; (ii) both SV5 and SeV inhibited the activation of an IFN-α/β-responsive promoter in human cells; and (iii) in both human and murine cells, SeV was a strong inducer of the IFN-β promoter, whereas SV5 was a poor inducer. The ability of SeV and SV5 to inhibit the activation of IFN-responsive genes in human cells was confirmed by RNase protection experiments. The importance of these results in terms of paramyxovirus pathogenesis is discussed.
Expression of EBNA-1 mRNA Is Regulated by Cell Cycle during Epstein-Barr Virus Type I Latency
Authors: Davenport Matthew G. , Pagano Joseph S.
Published: December 2019

Expression of EBNA-1 protein is required for the establishment and maintenance of the Epstein-Barr virus (EBV) genome during latent infection. During type I latency, the
Bam
HI Q promoter (Qp) gives rise to EBNA-1 expression. The dominant regulatory mechanism for Qp appears to be mediated through the Q locus, located immediately downstream of the transcription start site. Binding of EBNA-1 to the Q locus represses Qp constitutive activity, and repression has been reported to be overcome by an E2F family member that binds to the Q locus and displaces EBNA-1 (N. S. Sung, J. Wilson, M. Davenport, N. D. Sista, and J. S. Pagano, Mol. Cell. Biol. 14:7144–7152, 1994). These data suggest that the final outcome of Qp activity is reciprocally controlled by EBNA-1 and E2F. Since E2F activity is cell cycle regulated, Qp activity and EBNA-1 expression are predicted to be regulated in a cell cycle-dependent manner. Proliferation of the type I latently infected cell line, Akata, was synchronized with the use of the G
2
/M blocking agent nocodazole. From 65 to 75% of cells could be made to peak in S phase without evidence of viral reactivation. Following release from G
2
/M block, EBNA-1 mRNA levels declined as the synchronized cells entered the G
1
phase of the cell cycle. As cells proceeded into S phase, EBNA-1 mRNA levels increased parallel to the peak in cell numbers in S phase. However, EBNA-1 protein levels showed no detectable change during the cell cycle, most likely due to the protein’s long half-life as estimated by inhibition of protein synthesis by cycloheximide. Finally, in Qp luciferase reporter assays, the activity of Qp was shown to be regulated by cell cycle and to be dependent on the E2F sites within the Q locus. These findings demonstrate that transcriptional activity of Qp is cell cycle regulated and indicated that E2F serves as the stimulus for this regulation.
Biological Heterogeneity, Including Systemic Replication in Mice, of H5N1 Influenza A Virus Isolates from Humans in Hong Kong
Authors: Gao Peng , Watanabe Shinji , Ito Toshihiro , Goto Hideo , Wells Krisna , McGregor Martha , Cooley A. James , Kawaoka Yoshihiro
Published: December 2019

An H5N1 avian influenza A virus was transmitted to humans in Hong Kong in 1997. Although the virus causes systemic infection and is highly lethal in chickens because of the susceptibility of the hemagglutinin to furin and PC6 proteases, it is not known whether it also causes systemic infection in humans. The clinical outcomes of infection in Hong Kong residents ranged widely, from mild respiratory disease to multiple organ failure leading to death. Therefore, to understand the pathogenesis of influenza due to these H5N1 isolates, we investigated their virulence in mice. The results identified two distinct groups of viruses: group 1, for which the dose lethal for 50% of mice (MLD
50
) was between 0.3 and 11 PFU, and group 2, for which the MLD
50
was more than 10
3
PFU. One day after intranasal inoculation of mice with 100 PFU of group 1 viruses, the virus titer in lungs was 10
7
PFU/g or 3 log units higher than that for group 2 viruses. Both types of viruses had replicated to high titers (>10
6
PFU/g) in the lungs by day 3 and maintained these titers through day 6. More importantly, only the group 1 viruses caused systemic infection, replicating in nonrespiratory organs, including the brain. Immunohistochemical analysis demonstrated the replication of a group 1 virus in brain neurons and glial cells and in cardiac myofibers. Phylogenetic analysis of all viral genes showed that both groups of Hong Kong H5N1 viruses had formed a lineage distinct from those of other viruses and that genetic reassortment between H5N1 and H1 or H3 human viruses had not occurred. Since mice and humans harbor both the furin and the PC6 proteases, we suggest that the virulence mechanism responsible for the lethality of influenza viruses in birds also operates in mammalian hosts. The failure of some H5N1 viruses to produce systemic infection in our model indicates that multiple, still-to-be-identified, factors contribute to the severity of H5N1 infection in mammals. In addition, the ability of these viruses to produce systemic infection in mice and the clear differences in pathogenicity among the isolates studied here indicate that this system provides a useful model for studying the pathogenesis of avian influenza virus infection in mammals.
Interaction of Gli2 with CREB Protein on DNA Elements in the Long Terminal Repeat of Human T-Cell Leukemia Virus Type 1 Is Responsible for Transcriptional Activation by Tax Protein
Authors: Dan Shingo , Tanimura Akira , Yoshida Mitsuaki
Published: December 2019

The long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1) has two distinct DNA elements, one copy of TRE2S and three copies of a 21-bp sequence that respond to the viral
trans
-activator protein, Tax. Either multiple copies of the 21-bp sequence or a combination of one copy each of TRE2S and 21-bp sequence is required for efficient
trans
activation by Tax. In the
trans
activation of multiple copies of 21-bp sequence, CREB/ATF protein plays an essential role in forming a complex with Tax. To understand the role of TRE2S in
trans
activation of one copy of 21-bp sequence, we examined protein binding to the DNA elements by DNA affinity precipitation assay including Gli2 protein binding to TRE2S and CREB protein binding to 21-bp sequence. Binding of CREB to a DNA probe containing both elements, TRE2S-21bp probe, was dependent on Gli2 protein under restricted conditions and was enhanced in a dose-dependent fashion by the binding of Gli2 protein to the same probe. Mutation in either element abolished the efficient binding of CREB. A glutathione
S
-transferase fusion protein of a fragment of Gli2 was able to bind to CREB. Therefore, Gli2-CREB interaction on the DNA probe is proposed to stabilize CREB binding to DNA. Tax can bind to CREB protein on the DNA; therefore, stabilization of DNA binding of CREB results in more recruitment of Tax onto DNA. Conversely, Tax increased the DNA binding of CREB, although it had almost no effect on the binding of Gli2. These results suggest that Gli2 binds to the DNA element and interacts with CREB, resulting in more recruitment of Tax, which in turn stabilizes DNA binding of CREB. Similar cooperation of the protein binding to TRE2S-21bp probe was also observed in nuclear extract of an HTLV-1-infected T-cell line. Consistent with the Gli2-CREB interaction on the DNA elements, Tax-mediated
trans
activation was dependent on the size of the spacer between TRE2S and 21-bp sequence. The effective sizes of the spacer suggest that TRE2S in the LTR would cooperate with the second and third copies of the 21-bp sequence and contribute to
trans
activation of the viral gene transcription.
Mutations in the Carboxyl-Terminal Domain of the Small Hepatitis B Virus Envelope Protein Impair the Assembly of Hepatitis Delta Virus Particles
Authors: Jenna Sarah , Sureau Camille
Published: December 2019

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.
Role for Gamma Interferon in Control of Herpes Simplex Virus Type 1 Reactivation
Authors: Cantin Edouard , Tanamachi Becky , Openshaw Harry
Published: December 2019

Observation of chronic inflammatory cells and associated high-level gamma interferon (IFN-γ) production in ganglia during herpes simplex type 1 (HSV-1) latent infection in mice (E. M. Cantin, D. R. Hinton, J. Chen, and H. Openshaw, J. Virol. 69:4898–4905, 1995) prompted studies to determine a role of IFN-γ in maintaining latency. Mice lacking IFN-γ (GKO mice) or the IFN-γ receptor (RGKO mice) were inoculated with HSV-1, and the course of the infection was compared with that in IFN-γ-competent mice with the same genetic background (129/Sv//Ev mice). A time course study showed no significant difference in trigeminal ganglionic viral titers or the timing of establishment of latency. Spontaneous reactivation resulting in infectious virus in the ganglion did not occur during latency in any of the mice. However, 24 h after the application of hyperthermic stress to mice, HSV-1 antigens were detected in multiple neurons in the null mutant mice but in only a single neuron in the 129/Sv//Ev control mice. Mononuclear inflammatory cells clustered tightly around these reactivating neurons, and by 48 h, immunostaining was present in satellite cells as well. The incidence of hyperthermia-induced reactivation as determined by recovery of infectious virus from ganglia was significantly higher in the null mutant than in control mice: 11% in 129/Sv//Ev controls, 50% in GKO mice (P= 0.0002), and 33% in RGKO mice (P= 0.03). We concluded that IFN-γ is not involved in the induction of reactivation but rather contributes to rapid suppression of HSV once it is reactivated.
Protection against Establishment of Retroviral Persistence by Vaccination with a Live Attenuated Virus
Authors: Dittmer Ulf , Brooks Diane M. , Hasenkrug Kim J.
Published: December 2019

Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable.
A Novel Cellular Protein, p60, Interacting with both Herpes Simplex Virus 1 Regulatory Proteins ICP22 and ICP0 Is Modified in a Cell-Type-Specific Manner and Is Recruited to the Nucleus after Infection
Authors: Bruni Renato , Fineschi Beatrice , Ogle William O. , Roizman Bernard
Published: December 2019

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (γ
2
) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent
M
r
of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525–8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent
M
r
but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and U
L
13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent
M
r
. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the U
L
13 or U
S
3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and U
L
13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding U
L
13 or ICP22.
Virus Promoters Determine Interference by Defective RNAs: Selective Amplification of Mini-RNA Vectors and Rescue from cDNA by a 3′ Copy-Back Ambisense Rabies Virus
Authors: Finke Stefan , Conzelmann Karl-Klaus
Published: December 2019

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3′ copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.
Activation of Promoter P4 of the Autonomous Parvovirus Minute Virus of Mice at Early S Phase Is Required for Productive Infection
Authors: Deleu Laurent , Pujol Aurora , Faisst Steffen , Rommelaere Jean
Published: December 2019

Autonomous parvoviruses are tightly dependent on host cell factors for various steps of their life cycle. In particular, DNA replication and gene expression of the prototype strain of the minute virus of mice (MVMp) are closely linked to the onset of host cell DNA replication, pointing to the involvement of an S-phase-specific cellular factor(s) in parvovirus multiplication. The viral nonstructural protein NS-1 is absolutely required for parvovirus DNA replication and is able to transcriptionally regulate parvoviral and heterologous promoters. We previously showed that the promoter P4, which directs the transcription unit encoding the NS proteins, is activated at the onset of S phase. This activation is dependent on an E2F motif in the proximal region of promoter P4. An infectious MVM DNA clone was mutated in the E2F motif of P4. The wild type and the E2F mutant derivative were tested for their ability to produce progeny viruses after transfection of permissive cells. In the context of the whole MVMp genome, the E2F mutation abolished P4 induction in S phase and inactivated the infectious molecular clone, which failed to become amplified and generate progeny particles. The virus could be rescued when NS proteins were supplied in
trans
, showing that P4 hyperactivity in S is needed to reach a level of NS-1 expression that is sufficient to drive the viral replication cycle. These data show that E2F-mediated P4 activation at the early S phase is a limiting factor for parvovirus production. The primary barrier to parvovirus gene expression in G
1
is thought to be promoter formation rather than activation, due to the poor conversion of the parental single-strand genome to a duplex form. The S dependence of P4 activation may therefore be a sign of the virus adaptation to life in the S-phase host cell. If the conversion block in G
1
were to be leaky, the S induction of promoter P4 could be envisioned as a safeguard against the production of toxic NS proteins until cells reach the S phase and provide the full machinery for parvovirus replication.
Herpes Simplex Virus Transactivator VP16 Discriminates between HCF-1 and a Novel Family Member, HCF-2
Authors: Johnson Kristina M. , Mahajan Shahana S. , Wilson Angus C.
Published: December 2019

Herpes simplex virus infection is initiated by VP16, a viral transcription factor that activates the viral immediate-early (IE) genes. VP16 does not recognize the IE gene promoters directly but instead forms a multiprotein complex with Oct-1 and HCF-1, a ubiquitous nuclear protein required for progression through the G
1
phase of the cell cycle. The functional significance of recruiting HCF-1 to the VP16-induced complex is not understood. Here we describe the identification of a second HCF-like protein, designated HCF-2. HCF-2 is smaller than HCF-1 but shares three regions of strong amino acid sequence homology, including the β-propeller domain required for association with VP16. HCF-2 is expressed in many tissues, especially the testis, and shows a more dynamic pattern of subcellular localization than HCF-1. Although HCF-2 associates with VP16 and can support complex assembly with Oct-1 and DNA, it is significantly less efficient than HCF-1. A similar preference is shown by LZIP, a cellular counterpart of VP16. Analysis of chimeric proteins showed that differences between the fifth and sixth kelch repeats of the β-propeller domains from HCF-1 and HCF-2 dictate this selectivity. These results reveal an unexpected level of specificity in the recruitment of HCF-1 to the VP16-induced complex, paralleling the preferential selection of Oct-1 rather than the closely related POU domain protein Oct-2. Implications for regulation of the viral life cycle are discussed.
Simultaneous Infection with Retroviruses Pseudotyped with Different Envelope Proteins Bypasses Viral Receptor Interference Associated with Colocalization of gp70 and Target Cells on Fibronectin CH-296
Authors: MacNeill Emily C. , Hanenberg Helmut , Pollok Karen E. , van der Loo Johannes C. M. , Bierhuizen Marti F. A. , Wagemaker Gerard , Williams David A.
Published: December 2019

Several factors are thought to limit the efficiency of retroviral transduction in clinical gene therapy protocols that target hematopoietic stem cells. For example, the level of expression of the amphotropic receptor Pit-2, a phosphate symporter, appears to be low in human and murine hematopoietic stem cells. We have previously demonstrated that transduction of hematopoietic cells in the presence of the fibronectin (FN) fragment CH-296 is extremely efficient (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). To examine functionally whether the retrovirus receptor is a limiting factor in transduction of hematopoietic cells, we performed competition experiments in the presence of FN CH-296 with retrovirus vectors pseudotyped with the same or a different envelope protein. We demonstrate in both human erythroleukemia (HEL) cells and primary human CD34
+
hematopoietic cells inhibition of efficient infection due to receptor interference when two vectors targeting the amphotropic receptor are used simultaneously. Receptor interference lasted up to 24 h. No interference was demonstrated when vectors targeting the amphotropic receptor and the gibbon ape leukemia virus (GALV) receptor Pit-1 were used concurrently. In contrast, simultaneous infection with vectors targeting both Pit-1 and Pit-2 yielded transduction efficiencies consistently higher than with either vector alone in both HEL cells and human CD34
+
hematopoietic cells. These data demonstrate that the use of FN CH-296 leads to amphotropic receptor saturation in these cells. Simultaneous infection with vectors targeting both amphotropic and GALV receptors may prove to be of additional benefit in the design of gene therapy protocols.
Gene Therapy Vectors Based on Adeno-Associated Virus Type 1
Authors: Xiao Weidong , Chirmule Narendra , Berta Scott C. , McCullough Beth , Gao Guangping , Wilson James M.
Published: December 2019

The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is true in humans; and (ii) sera from 36% of nonhuman primates neutralized AAV-1 but not AAV-2, while sera from 8% of humans neutralized AAV-2 but not AAV-1. An infectious clone of AAV-1 was isolated from a replicated monomer form, and vectors were created with AAV-2 inverted terminal repeats and AAV-1 Rep and Cap functions. Both AAV-1- and AAV-2-based vectors transduced murine liver and muscle in vivo; AAV-1 was more efficient for muscle, while AAV-2 transduced liver more efficiently. Strong NAB responses were detected for each vector administered to murine skeletal muscle; these responses prevented readministration of the same serotype but did not substantially cross-neutralize the other serotype. Similar results were observed in the context of liver-directed gene transfer, except for a significant, but incomplete, neutralization of AAV-1 from a previous treatment with AAV-2. Vectors based on AAV-1 may be preferred in some applications of human gene therapy.
Human Herpesvirus 6 Infects Dendritic Cells and Suppresses Human Immunodeficiency Virus Type 1 Replication in Coinfected Cultures
Authors: Asada Hideo , Klaus-Kovtun Vera , Golding Hana , Katz Stephen I. , Blauvelt Andrew
Published: December 2019

Human herpesvirus 6 (HHV-6) has been implicated as a cofactor in the progressive loss of CD4+T cells observed in AIDS patients. Because dendritic cells (DC) play an important role in the immunopathogenesis of human immunodeficiency virus (HIV) disease, we studied the infection of DC by HHV-6 and coinfection of DC by HHV-6 and HIV. Purified immature DC (derived from adherent peripheral blood mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4) could be infected with HHV-6, as determined by PCR analyses, intracellular monoclonal antibody staining, and presence of virus in culture supernatants. However, HHV-6-infected DC demonstrated neither cytopathic changes nor functional defects. Interestingly, HHV-6 markedly suppressed HIV replication and syncytium formation in coinfected DC cultures. This HHV-6-mediated anti-HIV effect was DC specific, occurred when HHV-6 was added either before or after HIV, and was not due to decreased surface expression or function of CD4, CXCR4, or CCR5. Conversely, HIV had no demonstrable effect on HHV-6 replication. These findings suggest that HHV-6 may protect DC from HIV-induced cytopathicity in AIDS patients. We also demonstrate that interactions between HIV and herpesviruses are complex and that the observable outcome of dual infection is dependent on the target cell type.
Functional Dissection of CCR5 Coreceptor Function through the Use of CD4-Independent Simian Immunodeficiency Virus Strains
Authors: Edinger Aimee L. , Blanpain Cedric , Kunstman Kevin J. , Wolinsky Steven M. , Parmentier Marc , Doms Robert W.
Published: December 2019

With rare exceptions, all simian immunodeficiency virus (SIV) strains can use CCR5 as a coreceptor along with CD4 for viral infection. In addition, many SIV strains are capable of using CCR5 as a primary receptor to infect CD4-negative cells such as rhesus brain capillary endothelial cells. By using coupled fluorescence-activated cell sorter (FACS) and infection assays, we found that even very low levels of CCR5 expression could support CD4-independent virus infection. CD4-independent viruses represent valuable tools for finely dissecting interactions between Env and CCR5 which may otherwise be masked due to the stabilization of these contacts by Env-CD4 binding. Based on the ability of SIV Env to bind to and mediate infection of cells expressing CCR5 chimeras and mutants, we identified the N terminus of CCR5 as a critical domain for direct Env binding and for supporting CD4-independent virus infection. However, the activity of N-terminal domain CCR5 mutants could be rescued by the presence of CD4, indicating that other regions of CCR5 are important for post-binding events that lead to viral entry. Rhesus CCR5 supported CD4-independent infection and direct Env binding more efficiently than did human CCR5 due to a single amino acid difference in the N terminus. Interestingly, uncleaved, oligomeric SIV Env protein bound to both CD4 and CCR5 less efficiently than did monomeric gp120. Finally, several mutations present in chronically infected monkey populations are shown to decrease the ability of CCR5 to serve as a primary viral receptor for the SIV isolates examined.