bluuebunny/crossref_metadata_2025 huggingface dataset:
Variants from the Diverse Virus Population Identified at Seroconversion of a Clade A Human Immunodeficiency Virus Type 1-Infected Woman Have Distinct Biological Properties
Authors: Poss Mary , Overbaugh Julie
Published: December 2019

Development of effective therapeutics to prevent new infections with human immunodeficiency type 1 (HIV-1) is predicated on an understanding of the properties that provide a selective advantage to a transmitted viral population. In contrast to the homogeneous virus population that typifies early HIV-1 infection of men, the viral population in women recently infected with clade A HIV-1 is genetically diverse, based on evaluation of the envelope gene. A longitudinal study of viral envelope evolution in several women suggested that representative envelope variants detected at seroconversion had distinct biological properties that affected viral fitness. To test this hypothesis, a full-length, infectious molecular clone, Q23-17, was obtained from an infected woman 1 year following seroconversion, and chimeric viruses containing envelope genes representative of seroconversion and 27-month-postseroconversion populations were constructed. Dendritic cells (DC) could transfer infection of seroconversion variant Q23ScA, which dominated the viral population in the year following seroconversion, and the closely related 1-year isolate Q23-17 to resting peripheral blood mononuclear cells (PBMC). In contrast, resting PBMC exposed to DC pulsed with Q23ScB, which was detected infrequently in samples after seroconversion, or the 27-month chimeras were inconsistently infected. Additionally, quiescent PBMC infected with Q23ScA or Q23-17 proliferated more robustly than uninfected cells or cells infected with the other envelope chimeras in response to immobilized anti-CD3. Stimulation with tetanus toxoid led to an increased proportion of CD45RA+cells and a decreased expression of CD28 on CD45RO+cells in cultures of Q23-17-infected PBMC. These data demonstrate that variants from the heterogeneous seroconversion clade A HIV-1 population in a Kenyan woman have distinct biological features that may influence viral pathogenesis.
Measles Virus Spread by Cell-Cell Contacts: Uncoupling of Contact-Mediated Receptor (CD46) Downregulation from Virus Uptake
Authors: Firsching Ruth , Buchholz Christian J. , Schneider Urs , Cattaneo Roberto , ter Meulen Volker , Schneider-Schaulies Jürgen
Published: December 2019

CD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-d-Phe-l-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell.
Characterization of an Equine Arteritis Virus Replicase Mutant Defective in Subgenomic mRNA Synthesis
Authors: van Marle Guido , van Dinten Leonie C. , Spaan Willy J. M. , Luytjes Willem , Snijder Eric J.
Published: December 2019

Equine arteritis virus (EAV) is a positive-stranded RNA virus that synthesizes a 5′- and 3′-coterminal nested set of six subgenomic mRNAs. These mRNAs all contain a common leader sequence which is derived from the 5′ end of the genome. Subgenomic mRNA transcription and genome replication are directed by the viral replicase, which is expressed in the form of two polyproteins and subsequently processed into smaller nonstructural proteins (nsps). During the recent construction of an EAV infectious cDNA clone (pEAV030 [L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991–996, 1997]), a mutant cDNA clone (pEAV030F) which carries a single replicase point mutation was obtained. This substitution (Ser-2429→Pro) is located in the nsp10 subunit and renders the EAV030F virus deficient in subgenomic mRNA synthesis. To obtain more insight into the role of nsp10 in transcription and the nature of the transcriptional defect, we have now analyzed the EAV030F mutant in considerable detail. The Ser-2429→Pro mutation does not affect the proteolytic processing of the replicase but apparently affects the function of nsp10 in transcription. Furthermore, our study showed that EAV030F still produces subgenomic positive and negative strands, albeit at a very low level. Both subgenomic positive-strand synthesis and negative-strand synthesis are equally affected by the Ser-2429→Pro mutation, suggesting that nsp10 plays an important role in an early step of EAV mRNA transcription.
Immunogenicity of a Human Immunodeficiency Virus (HIV) Polytope Vaccine Containing Multiple HLA A2 HIV CD8+Cytotoxic T-Cell Epitopes
Authors: Woodberry T. , Gardner J. , Mateo L. , Eisen D. , Medveczky J. , Ramshaw I. A. , Thomson S. A. , Ffrench R. A. , Elliott S. L. , Firat H. , Lemonnier F. A. , Suhrbier A.
Published: December 2019

Compelling evidence now suggests that αβ CD8 cytotoxic T lymphocytes (CTL) have an important role in preventing human immunodeficiency virus (HIV) infection and/or slowing progression to AIDS. Here, we describe an HIV type 1 CTL polyepitope, or polytope, vaccine comprising seven contiguous minimal HLA A2-restricted CD8 CTL epitopes conjoined in a single artificial construct. Epitope-specific CTL lines derived from HIV-infected individuals were able to recognize every epitope within the construct, and HLA A2-transgenic mice immunized with a recombinant virus vaccine coding for the HIV polytope also generated CTL specific for different epitopes. Each epitope in the polytope construct was therefore processed and presented, illustrating the feasibility of the polytope approach for HIV vaccine design. By simultaneously inducing CTL specific for different epitopes, an HIV polytope vaccine might generate activity against multiple challenge isolates and/or preempt the formation of CTL escape mutants.
p53-Independent and -Dependent Requirements for E1B-55K in Adenovirus Type 5 Replication
Authors: Harada Josephine N. , Berk Arnold J.
Published: December 2019

The adenovirus type 5 mutant
dl
1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53
−
and some p53
+
tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639–645, 1997). It has been suggested that
dl
1520 might be used as a “magic bullet” that could selectively lyse tumor cells without harm to normal tissues. However, we report here that
dl
1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce
dl
1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653–8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109–124, 1982),
dl
1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.
Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3
Authors: Piron Maria , Delaunay Thierry , Grosclaude Jeanne , Poncet Didier
Published: December 2019

The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3′ end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in
Escherichia coli
, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.
Isolation of Recombinant Adeno-Associated Virus Vector-Cellular DNA Junctions from Mouse Liver
Authors: Nakai Hiroyuki , Iwaki Yuichi , Kay Mark A. , Couto Linda B.
Published: December 2019

Recombinant adeno-associated virus (rAAV) vectors allow for sustained expression of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.IX (hF.IX), AAV-EF1α-F.IX (hF.IX expression was controlled by the human elongation factor 1α [EF1α] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchyma, and the forms of rAAV vector DNA extracted from the liver were analyzed. Southern blot analyses suggested that rAAV vector integrated into the host genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences. To further confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, including ITR deletions and amplifications of the vector and cellular DNA sequences. The breakpoints of the vector were mostly located within the ITRs, and cellular DNA sequences were recombined with the vector genome in a nonhomologous manner. Two rAAV-targeted DNA sequences were identified as the mouse rRNA gene and the α1 collagen gene. These observations serve as direct evidence of rAAV integration into the host genome of mouse liver and allow us to begin to elucidate the mechanisms involved in rAAV integration into tissues in vivo.
Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys
Authors: Egan Michael A. , Kuroda Marcelo J. , Voss Gerald , Schmitz Jörn E. , Charini William A. , Lord Carol I. , Forman Meryl A. , Letvin Norman L.
Published: December 2019

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8+CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8+CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/β2m complexes. All SHIV-infected Mamu-A*01+rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8+CTL response is dominant and the p41A- and p68A-specific CD8+CTL responses are nondominant. These results indicate that CD8+CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8+CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.
Sequence Variations in Human Immunodeficiency Virus Type 1 Nef Are Associated with Different Stages of Disease
Authors: Kirchhoff Frank , Easterbrook Philippa J. , Douglas Nigel , Troop Maxine , Greenough Thomas C. , Weber Jonathan , Carl Silke , Sullivan John L. , Daniels Rod S.
Published: December 2019

nef
alleles derived from a large number of individuals infected with human immunodeficiency virus type 1 (HIV-1) were analyzed to investigate the frequency of disrupted
nef
genes and to elucidate whether specific amino acid substitutions in Nef are associated with different stages of disease. We confirm that deletions or gross abnormalities in
nef
are rarely present. However, a comparison of Nef consensus sequences derived from 41 long-term nonprogressors and from 50 individuals with progressive HIV-1 infection revealed that specific variations are associated with different stages of infection. Five amino acid variations in Nef (T15, N51, H102, L170, and E182) were more frequently observed among nonprogressors, while nine features (an additional N-terminal PxxP motif, A15, R39, T51, T157, C163, N169, Q170, and M182) were more frequently found in progressors. Strong correlations between the frequency of these variations in Nef and both the CD4
+
-cell count and the viral load were observed. Moreover, analysis of sequential samples obtained from two progressors revealed that several variations in Nef, which were more commonly observed in patients with low CD4
+
-T-cell counts, were detected only during or after progression to immunodeficiency. Our results indicate that sequence variations in Nef are associated with different stages of HIV-1 infection and suggest a link between
nef
gene function and the immune status of the infected individual.
The Anamnestic Neutralizing Antibody Response Is Critical for Protection of Mice from Challenge following Vaccination with a Plasmid Encoding the Japanese Encephalitis Virus Premembrane and Envelope Genes
Authors: Konishi Eiji , Yamaoka Masaoki , Khin-Sane-Win  , Kurane Ichiro , Takada Kazuo , Mason Peter W.
Published: December 2019

For Japanese encephalitis (JE), we previously reported that recombinant vaccine-induced protection from disease does not prevent challenge virus replication in mice. Moreover, DNA vaccines for JE can provide protection from high challenge doses in the absence of detectable prechallenge neutralizing antibodies. In the present study, we evaluated the role of postchallenge immune responses in determining the outcome of JE virus infection, using mice immunized with a plasmid, pcDNA3JEME, encoding the JE virus premembrane (prM) and envelope (E) coding regions. In the first experiment, 10 mice were vaccinated once (five animals) or twice (remainder) with 100 μg of pcDNA3JEME. All of these mice showed low (6 of 10) or undetectable (4 of 10) levels of neutralizing antibodies. Interestingly, eight of these animals showed a rapid rise in neutralizing antibody following challenge with 10,000 50% lethal doses of JE virus and survived for 21 days, whereas only one of the two remaining animals survived. No unimmunized animals exhibited a rise of neutralizing antibody or survived challenge. Levels of JE virus-specific immunoglobulin M class antibodies were elevated following challenge in half of the unimmunized mice and in the single pcDNA3JEME-immunized mouse that died. In the second experiment, JE virus-specific primary cytotoxic T-lymphocyte (CTL) activity was detected in BALB/c mice immunized once with 100 μg of pcDNA3JEME 4 days after challenge, indicating a strong postchallenge recall of CTLs. In the third experiment, evaluation of induction of CTLs and antibody activity by plasmids containing portions of the prM/E cassette demonstrated that induction of CTL responses alone were not sufficient to prevent death. Finally, we showed that antibody obtained from pcDNA3JEME-immunized mice 4 days following challenge could partially protect recipient mice from lethal challenge. Taken together, these results indicate that neutralizing antibody produced following challenge provides the critical protective component in pcDNA3JEME-vaccinated mice.
Core-Binding Factor Influences the Disease Specificity of Moloney Murine Leukemia Virus
Authors: Lewis Amy F. , Stacy Terryl , Green William R. , Taddesse-Heath Lekidelu , Hartley Janet W. , Speck Nancy A.
Published: December 2019

The core site in the Moloney murine leukemia virus (Moloney MLV) enhancer was previously shown to be an important determinant of the T-cell disease specificity of the virus. Mutation of the core site resulted in a significant shift in disease specificity of the Moloney virus from T-cell leukemia to erythroleukemia. We and others have since determined that a protein that binds the core site, one of the core-binding factors (CBF) is highly expressed in thymus and is essential for hematopoiesis. Here we test the hypothesis that CBF plays a critical role in mediating pathogenesis of Moloney MLV in vivo. We measured the affinity of CBF for most core sites found in MLV enhancers, introduced sites with different affinities for CBF into the Moloney MLV genome, and determined the effects of these sites on viral pathogenesis. We found a correlation between CBF affinity and the latent period of disease onset, in that Moloney MLVs with high-affinity CBF binding sites induced leukemia following a shorter latent period than viruses with lower-affinity sites. The T-cell disease specificity of Moloney MLV also appeared to correlate with the affinity of CBF for its binding site. The data support a role for CBF in determining the pathogenic properties of Moloney MLV.
A Recombinant Human Cytomegalovirus with a Large Deletion in
UL97
Has a Severe Replication Deficiency
Authors: Prichard Mark N. , Gao Ning , Jairath Sanju , Mulamba Gilbert , Krosky Paula , Coen Donald M. , Parker Breck O. , Pari Gregory S.
Published: December 2019

Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCΔ97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in
trans
in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, the
UL97
gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.
Both Neutralization Resistance and High Infectivity Phenotypes Are Caused by Mutations of Interacting Residues in the Human Immunodeficiency Virus Type 1 gp41 Leucine Zipper and the gp120 Receptor- and Coreceptor-Binding Domains
Authors: Park Eun Ju , Quinnan Gerald V.
Published: December 2019

Neutralization resistance of human immunodeficiency virus type 1 (HIV-1) is a major impediment to vaccine development. We have found that residues of HIV-1 MN strain in the C terminus of gp120 and the leucine zipper (LZ) region of gp41 viral envelope proteins interact cooperatively to determine neutralization resistance and modulate infectivity. Further, results demonstrate that this interaction, by which regions of gp120 are assembled onto the LZ, involves amino acid residues intimately related to those which participate in the binding of the envelope to its receptor and coreceptor. Variations in this critical assembly structure determine the concordant, interdependent evolution of increased infectivity efficiency and neutralization resistance phenotypes of the envelopes. The results elucidate important structure-function relationships among epitopes that are important targets of vaccine development.
The Mechanism of an Immature Secretion Phenotype of a Highly Frequent Naturally Occurring Missense Mutation at Codon 97 of Human Hepatitis B Virus Core Antigen
Authors: Yuan Thomas Ta-Tung , Sahu Gautam Kumar , Whitehead William E. , Greenberg Richard , Shih Chiaho
Published: December 2019

A very frequent missense mutation at codon 97 of human hepatitis B virus (HBV) core antigen (HBcAg) has been found in chronic carriers worldwide. Functional characterization of this mutant revealed one intracellular and two extracellular phenotypes in contrast to wild-type HBV: (i) a 6- to 12-fold decrease in the level of the full-length relaxed circular DNA, a 4- to 5-fold decrease in the plus-strand DNA, and an approximately 1.8-fold decrease in the minus-strand and overall DNA levels in the intracellular viral core particles; (ii) a 5.7-fold increase in the immature secretion of Dane particles, containing minus-strand, single-stranded virion DNA; and (iii) a significant reduction of nonenveloped core particles in the medium. The steady-state levels of mutant and wild-type core proteins expressed from the same vector appeared to be similar. Using a complementation assay and gradient centrifugation analysis, we demonstrated that this mutant core protein alone is necessary and sufficient for immature secretion. The decreased level of intracellular HBV DNA is caused by both thecisdefect of the mutant genome and thetransdefect of the mutant core protein. We have dissected further the relationship between the intracellular and extracellular phenotypes of mutant F97L. The pleiotropic effects of the HBcAg codon 97 mutation were observed consistently in several different experimental settings. The mechanism and biological significance of these findings are discussed.
A Mouse Model for the Evaluation of Pathogenesis and Immunity to Influenza A (H5N1) Viruses Isolated from Humans
Authors: Lu Xiuhua , Tumpey Terrence M. , Morken Timothy , Zaki Sherif R. , Cox Nancy J. , Katz Jacqueline M.
Published: December 2019

During 1997 in Hong Kong, 18 human cases of respiratory illness, including 6 fatalities, were caused by highly pathogenic avian influenza A (H5N1) viruses. Since H5 viruses had previously been isolated only from avian species, the outbreak raised questions about the ability of these viruses to cause severe disease and death in humans. To better understand the pathogenesis and immunity to these viruses, we have used the BALB/c mouse model. Four H5N1 viruses replicated equally well in the lungs of mice without prior adaptation but differed in lethality for mice. H5N1 viruses that were highly lethal for mice were detected in multiple organs, including the brain. This is the first demonstration of an influenza A virus that replicates systemically in a mammalian species and is neurotropic without prior adaptation. The mouse model was also used to evaluate a strategy of vaccination against the highly pathogenic avian H5N1 viruses, using an inactivated vaccine prepared from nonpathogenic A/Duck/Singapore-Q/F119-3/97 (H5N3) virus that was antigenically related to the human H5N1 viruses. Mice administered vaccine intramuscularly, with or without alum, were completely protected from lethal challenge with H5N1 virus. Protection from infection was also observed in 70% of animals administered vaccine alone and 100% of mice administered vaccine with alum. The protective effect of vaccination correlated with the level of virus-specific serum antibody. These results suggests a strategy of vaccine preparedness for rapid intervention in future influenza pandemics that uses antigenically related nonpathogenic viruses as vaccine candidates.
Human Herpesvirus 6 Open Reading Frame U83 Encodes a Functional Chemokine
Authors: Zou Ping , Isegawa Yuji , Nakano Kazusi , Haque M. , Horiguchi Yasuhiko , Yamanishi Koichi
Published: December 2019

Some viruses including herpesviruses have undergone evolution to benefit viral infection and propagation by pirating and modifying host genes such as chemokine genes. Human herpesvirus 6 (HHV-6), acutely or persistently infects mononuclear cells in vitro. DNA sequence analysis of HHV-6 has revealed that the putative protein encoded by an open reading frame (ORF) of the U83 gene in HHV-6 variant B resembled a human chemokine. We have cloned the U83 gene and analyzed the biological function of this gene. The U83 gene contained an ORF encoding a 113-amino-acid peptide, starting at the first methionine and containing a possible signal peptide and the typical cysteine residues characteristic of the chemokines. Reverse transcription-PCR analysis of mRNA and immunofluorescent-antibody testing of infected cells both indicated that the encoded protein was a late protein. The ORF U83 gene fused to the Fc gene was expressed as a fusion protein in COS-7 cells by transfection, and the fusion protein was purified from the supernatant of transfected cells to test its biological function. The purified protein was capable of inducing transient calcium mobilization in THP-1 cells and of chemotactically activating THP-1 cells. These findings suggested that the U83 protein might play an important role in HHV-6 propagation in vivo by activating and trafficking mononuclear cells to sites of viral replication, thus aiding the development of superbly efficient virus production mechanisms.
Neutralizing Antibodies Inhibit Axonal Spread of Herpes Simplex Virus Type 1 to Epidermal Cells In Vitro
Authors: Mikloska Zorka , Sanna Pietro Paolo , Cunningham Anthony L.
Published: December 2019

The ability of antibodies to interfere with anterograde transmission of herpes simplex virus (HSV) from neuronal axons to the epidermis was investigated in an in vitro model consisting of human fetal dorsal root ganglia innervating autologous skin explants in a dual-chamber tissue culture system. The number and size of viral cytopathic plaques in epidermal cells after axonal transmission from HSV type 1 (HSV-1)-infected dorsal root ganglionic neurons were significantly reduced by addition to the outer chamber of neutralizing polyclonal human sera to HSV-1, of a human recombinant monoclonal group Ib antibody to glycoprotein D (gD), and of rabbit sera to HSV-1 gB and gD but not by rabbit anti-gE or anti-gG. A similar pattern of inhibition of direct infection of epidermal cells by these antibodies was observed. High concentrations of the monoclonal anti-gD reduced transmission by 90%. Rabbit anti-gB was not taken up into neurons, and human anti-gD did not influence spread of HSV in the dorsal root ganglia or axonal transport of HSV antigens when applied to individual dissociated neurons. These results suggest that anti-gD and -gB antibodies interfere with axonal spread of HSV-1, possibly by neutralizing HSV during transmission across an intercellular gap between axonal termini and epidermal cells, and thus contribute to control of HSV spread and shedding. Therefore, selected human monoclonal antibodies to protective epitopes might even be effective in preventing epidermis-to-neuron transmission during primary HSV infection, especially neonatal infection.
Interaction of Peptides with Sequences from the Newcastle Disease Virus Fusion Protein Heptad Repeat Regions
Authors: Young John K. , Li Donghui , Abramowitz Matthew C. , Morrison Trudy G.
Published: December 2019

Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 μM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291–304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an α helix.
Expression of the Open Reading Frame 74 (G-Protein-Coupled Receptor) Gene of Kaposi’s Sarcoma (KS)-Associated Herpesvirus: Implications for KS Pathogenesis
Authors: Kirshner Jessica R. , Staskus Katherine , Haase Ashley , Lagunoff Michael , Ganem Don
Published: December 2019

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) encodes a G-protein-coupled receptor (GCR) homolog. This protein is a potent, constitutively active signalling molecule that can influence both proliferation and angiogenesis when ectopically expressed in fibroblasts in vitro. Here we have examined the expression of the KSHV GCR gene in virus-infected lymphoid cells and in KS tumors. Our results show that in both situations the gene is expressed primarily during lytic replication; its transcription is unaffected by inhibition of viral DNA synthesis, indicating that it is expressed in the early phases of the lytic program. The major transcript bearing GCR sequences is bicistronic, harboring coding sequences for another viral gene, K14, at its 5′ end. Extensive searches for monocistronic GCR mRNAs using nuclease mapping and reverse transcription-PCR failed to detect such species. The 5′ end of K14/GCR mRNA maps to nucleotide (nt) 127848, and its poly(A) addition site maps to nt 130546; a 149-nt intron is present in the K14/GCR intergenic region. These results suggest that the KSHV GCR is translated by unconventional mechanisms involving either translational reinitiation, internal ribosomal entry, or leaky ribosomal scanning. The restriction of GCR expression to the lytic cycle has important implications for the potential role(s) of the GCR in KS pathogenesis.
The Novel Structural Protein of Human Cytomegalovirus, pUL25, Is Localized in the Viral Tegument
Authors: Zini N. , Battista M. C. , Santi S. , Riccio M. , Bergamini G. , Landini M. P. , Maraldi N. M.
Published: December 2019

Human cytomegalovirus UL25 codes for a structural phosphoprotein of 85 kDa (C. J. Baldick and T. Shenk, J. Virol. 70:6097–6105, 1996; M. C. Battista et al., J. Virol. 73:3800–3809, 1999). In this study we analyzed the intracellular and intraviral localization of pUL25 by confocal and immunoelectron microscopy and found that pUL25 is a component of the viral tegument and the dense body matrix, acquired during the late cytoplasmic phase of virus maturation.
Use of Real-Time PCR and Molecular Beacons To Detect Virus Replication in Human Immunodeficiency Virus Type 1-Infected Individuals on Prolonged Effective Antiretroviral Therapy
Authors: Lewin S. R. , Vesanen M. , Kostrikis L. , Hurley A. , Duran M. , Zhang L. , Ho D. D. , Markowitz M.
Published: December 2019

We have designed a novel, precise, and sensitive assay to measure unspliced (US) human immunodeficiency virus type 1 (HIV-1) mRNA in peripheral blood mononuclear cells of HIV-1-infected individuals by using real-time PCR and molecular beacons. Individuals were classified as either well suppressed (WS) or partially suppressed, based on longitudinal measurements of plasma HIV-1 RNA. The proportion of individuals with US mRNA undetectable over time was significantly higher among WS individuals; however, 30% of WS subjects still had detectable US mRNA after 24 months of effective antiviral therapy.
Deletion Mutagenesis within the Dimerization Initiation Site of Human Immunodeficiency Virus Type 1 Results in Delayed Processing of the p2 Peptide from Precursor Proteins
Authors: Liang Chen , Rong Liwei , Cherry Elana , Kleiman Lawrence , Laughrea Michael , Wainberg Mark A.
Published: December 2019

Previous work has shown that deletions of genomic segments at nucleotide (nt) positions +238 to +253, i.e., construct BH10-LD3, or nt positions +261 to +274, i.e., construct BH10-LD4, within the human immunodeficiency virus type 1 (HIV-1) dimerization initiation site (DIS) destroyed DIS secondary structure and dramatically reduced viral replication capacity. Surprisingly, two point mutations located within the viral peptide 2 (p2) and nucleocapsid (NC) protein termed MP2 and MNC, respectively, were able to compensate for this defect. Since the MP2 mutation involves an amino acid substitution near the cleavage site between p2 and NC, we investigated the effects of the above-mentioned deletions on the processing of Gag proteins. Immunoprecipitation assays performed with monoclonal antibodies against viral capsid (CA) (p24) protein showed that p2 was cleaved from CA with less efficiency in viruses that contained the LD3 and LD4 deletions than in wild-type viruses. The presence of the two compensatory mutations, MP2 and MNC, increased the efficiency of the cleavage of p2 from CA, but neither mutation alone had this effect or was sufficient to compensate for the observed impairment in infectiousness. A virus that contained both of the above-mentioned deletions within the DIS was also impaired in regard to processing and infectiousness, and it could likewise be compensated by the MP2 and MNC point mutations. These results suggest that the DIS region of HIV-1 RNA plays an important role in the processing of Gag proteins.
Contributions of Viral Splice Sites and
cis
-Regulatory Elements to Lentivirus Vector Function
Authors: Cui Yan , Iwakuma Tomoo , Chang Lung-Ji
Published: December 2019

The mobile transgene constructs of most human immunodeficiency virus (HIV)-based lentivirus vectors currently in use contain viral long terminal repeats, a 5′ untranslated region,
gag
sequences, and
env
sequences that include the Rev-responsive element (RRE). In this study, we examined the possibility of deleting HIV splice sites and
gag
and
env
sequences from an HIV type 1 recombinant vector established in our laboratory as part of our ongoing efforts to improve this vector system. Mutations in the major splice donor site (SD) markedly reduced viral RNA expression but had little effect on vector titer. Deletion of
gag
or
env
sequences, excluding RRE, led to a moderate reduction in vector titer. Interestingly, deletion of RRE slightly reduced viral RNA expression but markedly impaired vector function. Combined deletions of RRE,
gag
(except for the first 40 nucleotides),
env
, and the SD mutation resulted in a twofold increase in cytoplasmic viral RNA expression and a recovery of vector efficiency to ∼50% of the wild-type level. This increase in cytoplasmic RNA levels is likely to be due, at least in part, to effects of the TE671 host cells, a human rhabdomyosarcoma cell line used for vector production in our system, on the cytoplasmic distribution of spliced and unspliced viral RNA. These results show that optimal lentivirus vector function can be maintained in the absence of multiple essential viral elements.
Depletion of Blood-Borne Macrophages Does Not Reduce Demyelination in Mice Infected with a Neurotropic Coronavirus
Authors: Xue Shurong , Sun Ning , Van Rooijen Nico , Perlman Stanley
Published: December 2019

Mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating disease with symptoms of hindlimb paralysis. Histological examination of the brains and spinal cords of these animals reveals the presence of large numbers of activated macrophages/microglia. In two other experimental models of demyelination, experimental allergic encephalomyelitis and Theiler’s murine encephalomyelitis virus-induced demyelination, depletion of hematogenous macrophages abrogates the demyelinating process. In both of these diseases, early events in the demyelinating process are inhibited by macrophage depletion. From these studies, it was not possible to determine whether infiltrating macrophages were required for late steps in the process, such as myelin removal. In this study, we show that when macrophages are depleted with either unmodified or mannosylated liposomes encapsulating dichloromethylene diphosphate, the amount of demyelination detected in MHV-infected mice is not affected. At a time when these cells were completely depleted from the liver, approximately equivalent numbers of macrophages were present in the spinal cords of control and drug-treated animals. These results suggest that blood-borne macrophages are not required for MHV-induced demyelination and also suggest that other cells, such as perivascular macrophages or microglia, perform the function of these cells in the presence of drug.
Construction and Transposon Mutagenesis in
Escherichia coli
of a Full-Length Infectious Clone of Pseudorabies Virus, an Alphaherpesvirus
Authors: Smith Gregory A. , Enquist Lynn W.
Published: December 2019

A full-length clone of the 142-kb pseudorabies virus (PRV) genome was constructed as a stable F plasmid in
Escherichia coli
. The clone, pBecker1, was colinear with PRV-Becker genomic DNA, lacking detectable rearrangements, deletions, or inversions. The transfection of pBecker1 into susceptible eukaryotic cells resulted in productive viral infection. Virus isolated following transfection was indistinguishable from wild-type virus in a rodent model of infection and spread to retinorecipient regions of the brain following inoculation in the vitreous body of the eye. Mutagenesis of pBecker1 in
E. coli
with a mini-Tn
5
-derived transposon enabled the rapid isolation of insertion mutants, identification of essential viral genes, and simplified construction of viral revertants. The serial passage of a viral insertion mutant demonstrated the transposon insertion to be stable. However, the F-plasmid insertion present in the viral gG locus was found to undergo a spontaneous deletion following transfection into eukaryotic cells. The implications of F-plasmid insertion into the viral genome with regard to phenotype and genomic stability are discussed.
A Lysine-to-Arginine Change Found in Natural Alleles of the Human T-Cell Lymphotropic/Leukemia Virus Type 1 p12
I
Protein Greatly Influences Its Stability
Authors: Trovato Raffaella , Mulloy James C. , Johnson Julie M. , Takemoto Shigeki , de Oliveira Maria Pombo , Franchini Genoveffa
Published: December 2019

The HTLV-1 singly spliced open reading frame I protein, p12
I
, is highly unstable and appears to be necessary for persistent infection in rabbits. Here we demonstrate that p12
I
forms dimers through two putative leucine zipper domains and that its stability is augmented by specific proteasome inhibitors. p12
I
is ubiquitylated, and mutations of its unique carboxy-terminus lysine residue to an arginine greatly enhance its stability. Interestingly, analysis of 53 independent HTLV-1 strains revealed that the natural p12
I
alleles found in ex vivo samples of tropical spastic paraparesis-HTLV-1-associated myelopathy patients contain a Lys at position 88 in some cases, whereas arginine is consistently found at position 88 in HTLV-1 strains from all adult T-cell leukemia-lymphoma (ATLL) cases and healthy carriers studied. This apparent segregation of different alleles in tropical spastic paraparesis-HTLV-associated myelopathy and ATLL or healthy carriers may be relevant in vivo, since p12
I
binds the interleukin-2 receptor β and γ
c
chains, raising the possibility that the two natural alleles might affect differently the regulation of these molecules.
Cell Surface Expression of H2 Antigens on Primary Sensory Neurons in Response to Acute but Not Latent Herpes Simplex Virus Infection In Vivo
Authors: Pereira Rosemarie A. , Simmons Anthony
Published: December 2019

CD8
+
T lymphocytes and class I major histocompatibility complex (MHC-I) molecules profoundly influence the severity of neuronal herpes simplex virus (HSV) infection in experimentally infected mice. Paradoxically, neurons are classically regarded as MHC-I deficient. However, it is shown here that H2-encoded heavy chains (αCs) and their associated light chain, β2 microglobulin, are present on the surfaces of primary sensory neurons recovered from sensory ganglia within 1 to 2 weeks of HSV infection. During this time, some neurons are found to be tightly associated with T cells in vivo. Prior data showed that termination of productive HSV infection in the peripheral nervous system is not dependent on cell-mediated lysis of infected neurons. Consistent with these data, immunogold electron microscopy showed that the density of cell surface H2 on neurons is an order of magnitude lower than on satellite glia, which is predicted to favor a noncytolytic CD8 cell response.
Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase
Authors: Gale Michael , Kwieciszewski Bart , Dossett Michelle , Nakao Haruhisa , Katze Michael G.
Published: December 2019

Hepatitis C virus (HCV) is prevalent worldwide and has become a major cause of liver dysfunction and hepatocellular carcinoma. The high prevalence of HCV reflects the persistent nature of infection and the large frequency of cases that resist the current interferon (IFN)-based anti-HCV therapeutic regimens. HCV resistance to IFN has been attributed, in part, to the function of the viral nonstructural 5A (NS5A) protein. NS5A from IFN-resistant strains of HCV can repress the PKR protein kinase, a mediator of the IFN-induced antiviral and apoptotic responses of the host cell and a tumor suppressor. Here we examined the relationship between HCV persistence and resistance to IFN therapy. When expressed in mammalian cells, NS5A from IFN-resistant HCV conferred IFN resistance to vesicular stomatitis virus (VSV), which normally is sensitive to the antiviral actions of IFN. NS5A blocked viral double-stranded RNA (dsRNA)-induced PKR activation and phosphorylation of eIF-2α in IFN-treated cells, resulting in high levels of VSV mRNA translation. Mutations within the PKR-binding domain of NS5A restored PKR function and the IFN-induced block to viral mRNA translation. The effects due to NS5A inhibition of PKR were not limited to the rescue of viral mRNA translation but also included a block in PKR-dependent host signaling pathways. Cells expressing NS5A exhibited defective PKR signaling and were refractory to apoptosis induced by exogenous dsRNA. Resistance to apoptosis was attributed to an NS5A-mediated block in eIF-2α phosphorylation. Moreover, cells expressing NS5A exhibited a transformed phenotype and formed solid tumors in vivo. Disruption of apoptosis and tumorogenesis required the PKR-binding function of NS5A, demonstrating that these properties may be linked to the IFN-resistant phenotype of HCV.
Herpes Simplex Virus Latency-Associated Transcript Encodes a Protein Which Greatly Enhances Virus Growth, Can Compensate for Deficiencies in Immediate-Early Gene Expression, and Is Likely To Function during Reactivation from Virus Latency
Authors: Thomas S. K. , Gough G. , Latchman D. S. , R. S.  , Coffin
Published: December 2019

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) enter and reactivate from latency in sensory neurons, although the events governing these processes are little understood. During latency, only the latency-associated transcripts (LATs) are produced. However, although the LAT RNAs were described ≈10 years ago, their function remains ambiguous. Mutations affecting the LATs have minimal effects other than a small reduction in establishment of and reactivation from latency in some cases. Mutations in putative LAT-contained open reading frames (ORFs) have so far shown no effect. The LATs consist of a large species from which smaller (≈2 kb), nuclear, nonlinear LATs which are abundant during latency are spliced. Thus, translation of ORFs in these smaller LATs would not usually be expected to be possible, and if expressed at all, their expression might be tightly regulated. Here we show that deregulated expression of the largest HSV1 2-kb LAT-contained ORF in various cells of neuronal and nonneuronal origin greatly enhances virus growth in a manner specific to HSV1—the HSV1 LAT ORF has no effect on the growth of HSV2. Similar results of enhanced growth were found when the HSV1 LAT ORF was constitutively expressed from within the HSV1 genome. The mechanism of LAT ORF action was strongly suggested to be by substituting for deficiencies in immediate-early (IE) gene expression (particularly ICP0), because deregulated LAT ORF expression, as well as enhancing wild-type virus growth, was also found to allow efficient growth of viruses with mutations in ICP0 or VMW65. Such viruses otherwise exhibit considerable growth defects. IE gene expression deficiencies are often the block to productive infection in nonpermissive cells and are also evident during latency. These results, which we show to be protein- rather than RNA-mediated effects, strongly suggest a function of the tightly regulated expression of a LAT ORF-encoded protein in the reactivation from HSV latency.