bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolution of Syncytium-Inducing and Non-Syncytium-Inducing Biological Virus Clones in Relation to Replication Kinetics during the Course of Human Immunodeficiency Virus Type 1 Infection
Authors: van ’t Wout Angélique B. , Blaak Hetty , Ran Leonie J. , Brouwer Margreet , Kuiken Carla , Schuitemaker Hanneke
Published: December 2019

To investigate the temporal relationship between human immunodeficiency virus type 1 (HIV-1) replicative capacity and syncytium-inducing (SI) phenotype, biological and genetic characteristics of longitudinally obtained virus clones from two HIV-1-infected individuals who developed SI variants were studied. In one individual, the emergence of rapidly replicating SI and non-syncytium-inducing (NSI) variants was accompanied by a loss of the slowly replicating NSI variants. In the other subject, NSI variants were always slowly replicating, while the coexisting SI variants showed an increase in the rate of replication. Irrespective their replicative capacity, the NSI variants remained present throughout the infection in both individuals. Phylogenetic analysis of the V3 region showed early branching of the SI variants from the NSI tree. Successful SI conversion seemed a unique event since no SI variants were found among later-stage NSI variants. This was also confirmed by the increasing evolutionary distance between the two subpopulations. At any time point during the course of the infection, the variation within the coexisting SI and NSI populations did not exceed 2%, indicating continuous competition within each viral subpopulation.
Human Immunodeficiency Virus Inhibits Multilineage Hematopoiesis In Vivo
Authors: Koka Prasad S. , Fraser John K. , Bryson Yvonne , Bristol Gregory C. , Aldrovandi Grace M. , Daar Eric S. , Zack Jerome A.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often exhibit multiple hematopoietic abnormalities reaching far beyond loss of CD4+lymphocytes. We used the SCID-hu (Thy/Liv) mouse (severe combined immunodeficient mouse transplanted with human fetal thymus and liver tissues), which provides an in vivo system whereby human pluripotent hematopoietic progenitor cells can be maintained and undergo T-lymphoid differentiation and wherein HIV-1 infection causes severe depletion of CD4-bearing human thymocytes. Herein we show that HIV-1 infection rapidly and severely decreases the ex vivo recovery of human progenitor cells capable of differentiation into both erythroid and myeloid lineages. However, the total CD34+cell population is not depleted. Combination antiretroviral therapy administered well after loss of multilineage progenitor activity reverses this inhibitory effect, establishing a causal role of viral replication. Taken together, our results suggest that pluripotent stem cells are not killed by HIV-1; rather, a later stage important in both myeloid and erythroid differentiation is affected. In addition, a primary virus isolated from a patient exhibiting multiple hematopoietic abnormalities preferentially depleted myeloid and erythroid colony-forming activity rather than CD4-bearing thymocytes in this system. Thus, HIV-1 infection perturbs multiple hematopoietic lineages in vivo, which may explain the many hematopoietic defects found in infected patients.
Immunoglobulin G, Plasma Cells, and Lymphocytes in the Murine Vagina after Vaginal or Parenteral Immunization with Attenuated Herpes Simplex Virus Type 2
Authors: Parr Earl L. , Parr Margaret B.
Published: December 2019

This investigation evaluated immunity to vaginal herpes simplex virus type 2 (HSV-2) infection after local or parenteral immunization with attenuated HSV-2. Vaginal immunization induced sterilizing immunity against challenge with a high dose of wild-type virus, whereas parenteral immunizations protected against neurologic disease but did not entirely prevent infection of the vagina. Vaginal immunization caused 86- and 31-fold increases in the numbers of immunoglobulin G (IgG) plasma cells in the vagina at 6 weeks and 10 months after immunization, whereas parenteral immunizations did not increase plasma cell numbers in the vagina. Vaginal secretion/serum titer ratios and specific antibody activities in vaginal secretions and serum indicated that IgG viral antibody was produced in the vagina and released into vaginal secretions at 6 weeks and 10 months after vaginal immunization but not after parenteral immunizations. In contrast to the case for plasma cells, the numbers of T and B lymphocytes in the vagina were similar in vaginally and parenterally immunized mice. Also, lymphocyte numbers in the vagina were markedly but similarly increased by vaginal challenge with HSV-2 in both vaginally and parenterally immunized mice. Lymphocyte recruitment to the vagina after virus challenge appeared to involve memory lymphocytes, because it was not observed in nonimmunized mice. Thus, local vaginal immunization with attenuated HSV-2 increased the number of IgG plasma cells in the vagina and increased vaginal secretion/serum titer ratios to 3.0- to 4.7-fold higher than in parenterally immunized groups but caused little if any selective homing of T and B lymphocytes to the vagina.
Similar Levels of Human Immunodeficiency Virus Type 1 Replication in Human TH1 and TH2 Clones
Authors: Mikovits Judy A. , Taub Dennis D. , Turcovski-Corrales Susan M. , Ruscetti Francis W.
Published: December 2019

Studies on the development and function of CD4+TH1 and TH2 cells during the progression to AIDS may increase the understanding of AIDS pathogenesis. The preferential replication of human immunodeficiency virus (HIV) in either TH1 or TH2 cells could alter the delicate balance of the immune response. TH1 (gamma interferon [IFN-γ] positive, interleukin-4 [IL-4] and IL-5 negative) and TH2 (IFN-γ negative, IL-4 and IL-5 positive) clones, developed from several healthy donors, pedigreed by reverse transcriptase PCR (RT-PCR) and enzyme linked immunosorbent assay have similar levels of cell surface expression of CD4 and several chemokine receptor cofactors necessary for viral entry. After activation by specific antigens and infection with T-cell-tropic strains of HIV type 1 (HIV-1), TH1 and TH2 clones showed similar levels of viral entry and reverse transcription. At days 3 through 14 postinfection, HIV replicated to similar levels in several TH1 and TH2 clones as measured by release of HIV p24 and total number of copies ofgagRNA/total cell RNA as measured by RT-PCR. When values were normalized for viable cell number in three clones of each type, there was up to twofold more HIV RNA in TH1 than TH2 cells. In addition, several primary monocytotropic HIV-1 strains were able to replicate to similar levels in TH1 and TH2 cells. These studies suggest that the importance of TH1 and TH2 subsets in AIDS pathogenesis transcends clonal differences in their ability to support HIV replication.
Isolation and Propagation of Human Papillomavirus Type 16 in Human Xenografts Implanted in the Severe Combined Immunodeficiency Mouse
Authors: Bonnez William , DaRin Carrie , Borkhuis Christine , de Mesy Jensen Karen , Reichman Richard C. , Rose Robert C.
Published: December 2019

We report the isolation and propagation of human papillomavirus type 16, the main agent of cervical cancer, using human foreskin fragments implanted in severe combined immunodeficiency mice. The infection produced viral particles, and with each passage of the virus it caused lesions identical to intraepithelial neoplasia, the precursor to carcinoma.
Nucleotide Sequence of Porcine Circovirus Associated with Postweaning Multisystemic Wasting Syndrome in Pigs
Authors: Hamel Andre L. , Lin Lihua L. , Nayar Gopi P. S.
Published: December 2019

This article describes the nucleotide sequence of a porcine circovirus (PCV) which possesses a high degree of association with postweaning multisystemic wasting syndrome (PMWS), a newly described disease of young pigs. The DNA sequence of this PMWS-associated PCV (pmws PCV) has 68% homology with that of a previously published nonpathogenic strain of PCV. The strains appear to be closely related yet distinct from one another.
A Recombinant Classical Swine Fever Virus Stably Expresses a Marker Gene
Authors: Moser Christian , Tratschin Jon-Duri , Hofmann Martin A.
Published: December 2019

The gene coding for bacterial chloramphenicol acetyltransferase (CAT) was inserted in frame into the viral N
pro
gene of the full-length cDNA clone pA187-1 of the classical swine fever virus (CSFV) strain Alfort/187. RNA transcribed in vitro from the resulting plasmid was transfected into SK-6 porcine kidney cells. Infectious progeny virus vA187-CAT recovered from transfected cells had growth characteristics indistinguishable from those of parental virus vA187-1. In cells infected with vA187-CAT the predicted fusion protein, CAT-N
pro
, was detected, and it retained the enzymatic activities of both CAT and N
pro
. The CAT gene remained stably inserted in the viral genome after 10 virus passages. Thus, marker virus vA187-CAT represents a useful tool for quantitative analysis of viral replication and gene expression.
Archetype JC Virus Efficiently Replicates in COS-7 Cells, Simian Cells Constitutively Expressing Simian Virus 40 T Antigen
Authors: Hara Kazuya , Sugimoto Chie , Kitamura Tadaichi , Aoki Naoto , Taguchi Fumiaki , Yogo Yoshiaki
Published: December 2019

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), is ubiquitous in humans, infecting children asymptomatically and then persisting in the kidney. Renal JCV is not latent but replicates to excrete progeny in the urine. The renal-urinary JCV DNAs carry the archetype regulatory region that generates various rearranged regulatory regions occurring in JCVs derived from the brains of PML patients. Tissue cultures that support the efficient growth of archetype JCV have not been reported. We studied whether archetype JCV could replicate in COS-7 cells, simian cells transformed with an origin-defective mutant of simian virus 40 (SV40). Efficient JCV replication, as detected by a hemagglutination assay, was observed in cultures transfected with five of the six archetype DNAs. The progeny JCVs could be passaged to fresh COS-7 cells. However, when the parental cells of COS-7 not expressing T antigen were transfected with archetype JCV DNAs, no viral replication was detected, indicating that SV40 T antigen is essential for the growth of JCV in COS-7 cells. The archetype regulatory region was conserved during viral growth in COS-7 cells, although a small proportion of JCV DNAs underwent rearrangements outside the regulatory region. We then attempted to recover archetype JCV from urine by viral culture in COS-7 cells. Efficient JCV production was observed in COS-7 cells infected with five of the six JCV-positive urine samples examined. Thus, COS-7 cells should be of use not only for the production of archetype JCV on a large scale but also for the isolation of archetype JCV from urine.
The Latent Herpes Simplex Virus Type 1 Genome Copy Number in Individual Neurons Is Virus Strain Specific and Correlates with Reactivation
Authors: Sawtell N. M. , Poon D. K. , Tansky C. S. , Thompson R. L.
Published: December 2019

The viral genetic elements that determine the in vivo reactivation efficiencies of fully replication competent wild-type herpes simplex virus (HSV) strains have not been identified. Among the common laboratory strains, KOS reactivates in vivo at a lower efficiency than either strain 17syn+ or strain McKrae. An important first step in understanding the molecular basis for this observation is to distinguish between viral genetic factors that regulate the establishment of latency from those that directly regulate reactivation. Reported here are experiments performed to determine whether the reduced reactivation of KOS was associated with a reduced ability to establish or maintain latent infections. For comparative purposes, latent infections were quantified by (i) quantitative PCR on DNA extracted from whole ganglia, (ii) the number of latency-associated transcript (LAT) promoter-positive neurons, using KOS and 17syn+ LAT promoter–β-galactosidase reporter mutants, and (iii) contextual analysis of DNA. Mice latently infected with 17syn+-based strains contained more HSV type 1 (HSV-1) DNA in their ganglia than those infected with KOS strains, but this difference was not statistically significant. The number of latently infected neurons also did not differ significantly between ganglia latently infected with either the low- or high-reactivator strains. In addition to the number of latent sites, the number of viral genome copies within the individual latently infected neurons has recently been demonstrated to be variable. Interestingly, neurons latently infected with KOS contained significantly fewer viral genome copies than those infected with either 17syn+ or McKrae. Thus, the HSV-1 genome copy number profile is viral strain specific and positively correlates with the ability to reactivate in vivo. This is the first demonstration that the number of HSV genome copies within individual latently infected neurons is regulated by viral genetic factors. These findings suggest that the latent genome copy number may be an important parameter for subsequent induced reactivation in vivo.
Characterization of the Proline-Rich Region of Murine Leukemia Virus Envelope Protein
Authors: Weimin Wu Bonnie , Cannon Paula M. , Gordon Erlinda M. , Hall Frederick L. , Anderson W. French
Published: December 2019

Mammalian type C retroviral envelope proteins contain a variable proline-rich region (PRR), located between the N-terminal receptor-binding domain and the more highly conserved C-terminal portion of the surface (SU) subunit. We have investigated the role of the PRR in the function of murine leukemia virus (MuLV) envelope protein. In the MuLVs, the PRR contains a highly conserved N-terminal sequence and a hypervariable C-terminal sequence. Despite this variability, the amphotropic PRR could functionally substitute for the ecotropic PRR. The hypervariable region of the PRR was not absolutely required for envelope protein function. However, truncations in this region resulted in decreased levels of both the SU and TM proteins in viral particles and increased amounts of the uncleaved precursor protein, Pr85. In contrast, the N-terminal conserved region was essential for viral infectivity. Deletion of this region prevented the stable incorporation of envelope proteins into viral particles in spite of normal envelope protein processing, wild-type levels of cell surface expression, and a wild-type ability to induce syncytia in an XC cell cocultivation assay. However, higher levels of the SU protein were shed into the supernatant, suggesting a defect in SU-TM interactions. Our data are most consistent with a role for the PRR in stabilizing the overall structure of the protein, thereby affecting the proper processing of Pr85, SU-TM interactions, and the stable incorporation of envelope proteins into viral particles. In addition, we have demonstrated that the PRR can tolerate the insertion of a peptide-binding domain, making this a potentially useful site for constructing targetable retroviral vectors.
Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins
Authors: Biswas Siddhartha K. , Boutz Paul L. , Nayak Debi P.
Published: December 2019

Influenza virus nucleoprotein (NP) is a critical factor in the viral infectious cycle in switching influenza virus RNA synthesis from transcription mode to replication mode. In this study, we investigated the interaction of NP with the viral polymerase protein complex. Using coimmunoprecipitation with monospecific or monoclonal antibodies, we observed that NP interacted with the RNP-free polymerase protein complex in influenza virus-infected cells. In addition, coexpression of the components of the polymerase protein complex (PB1, PB2, or PA) with NP either together or pairwise revealed that NP interacts with PB1 and PB2 but not PA. Interaction of NP with PB1 and PB2 was confirmed by both coimmunoprecipitation and histidine tagging of the NP-PB1 and NP-PB2 complexes. Further, it was observed that NP-PB2 interaction was rather labile and sensitive to dissociation in 0.1% sodium dodecyl sulfate and that the stability of NP-PB2 interaction was regulated by the sequences present at the COOH terminus of NP. Analysis of NP deletion mutants revealed that at least three regions of NP interacted independently with PB2. A detailed analysis of the COOH terminus of NP by mutation of serine-to-alanine (SA) residues either individually or together demonstrated that SA mutations in this region did not affect the binding of NP to PB2. However, some SA mutations at the COOH terminus drastically affected the functional activity of NP in an in vivo transcription-replication assay, whereas others exhibited a temperature-sensitive phenotype and still others had no effect on the transcription and replication of the viral RNA. These results suggest that a direct interaction of NP with polymerase proteins may be involved in regulating the switch of viral RNA synthesis from transcription to replication.
Differential Selection of Cells with Proviral c-
myc
and c-
erbB
Integrations after Avian Leukosis Virus Infection
Authors: Gong Min , Semus Helen L. , Bird Kelly J. , Stramer Brian J. , Ruddell Alanna
Published: December 2019

Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-
myc
proto-oncogene and induces erythroblastosis after integration within the c-
erbB
proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-
myc
integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-
myc
integrations. These integrations were clustered within the 3′ half of c-
myc
intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3′ c-
myc
intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-
myc
integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-
myc
integrations are preferentially expanded within the bursal environment. Proviral integration within the c-
erbB
gene was also analyzed, to detect clustered c-
erbB
intron 14 integrations associated with erythroblastosis. Proviral c-
erbB
integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-
erbB
exon 15, indicating that cells carrying 3′ intron 14 integrations must be selected during induction of erythroblastosis.
Sequences Just Upstream of the Simian Immunodeficiency Virus Core Enhancer Allow Efficient Replication in the Absence of NF-κB and Sp1 Binding Elements
Authors: Pöhlmann Stefan , Flöss Stefan , Ilyinskii Petr O. , Stamminger Thomas , Kirchhoff Frank
Published: December 2019

Large deletions of the upstream U3 sequences in the long terminal repeats (LTRs) of human immunodeficiency virus and simian immunodeficiency virus (SIV) accumulate in vivo in the absence of an intact
nef
gene. In the SIV U3 region, about 65 bp just upstream of the single NF-κB binding site always remained intact, and some evidence for a novel enhancer element in this region exists. We analyzed the transcriptional and replicative capacities of SIVmac239 mutants containing deletions or mutations in these upstream U3 sequences and/or the NF-κB and Sp1 binding sites. Even in the absence of 400 bp of upstream U3 sequences, the NF-κB site and all four Sp1 binding sites, the SIV promoter maintained about 15% of the wild-type LTR activity and was fully responsive to Tat activation in transient reporter assays. The effects of these deletions on virus production after transfection of COS-1 cells with full-length proviral constructs were much greater. Deletion of the upstream U3 sequences had no significant influence on viral replication when either the single NF-κB site or the Sp1 binding sites were intact. In contrast, the 26 bp of sequence located immediately upstream of the NF-κB site was essential for efficient replication when all core enhancer elements were deleted. A purine-rich site in this region binds specifically to the transcription factor Elf-1, a member of the
ets
proto-oncogene-encoded family. Our results indicate a high degree of functional redundancy in the SIVmac U3 region. Furthermore, we defined a novel regulatory element located immediately upstream of the NF-κB binding site that allows efficient viral replication in the absence of the entire core enhancer region.
NF-κB-Mediated Inhibition of Apoptosis Is Required for Encephalomyocarditis Virus Virulence: a Mechanism of Resistance in p50 Knockout Mice
Authors: Schwarz Edward M. , Badorff Cornel , Hiura Timothy S. , Wessely Rainer , Badorff Annette , Verma Inder M. , Knowlton Kirk U.
Published: December 2019

Apoptosis is a central host defense mechanism to eliminate virus-infected cells. Activation of NF-κB suppresses apoptosis following some types of stimulation in vitro. To test the physiological importance of this pathway in vivo, we studied murine encephalomyocarditis virus (EMCV) infection in mice and cell lines defective in NF-κB1 (p50) signaling. As previously reported, we find that all p50 knockout (p50 −/−) mice survive an EMCV infection that readily kills normal mice. By introducing the p50 mutation into interferon (IFN) type I receptor knockout (IFNRI −/−) mice, we find that this resistance is not mediated by IFN-β as previously thought. While no IFNRI −/− mice survive, the double-knockout mice survive 60% of the time. The survival is tightly linked to the animals’ ability to clear the virus from the heart in vivo. Using murine embryonic fibroblasts (MEF) derived from wild-type, p50 −/−, and p65 −/− embryos, we found that NF-κB is not required for the replication cycle of EMCV. However, during these experiments we observed that p50 −/− and p65 −/− MEF infected with EMCV undergo enhanced, premature cytotoxicity. Upon examination of this cell death, we found that EMCV infection induced both plasma membrane and nuclear changes typical of apoptosis in all cell lines. These apoptotic processes occurred in an accelerated and pronounced way in the NF-κB-defective cells, as soon as 6 h after infection, when virus is beginning to be released. Previously, only the RelA (p65) subunit of NF-κB has been shown to play a role in suppressing apoptosis. In our studies, we find that p50 is equally important in suppressing apoptosis during EMCV infection. Additionally, we show that suppression of apoptosis by NF-κB1 is required for EMCV virulence in vivo. The attenuation in p50 −/− mice can be explained by rapid apoptosis of infected cells which allows host phagocytes to clear infected cells before the viral burst leading to a reduction of the viral burden and survival of the mice.
B-Cell Lymphoma Induction by Akv Murine Leukemia Viruses Harboring One or Both Copies of the Tandem Repeat in the U3 Enhancer
Authors: Lovmand Jette , Sørensen Annette B. , Schmidt Jörg , Østergaard Mette , Luz Arne , Pedersen Finn Skou
Published: December 2019

Akv is an endogenous, ecotropic murine leukemia virus (MuLV) of the AKR strain. It has served as a prototype nonpathogenic or weakly pathogenic reference virus for studies of closely related potent lymphomagenic viruses such as the T-lymphomagenic SL3-3. We here report that Akv and an Akv mutant (Akv1-99) with only one copy of the 99-bp transcriptional enhancer induce malignant lymphomas with nearly 100% incidence and mean latency periods of 12 months after injection into newborn NMRI mice. Molecular analysis of tumor DNA showed that the majority of the tumors were of the B-cell type. Sequence analysis of proviral transcriptional enhancers in DNA of B-cell lymphomas revealed conservation of the enhancer sequence, as well as a lack of sequence duplications of the Akv1-99 variant, while the repeat copy number in Akv was subject to fluctuations. In support of a B-cell specificity of the Akv enhancer, a murine plasmacytoma cell line was found to sustain three- to fivefold-higher transient transcriptional activity upon the Akv and Akv1-99 enhancers than upon the enhancer of the T-lymphomagenic SL3-3 MuLV. Thus, the overall picture is that Akv MuLV possesses a B- lymphomagenic potential and that the second copy of the 99-bp sequence seems to be of minor importance for this potential. However, in one animal the lymphomas induced by Akv1-99 were of the T-cell type. Among the 24 tumors analyzed only this one harbored a clonal proviral integration in the c-
myc
locus. This provirus had undergone a duplication of a 113-bp sequence of the enhancer region, partly overlapping with the 99-bp repeat of Akv, as well as a few single nucleotide alterations within and outside the repeats. Taken together with previous studies, our results suggest that T- versus B-lymphomagenic specificity of the enhancer is governed by more than one nucleotide difference and that alterations in binding sites for transcription factors of the AML1 and nuclear-factor-1 families may contribute to this specificity.
The Interleukin-17 Gene of Herpesvirus Saimiri
Authors: Knappe Andrea , Hiller Christian , Niphuis Henk , Fossiez François , Thurau Mathias , Wittmann Sabine , Kuhn Eva-Maria , Lebecque Serge , Banchereau Jacques , Rosenwirth Brigitte , Fleckenstein Bernhard , Heeney Jonathan , Fickenscher Helmut
Published: December 2019

In comparison to wild-type herpesvirus saimiri, viral interleukin-17 gene knockout mutants have unaltered behavior regarding viral replication, T-cell transformation in vitro, and pathogenicity in cottontop tamarins. Thus, this gene is not required for T-cell lymphoma induction but may contribute to apathogenic viral persistence in the natural host, the squirrel monkey.
HveA (Herpesvirus Entry Mediator A), a Coreceptor for Herpes Simplex Virus Entry, also Participates in Virus-Induced Cell Fusion
Authors: Terry-Allison Tracy , Montgomery Rebecca I. , Whitbeck J. Charles , Xu Ruliang , Cohen Gary H. , Eisenberg Roselyn J. , Spear Patricia G.
Published: December 2019

The purpose of this study was to determine whether a cell surface protein that can serve as coreceptor for herpes simplex virus type 1 (HSV-1) entry, herpesvirus entry mediator (previously designated HVEM but renamed HveA), also mediates HSV-1-induced cell-cell fusion. We found that transfection of DNA from KOS-804, a previously described HSV-1 syncytial (Syn) strain whose Syn mutation was mapped to an amino acid substitution in gK, induced numerous large syncytia on HveA-expressing Chinese hamster ovary cells (CHO-HVEM12) but not on control cells (CHO-C8). Antibodies specific for gD as well as for HveA were effective inhibitors of KOS-804-induced fusion, consistent with previously described direct interactions between gD and HveA. Since mutations in gD determine the ability of HSV-1 to utilize HveA for entry, we examined whether the form of virally expressed gD also influenced the ability of HveA to mediate fusion. We produced a recombinant virus carrying the KOS-804 Syn mutation and the KOS-Rid1 gD mutation, which significantly reduces viral entry via HveA, and designated it KOS-SR1. KOS-SR1 DNA had a markedly reduced ability to induce syncytia on CHO-HVEM12 cells and a somewhat enhanced ability to induce syncytia on CHO-C8 cells. These results support previous findings concerning the relative abilities of KOS and KOS-Rid1 to infect CHO-HVEM12 and CHO-C8 cells. Thus, HveA mediates cell-cell fusion as well as viral entry and both activities of HveA are contingent upon the form of gD expressed by the virus.
Substitutions in a Major Histocompatibility Complex Class II-Restricted Human Immunodeficiency Virus Type 1 gp120 Epitope Can Affect CD4+T-Helper-Cell Function
Authors: Lekutis Christine , Letvin Norman L.
Published: December 2019

It has been suggested that the inability of the immune response to control human immunodeficiency virus type 1 (HIV-1) replication may be due, at least in part, to the capacity of this virus to escape from immune recognition through mutation. While there is increasing evidence for the importance of HIV-1-specific CD4+T cells in containing HIV-1 spread in the infected individual, little is known about the consequences of HIV-1 mutation on virus-specific CD4+T-cell function. The impact of HIV-1 sequence variation on CD4+T-helper (Th)- cell function was assessed with a rhesus monkey model for immune recognition of the HIV-1 envelope (Env) glycoprotein. A series of HIV-1 Env(484-496) variant peptides were shown to retain the ability to bind to the appropriate rhesus monkey major histocompatibility complex class II DR molecule. Peptides bearing substitutions at position 490, however, failed to drive the proliferation or cytokine secretion of two well-characterized HXBc2 Env-specific rhesus monkey CD4+Th-cell lines. Exogenous costimulation was ineffective in complementing the ability of the nonstimulatory peptides to induce [3H]thymidine incorporation by these cells. Finally, HIV-1 Env(484-496) variant peptides with substitutions at position 490 antagonized the HXBc2 Env peptide-induced proliferative response of the CD4+Th-cell lines. Thus, HIV-1 variants appear to have the capacity to neutralize the function of virus-specific CD4+T lymphocytes.
Characterization of Drug Resistance-Associated Mutations in the Human Cytomegalovirus DNA Polymerase Gene by Using Recombinant Mutant Viruses Generated from Overlapping DNA Fragments
Authors: Cihlar Tomas , Fuller Michael D. , Cherrington Julie M.
Published: December 2019

A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and δ-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.
Retroviral Diversity and Distribution in Vertebrates
Authors: Herniou Elisabeth , Martin Joanne , Miller Karen , Cook James , Wilkinson Mark , Tristem Michael
Published: December 2019

We used the PCR to screen for the presence of endogenous retroviruses within the genomes of 18 vertebrate orders across eight classes, concentrating on reptilian, amphibian, and piscine hosts. Thirty novel retroviral sequences were isolated and characterized by sequencing approximately 1 kb of their encoded protease and reverse transcriptase genes. Isolation of novel viruses from so many disparate hosts suggests that retroviruses are likely to be ubiquitous within all but the most basal vertebrate classes and, furthermore, gives a good indication of the overall retroviral diversity within vertebrates. Phylogenetic analysis demonstrated that viruses clustering with (but not necessarily closely related to) the spumaviruses and murine leukemia viruses are widespread and abundant in vertebrate genomes. In contrast, we were unable to identify any viruses from hosts outside of mammals and birds which grouped with the other five currently recognized retroviral genera: the lentiviruses, human T-cell leukemia-related viruses, avian leukemia virus-related retroviruses, type D retroviruses, and mammalian type B retroviruses. There was also some indication that viruses isolated from individual vertebrate classes tended to cluster together in phylogenetic reconstructions. This implies that the horizontal transmission of at least some retroviruses, between some vertebrate classes, occurs relatively infrequently. It is likely that many of the retroviral sequences described here are distinct enough from those of previously characterized viruses to represent novel retroviral genera.
Encapsidation of the Flavivirus Kunjin Replicon RNA by Using a Complementation System Providing Kunjin Virus Structural Proteins in
trans
Authors: Khromykh Alexander A. , Varnavski Andrei N. , Westaway Edwin G.
Published: December 2019

Kunjin virus (KUN) replicon RNA was encapsidated by a procedure involving two consecutive electroporations of BHK-21 cells, first with KUN replicon RNA C20DXrep (with prME and most of C deleted) and about 24 h later with a recombinant Semliki Forest virus (SFV) replicon RNA(s) expressing KUN structural proteins. The presence of KUN replicon RNA in encapsidated particles was demonstrated by its amplification and expression in newly infected BHK-21 cells, detected by Northern blotting with a KUN-specific probe and by immunofluorescence analysis with anti-NS3 antibodies. No infectious particles were produced when C20DXrep RNA and recombinant SFV RNAs were electroporated simultaneously. When the second electroporation was performed with a single SFV replicon RNA expressing the KUN contiguous prME genes and the KUN C gene together but under control of two separate 26S subgenomic promoters (SFV-prME-C107), a 10-fold-higher titer of infectious particles was achieved than when two different SFV replicon RNAs expressing the KUN C gene (SFV-C107) and prME genes (SFV-prME) separately were used. No SFV replicon RNAs expressing KUN structural proteins were encapsidated in secreted particles. Infectious particles pelleted by ultracentrifugation of the culture fluid from cells sequentially transfected with C20DXrep and SFV-prME-C107 RNAs were neutralized by preincubation with monoclonal antibodies to KUN E protein. Radioimmunoprecipitation analysis with anti-E antibodies of the culture fluid of the doubly transfected cells showed the presence of C, prM/M, and E proteins in the immunoprecipitated particles. Reverse transcription-PCR analysis showed that the immunoprecipitated particles also contained KUN-specific RNA. The encapsidated replicon particles sedimented more slowly than KUN virions in a 5 to 25% sucrose density gradient and were uniformly spherical, with an ∼35-nm diameter, compared with ∼50 nm for KUN virions. The results of this study demonstrate for the first time packaging of flavivirus RNA in
trans
, and they exclude a role in packaging for virtually all of the structural region. Possible applications of the developed packaging system include the definition of the packaging signal(s) in flavivirus RNA as well as the amino acid motif(s) in the structural proteins involved in RNA encapsidation, virion assembly, and secretion. Furthermore, it could facilitate the development of a noninfectious vaccine delivery system based on encapsidation of a noncytopathic flavivirus replicon expressing heterologous genes.
The Various Sendai Virus C Proteins Are Not Functionally Equivalent and Exert both Positive and Negative Effects on Viral RNA Accumulation during the Course of Infection
Authors: Latorre Patrizia , Cadd Tamarra , Itoh Masae , Curran Joseph , Kolakofsky Daniel
Published: December 2019

Recombinant Sendai viruses were prepared which cannot express their Cprime, C, or Cprimeplus C proteins due to mutation of their respective start codons ([Cprime-minus], [C-minus] and [double mutant], respectively). The [Cprime-minus] and [C-minus] stocks were similar to that of wild-type (wt) virus in virus titer and plaque formation, whereas the double-mutant stock had a much-reduced PFU or 50% egg infective dose/particle ratio and produced very small plaques. Relative to the wt virus infection, the [Cprime-minus] and [C-minus] infections of BHK cells resulted in significantly greater accumulation of viral RNAs, consistent with the known inhibitory effects of the Cprimeand C proteins. The double-mutant infection, in contrast, was delayed in its accumulation of viral RNAs; however, once accumulation started, overaccumulation quickly occurred, as in the single-mutant infections. Our results suggest that the Cprimeand C proteins both provide a common positive function early in infection, so that only the double mutant undergoes delayed RNA accumulation and exhibits the highly debilitated phenotype. Later in infection, the same proteins appear to act as inhibitors of RNA accumulation. In infections of mice, [Cprime-minus] was found to be as virulent as wt virus whereas [C-minus] was highly attenuated. These results suggest that the Cprimeand C proteins cannot be functionally equivalent, since C can replace Cprimefor virulence in mice whereas Cprimecannot replace C.
Limited Entry of Adenovirus Vectors into Well-Differentiated Airway Epithelium Is Responsible for Inefficient Gene Transfer
Authors: Pickles Raymond J. , McCarty Douglas , Matsui Hirotoshi , Hart Pádraig J. , Randell Scott H. , Boucher Richard C.
Published: December 2019

Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of α
v
β
3/5
integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.
Mouse Mammary Tumor Virus Superantigen Expression in B Cells Is Regulated by a Central Enhancer within thepolGene
Authors: Reuss Frank U. , Coffin John M.
Published: December 2019

Expression of mouse mammary tumor virus (MMTV)-encoded superantigens in B lymphocytes is required for viral transmission and pathogenesis. The mechanism of superantigen expression from the viralsaggene in B cells is largely unknown, due to problems with detection and quantification of these low-abundance proteins. We have established a sensitive superantigen-luciferase reporter assay to study the expression and regulation of the MMTVsaggene in B-cell lymphomas. The regulatory elements for retroviral gene expression are generally located in the 5′ long terminal repeat (LTR) of the provirus. However, we found that neither promoters nor enhancers in the MMTV 5′ LTR play a significant role in superantigen expression in these cells. Instead, the essential regulatory regions are located in thepolandenvgenes of MMTV. We report here that maximalsagexpression in B-cell lines depends on an enhancer within the viralpolgene which can be localized to a minimal 183-bp region. Regulation ofsaggene expression differs between B-cell lymphomas and pro-B cells, where an enhancer within the viral LTRs is involved. Thus, MMTVsagexpression during B-cell development is achieved through the use of two separate enhancer elements.
A Human Endogenous Retrovirus Suppresses Translation of an Associated Fusion Transcript, PLA2L
Authors: Kowalski Paul E. , Mager Dixie L.
Published: December 2019

Human endogenous retroviruses (HERVs) are repetitive, noninfectious chromosomal elements degenerated from exogenous retroviruses. The HERV-H family is composed of approximately 1,000 elements which are dispersed throughout the human genome. We have shown previously that an HERV-H element splices into a downstream locus, termed PLA2L, which has a large open reading frame (ORF) containing two domains with phospholipase A2 homology. Over half of the putative 5′ untranslated region (5′ UTR) of the resulting fusion transcript is derived from HERV-H long-terminal-repeat and internal sequences. As 5′ UTRs are known to modulate translation initiation, we tested for possible effects upon gene expression at the translation level due to the 5′ fusion with HERV-H sequences. No PLA2L protein was detected in teratocarcinoma cell lines in which PLA2L mRNA is abundantly expressed. In addition, despite a high level of transcription, no protein synthesis was detected when the full-length PLA2L cDNA was expressed in COS cells. Upon removal of the 5′-terminal HERV-H sequences, PLA2L protein was seen in transfectants. The 5′ UTR contains both small ORFs and a strong predicted RNA secondary structure, both of which have been shown to contribute to translation suppression. The HERV-H sequences, combined with a unique PLA2L 5′ UTR sequence, form a predicted RNA stem-loop that has a stability greater than that proposed to negatively affect translation. Interestingly, this stem-loop is abolished when the HERV-H sequences are removed. We hypothesize that the PLA2L 5′ HERV-H sequences function as an abnormally long and complex 5′ UTR, resulting in suppression of translation in both teratocarcinoma cell lines and full-length cDNA transfectants. This is the first known example of a endogenous retrovirus integration affecting expression of a heterologous human gene at the translational level.
Up to 100-Fold Increase of Apparent Gene Expression in the Presence of Epstein-Barr Virus
oriP
Sequences and EBNA1: Implications of the Nuclear Import of Plasmids
Authors: Längle-Rouault Françoise , Patzel Volker , Benavente Annie , Taillez Martine , Silvestre Nathalie , Bompard Albine , Sczakiel Georg , Jacobs Eric , Rittner Karola
Published: December 2019

A 100-fold increase in luciferase activity was observed in 293 cells, stably expressing Epstein-Barr nuclear antigen 1 (EBNA1; 293-EBNA1 cells), that had been transiently transfected with plasmids carrying Epstein-Barr virus (EBV)
oriP
sequences. This increase was observed in comparison to reporter gene activity obtained after transfection with a plasmid carrying no
oriP
sequences. The luciferase gene on these plasmids was under the control of either the cytomegalovirus immediate-early 1 gene enhancer-promoter (CMV IE1) or the Rous sarcoma virus promoter. The increase of reporter gene activity was not due to plasmid replication, since a similar enhancement was observed in the presence of aphidicolin, an inhibitor of replicative DNA synthesis, or after deletion of the dyad symmetry (
DS
) element within
oriP
. Luciferase production was not increased in the presence of only the
DS
element. Microinjection of plasmids carrying the CMV IE1 promoter-driven luciferase gene with or without
oriP
sequences into the nuclei of 293-EBNA1 cells resulted in a 17-fold increase in luciferase activity. Cytoplasmic injection of these plasmids led to an enhancement of luciferase activity of up to 100-fold. This difference in the factor of activation after nuclear or cytoplasmic injection could be ascribed to increased transport of plasmids carrying
oriP
from the cytoplasm to the nucleus in the presence of EBNA1. These data suggest the possibility of substantially increasing the apparent expression of a gene under the control of a strong constitutive promoter in the presence of
oriP
sequences and EBNA1. This improvement in expression is due to intranuclear enhancement of gene expression.
oriP
-specific transport of plasmid DNA from the cytoplasm of 293-EBNA1 cells to the nucleus seems to contribute to the observed effect.
CXCR4 and CCR5 Genetic Polymorphisms in Long-Term Nonprogressive Human Immunodeficiency Virus Infection: Lack of Association with Mutations other than CCR5-Δ32
Authors: Cohen Oren J. , Paolucci Stefania , Bende Steven M. , Daucher MaryBeth , Moriuchi Hiroyuki , Moriuchi Masako , Cicala Claudia , Davey Richard T. , Baird Barbara , Fauci Anthony S.
Published: December 2019

Polymorphisms in the coding sequences of CCR5 and CXCR4 were studied in a group of human immunodeficiency virus (HIV)-infected long-term nonprogressors. Two different point mutations were found in the CXCR4 coding sequence. One of these CXCR4 mutations was silent, and each was unique to two nonprogressors. The well-described 32-bp deletion within the CCR5 coding sequence (CCR5-Δ32) was found in 4 of 13 nonprogressors, and 12 different point mutations were found scattered over the CCR5 coding sequence from 8 nonprogressors. Most of the mutations created either silent or conservative changes in the predicted amino acid sequence: only one of these mutations was found in more than a single nonprogressor. All nonsilent mutations were tested in an HIV envelope-dependent fusion assay, and all functioned comparably to wild-type controls. Polymorphisms in the CXCR4 and CCR5 coding sequences other than CCR5-Δ32 do not appear to play a dominant mechanistic role in nonprogression among HIV-infected individuals.
Coreceptor Usage of Human Immunodeficiency Virus Type 2 Primary Isolates and Biological Clones Is Broad and Does Not Correlate with Their Syncytium-Inducing Capacities
Authors: Guillon Christophe , van der Ende Marchina E. , Boers Patrick H. M. , Gruters Rob A. , Schutten Martin , Osterhaus Albert D. M. E.
Published: December 2019

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by binding of the surface envelope glycoprotein to the CD4 molecule. Interaction of the resulting CD4-glycoprotein complex with α- or β-chemokine receptors, depending on the biological phenotype of the virus, then initiates the fusion process. Here, we show that primary HIV-2 isolates and biological clones, in contrast to those of HIV-1, may use a broad range of coreceptors, including CCR-1, CCR-3, CCR-5, and CXCR-4. The syncytium-inducing capacity of these viruses did not correlate with the ability to infect via CXCR-4 or any other coreceptor. One cell-free passage of the intermediate isolates in mitogen-stimulated, CD8
+
cell-depleted peripheral blood mononuclear cells resulted in the outgrowth of variants with CCR-5 only, whereas the coreceptor usage of late and early isolates did not change. Since HIV-2 is less pathogenic in vivo than HIV-1, these data suggest that HIV pathogenicity in vivo is not directly related to the spectrum of coreceptors used in in vitro systems.