bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus
Authors: Clements J E , Montelaro R C , Zink M C , Amedee A M , Miller S , Trichel A M , Jagerski B , Hauer D , Martin L N , Bohm R P
Published: January 2020

The simian immunodeficiency virus (SIV) macaque model of AIDS has provided a valuable system with which to investigate vaccine approaches for protection against human immunodeficiency virus type 1 (HIV-1) infection. In particular, the ability of macaques persistently infected with attenuated infectious molecular clones of SIV to resist challenge with the pathogenic parental swarm has conclusively demonstrated that protective immunity can be achieved by immunization prior to exposure. The breadth of these protective responses and the immunological correlates of protection, however, have not been identified. In addition, vaccine studies have mainly employed lymphocyte-tropic strains of HIV-1 and SIV. Recent studies have implicated macrophage-tropic strains in the transmission of HIV-1 and have suggested that these virus strains should be examined in vaccine strategies. Macrophage-tropic viruses may confer additional advantages in the induction of protective immunity by replication in antigen-presenting cells. In this study, the immune response of rhesus macaques inoculated with an attenuated macrophage-tropic recombinant of SIVmac239 (SIV/17E-Cl) was evaluated with respect to protective immunity by heterologous challenge at various times after infection. Vigorous type-specific neutralizing-antibody responses restricted to SIV/17E-Cl were evident by 2 weeks postinfection. By 7 months, however, cross-reactive neutralizing antibodies emerged which neutralized not only SIV/17E-Cl but also the heterologous primary isolate SIV/DeltaB670. Challenge of SIV/17E-Cl-infected monkeys with SIV/DeltaB670 at various times postinfection demonstrated that protective responses were associated with the appearance of cross-reactive neutralizing antibodies. Furthermore, passive transfer of sera from SIV/17E-Cl-infected animals passively protected two of four naive recipients.
Preferential recognition of hepatitis B nucleocapsid antigens by Th1 or Th2 cells is epitope and major histocompatibility complex dependent
Authors: Milich D R , Peterson D L , Schödel F , Jones J E , Hughes J L
Published: January 2020

Regulatory T-helper (Th) cells have been categorized into two functional subsets, Th1 and Th2 cells, which produce distinct lymphokines. In general, Th1 cells mediate cellular immune responses and Th2 cells mediate humoral immunity. Recent serological studies suggest that the Th1-Th2 balance may be relevant in acute and chronic hepatitis B virus (HBV) infections. The purpose of this study was to determine the potential of the nucleocapsid antigens (Ags) (hepatitis B core and e Ags [HBc/eAg]) of HBV to preferentially elicit either a Th1 or a Th2 dominant response. For this purpose, H-2 congenic B10.S and B10 mice were immunized with HBc/eAg, and Ag-specific T-cell proliferative responses, T-cell helper function, and T-cell cytokine production were analyzed. The results indicated that B10.S mice preferentially develop a Th1-like response whereas B10 mice preferentially develop a Th2-like response after immunization with HBc/eAg. Furthermore, the preferential Th1 and Th2 response patterns were reproduced when 12-residue peptides representing the dominant HBc/eAg-specific T-cell sites for B10.S (peptide 120-131) and B10 (peptide 129-140) mice were used as immunogens. Therefore, the combination of the T-cell site recognized and the major histocompatibility complex restricting element can in large part determine the Th phenotype of the HBc/eAg-specific T-cell response. Other factors that influenced Th phenotype were the presence of exogenous cytokines, Ag structure, and tissue distribution.
The Rev protein of human immunodeficiency virus type 1 counteracts the effect of an AU-rich negative element in the human papillomavirus type 1 late 3' untranslated region
Authors: Tan W , Schwartz S
Published: January 2020

We have identified a sequence in the late 3' untranslated region of human papillomavirus type 1 mRNAs that acts posttranscriptionally to repress gene expression. Deletion analysis localized the inhibitory element to an AU-rich sequence between nucleotides 6958 and 6984 on the human papillomavirus type 1 genome. This sequence inhibits gene expression in an orientation-dependent manner. Upon transfection of eucaryotic cells with plasmids containing this sequence, approximately 4-fold-lower cytoplasmic mRNA levels and 64- to 128-fold-lower protein levels were produced compared with those produced by plasmids lacking the inhibitory sequence. Interestingly, providing the constitutive transport element of simian retrovirus type 1 in sense orientation counteracted inhibition exerted by the human papillomavirus type 1 sequence. Inhibition could also be overcome by the presence of human immunodeficiency virus type 1 Rev protein in trans and its target sequence, the Rev-responsive element, in cis. Rev is a nuclear protein and acts by promoting nuclear export of human immunodeficiency virus type 1 mRNAs encoding structural proteins. Our results are consistent with a model for human papillomavirus type 1 late-gene expression in which mRNAs containing human papillomavirus type 1 inhibitory sequences enter a nonproductive route in the nucleus, resulting in inefficient mRNA utilization. Rev directs mRNA containing inhibitory sequences to a productive route by interacting with the Rev-responsive element.
An avirulent ICP34.5 deletion mutant of herpes simplex virus type 1 is capable of in vivo spontaneous reactivation
Authors: Perng G C , Thompson R L , Sawtell N M , Taylor W E , Slanina S M , Ghiasi H , Kaiwar R , Nesburn A B , Wechsler S L
Published: January 2020

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 x 10(7) PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 x 10(5) PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10(6) PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.
Control of baculovirus gp64-induced syncytium formation by membrane lipid composition
Authors: Chernomordik L , Leikina E , Cho M S , Zimmerberg J
Published: January 2020

We have investigated the effects of membrane lipid composition on biological membrane fusion triggered by low pH and mediated by the baculovirus envelope glycoprotein gp64. Lysolipids, either added exogenously or produced in situ by phospholipase A2 treatment of cell membranes, reversibly inhibited syncytium formation. Lysolipids also decreased the baculovirus infection rate. In contrast, oleic and arachidonic acids and monoolein promoted cell-cell fusion. Membrane lipid composition affected pH-independent processes which followed the low-pH-induced change in fusion protein conformation. Inhibition and promotion of membrane fusion by a number of lipids could not be explained by mere binding or incorporation into membranes, but rather was correlated with the effective molecular shape of exogenous lipids. Our data are consistent with the hypothesis that membrane fusion proceeds through highly bent membrane intermediates (stalks) having a net negative curvature. Consequently, inverted cone-shaped lysolipids inhibit and cone-shaped cis-unsaturated fatty acids promote stalk formation and, ultimately, membrane fusion.
Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy
Authors: Chan S Y , Delius H , Halpern A L , Bernard H U
Published: January 2020

Our aim was to study the phylogenetic relationships of all known papillomaviruses (PVs) and the possibility of establishing a supratype taxonomic classification based on this information. Of the many detectably homologous segments present in PV genomes, a 291-bp segment of the L1 gene is notable because it is flanked by the MY09 and MY11 consensus primers and contains highly conserved amino acid residues which simplify sequence alignment. We determined the MY09-MY11 sequences of human PV type 20 (HPV-20), HPV-21, HPV-22, HPV-23, HPV-24, HPV-36, HPV-37, HPV-38, HPV-48, HPV-50, HPV-60, HPV-70, HPV-72, HPV-73, ovine (sheep) PV, bovine PV type 3 (BPV-3), BPV-5, and BPV-6 and created a database which now encompasses HPV-1 to HPV-70, HPV-72, HPV-73, seven yet untyped HPV genomes, and 15 animal PV types. Three additional animal PVs were analyzed on the basis of other sequence data. We constructed phylogenies based on partial L1 and E6 gene sequences and distinguished five major clades that we call supergroups. One of them unites 54 genital PV types, which can be further divided into eleven groups. The second supergroup has 24 types and unites most PVs that are typically found in epidermodysplasia verruciformis patients but also includes several types typical of other cutaneous lesions, like HPV-4. The third supergroup unites the six known ungulate fibropapillomaviruses, the fourth includes the cutaneous ungulate PVs BPV-3, BPV-4, and BPV-6, and the fifth includes HPV-1, HPV-41, HPV-63, the canine oral PV, and the cottontail rabbit PV. The chaffinch PV and two rodent PVs, Micromys minutus PV and Mastomys natalensis PV, are left ungrouped because of the relative isolation of each of their lineages. Within most supergroups, groups formed on the basis of cladistic principles unite phenotypically similar PV types. We discuss the basis of our classification, the concept of the PV type, speciation, PV-host evolution, and estimates of their rates of evolution.
Functional analysis of amino acid residues encompassing and surrounding two neighboring H-2Db-restricted cytotoxic T-lymphocyte epitopes in simian virus 40 tumor antigen
Authors: Lippolis J D , Mylin L M , Simmons D T , Tevethia S S
Published: January 2020

Simian virus 40 tumor (T) antigen contains three H-2Db-and one H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes (sites). Two of the H-2Db-restricted CTL epitopes, I and II/III, are separated by 7 amino acids in the amino-terminal one third of T antigen. In this study, we determine if the amino acids separating these two H-2Db-restricted CTL epitopes are dispensable for efficient processing and presentation. In addition, the importance of amino acid residues lying within and flanking the H-2Db-restricted epitopes I and II/III for efficient processing, presentation, and recognition by site-specific CTL clones was determined by using T-antigen mutants containing single-amino-acid substitutions between residues 200 and 239. Using synthetic peptides in CTL lysis and major histocompatibility complex class I stabilization assays, CTL recognition site I has been redefined to include residues 206 to 215. Substitutions in amino acids flanking either site I or site II/III did not affect recognition by any of the T-antigen-specific CTL clones. Additionally, the removal of the 7 residues separating site I and site II/III did not affect CTL recognition, thus demonstrating that these two epitopes when arranged in tandem in the native T antigen can be efficiently processed and presented to CTL clones. Differences in fine specificities of two CTL clones which recognize the same epitope (Y-1 and K-11 for site I and Y-2 and Y-3 for site II/III) have been used in conjunction with synthetic peptide variants to assign roles for residues within epitopes I and II/III with respect to TCR recognition and/or peptide-major histocompatibility complex association.
5' termini of Pichinde arenavirus S RNAs and mRNAs contain nontemplated nucleotides
Authors: Polyak S J , Zheng S , Harnish D G
Published: January 2020

Primer extension of Pichinde arenavirus purified virion RNA suggests that genomes have at least a single nontemplated base at the 5' end which is a G in all cDNA clones having one such single base. On the other hand, the predominant products of primer extension on total virus-infected-cell RNA are at positions -1 and -2. The primer extension product at position -2 is not represented in virion RNA, and neither of these products is proportionally represented in mRNA. mRNA is predominantly 3 or 4 bases longer than genomes and antigenomes, but primer extension products as long as 7 bases were observed. The sequence of nontemplated bases reported here is unambiguous with respect to the 5'-terminal base and supports the view that there is a sequence preference for a G at the 5' termini of mRNAs. Assessment of our sequence data in the context of the sequences of Tacaribe and lymphocytic choriomeningitis viruses suggests that the mechanism of initiation of arenavirus transcription is fundamentally different from that of members of the families Orthomyxoviridae and Bunyaviridae.
Echovirus 1 interaction with the isolated VLA-2 I domain
Authors: King S L , Cunningham J A , Finberg R W , Bergelson J M
Published: January 2020

The isolated I domain of the integrin VLA-2, produced as a bacterial fusion protein, specifically bound echovirus 1 and prevented virus attachment to cells. These results demonstrate that the receptor structures critical for virus attachment are contained solely within the VLA-2 I domain and that soluble receptor fragments are capable of preventing infection.
The spacer peptide between human immunodeficiency virus capsid and nucleocapsid proteins is essential for ordered assembly and viral infectivity
Authors: Kräusslich H G , Fäcke M , Heuser A M , Konvalinka J , Zentgraf H
Published: January 2020

Morphogenesis of retroviruses involves ordered assembly of the structural Gag- and Gag-Pol polyproteins, with subsequent budding from the plasma membrane and proteolytic cleavage by the viral proteinase (PR). Two cleavage sites exist between the capsid (CA) and nucleocapsid (NC) domains of the human immunodeficiency virus (HIV) type 1 Gag polyprotein which are separated by a 14-amino-acid spacer peptide of unknown function. To analyze the role of the two cleavage sites and the spacer peptide, both sites were individually mutated and a deletion mutation that precisely removes the spacer peptide was constructed. Following transfection of proviral DNA carrying the point mutations, mutant polyproteins were synthesized and assembled like wild-type polyprotein, and release of particles was not significantly altered. Both mutations abolished cleavage at the respective site and reduced or abolished viral infectivity. Deletion of the spacer peptide severely affected ordered assembly and reduced particle release. The extracellular particles that were released exhibited normal density but were heterogeneous in size. Electron micrographs revealed large electron-dense plaques underneath the plasma membrane of transfected cells which appeared like confluent ribonucleoprotein complexes arrested early in the budding process. Extracellular particles exhibited very aberrant and heterogeneous morphology and were incapable of inducing viral spread. These particles may correspond to membrane vesicles sequestered by the rigid structures underneath the cell membrane and not released by a regular budding process.
Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs
Authors: Ou S H , Wu F , Harrich D , García-Martínez L F , Gaynor R B
Published: January 2020

Human immunodeficiency virus type 1 (HIV-1) gene expression is modulated by both viral and cellular factors. A regulatory element in the HIV-1 long terminal repeat known as TAR, which extends from nucleotides -18 to +80, is critical for the activation of gene expression by the transactivator protein, Tat. RNA transcribed from TAR forms a stable stem-loop structure which serves as the binding site for both Tat and cellular factors. Although TAR RNA is critical for Tat activation, the role that TAR DNA plays in regulating HIV-1 gene expression is not clear. Several studies have demonstrated that TAR DNA can bind cellular proteins, such as UBP-1/LBP-1, which repress HIV-1 gene expression and other factors which are involved in the generation of short, nonprocessive transcripts. In an attempt to characterize additional cellular factors that bind to TAR DNA, a lambda gt11 expression cloning strategy involving the use of a portion of TAR DNA extending from -18 to +28 to probe a HeLa cDNA library was used. We identified a cDNA, designated TAR DNA-binding protein (TDP-43), which encodes a cellular factor of 43 kDa that binds specifically to pyrimidine-rich motifs in TAR. Antibody to TDP-43 was used in gel retardation assays to demonstrate that endogenous TDP-43, present in HeLa nuclear extract, also bound to TAR DNA. Although TDP-43 bound strongly to double-stranded TAR DNA via its ribonucleoprotein protein-binding motifs, it did not bind to TAR RNA extending from +1 to +80. To determine the function of TDP-43 in regulating HIV-1 gene expression, in vitro transcription analysis was performed. TDP-43 repressed in vitro transcription from the HIV-1 long terminal repeat in both the presence and absence of Tat, but it did not repress transcription from other promoters such as the adenovirus major late promoter. In addition, transfection of a vector which expressed TDP-43 resulted in the repression of gene expression from an HIV-1 provirus. These results indicate that TDP-43 is capable of modulating both in vitro and in vivo HIV-1 gene expression by either altering or blocking the assembly of transcription complexes that are capable of responding to Tat.
RNA-protein interactions directed by the 3' end of human rhinovirus genomic RNA
Authors: Todd S , Nguyen J H , Semler B L
Published: January 2020

The replication of a picornavirus genomic RNA is a template-specific process involving the recognition of viral RNAs as target replication templates for the membrane-bound viral replication initiation complex. The virus-encoded RNA-dependent RNA polymerase, 3Dpol, is a major component of the replication complex; however, when supplied with a primed template, 3Dpol is capable of copying polyadenylated RNAs which are not of viral origin. Therefore, there must be some other molecular mechanism to direct the specific assembly of the replication initiation complex at the 3' end of viral genomic RNAs, presumably involving cis-acting binding determinants within the 3' noncoding region (3' NCR). This report describes the use of an in vitro UV cross-linking assay to identify proteins which interact with the 3' NCR of human rhinovirus 14 RNA. A cellular protein(s) was identified in cytoplasmic extracts from human rhinovirus 14-infected cells which had a marked binding preference for RNAs containing the rhinovirus 3' NCR sequence. This protein(s) showed reduced cross-linking efficiency for a 3' NCR with an engineered deletion. Virus recovered from RNA transfections with in vitro transcribed RNA containing the same 3' NCR deletion demonstrated a defective replication phenotype in vivo. Cross-linking experiments with RNAs containing the poliovirus 3' NCR and cytoplasmic extracts from poliovirus-infected cells produced an RNA-protein complex with indistinguishable electrophoretic properties, suggesting that the appearance of the cellular protein(s) may be a common phenomenon of picornavirus infection. We suggest that the observed cellular protein(s) is sequestered or modified as a result of rhinovirus or poliovirus infection and is utilized in viral RNA replication, perhaps by binding to the 3' NCR as a prerequisite for replication complex assembly at the 3' end of the viral genomic RNA.
Tissue- and transformation-specific phosphotyrosyl proteins in v-erbB-transformed cells
Authors: McManus M J , Connolly D C , Maihle N J
Published: January 2020

To understand the mechanism of tissue-specific and transformation-specific signaling by the v-ErbB oncoprotein, we have investigated signaling pathways downstream of this transmembrane tyrosine kinase. In this report, we describe tissue-specific patterns of phosphotyrosyl proteins in three distinct cell types transformed by the v-erbB oncogene: fibroblasts, erythroblasts, and endothelial cells. In addition, we describe transformation-specific tyrosine phosphorylation events and signal complex formation in v-erbB-transformed fibroblasts. Two patterns of phosphotyrosyl proteins have been detected in v-erbB-transformed cells. The first is a fibroblast-specific pattern which includes unique phosphotyrosyl proteins of 170 kDa (c-ErbB1), 158 kDa, and 120 kDa (the catenin-like protein p120cas). The second is an erythroblast/endothelial cell-specific pattern which includes a prominent unidentified phosphotyrosyl protein of 120 kDa. Evaluation of the phosphotyrosyl proteins p120cas and SHC in chicken embryo fibroblasts infected with transforming and nontransforming v-erbB mutants reveals transformation-specific patterns of tyrosine phosphorylation. One corollary of these phosphorylation events in v-erbB-transformed fibroblasts is the formation of a complex involving SHC, growth factor receptor-bound protein 2, and a novel 75-kDa phosphotyrosyl protein. The results of these studies suggest that the v-ErbB oncoprotein can couple to multiple signal transduction pathways, that these pathways are tissue specific, and that v-erbB-mediated transformation involves specific tyrosine phosphorylation events.
Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region
Authors: Kerry J A , Sehgal A , Barlow S W , Cavanaugh V J , Fish K , Nelson J A , Stenberg R M
Published: January 2020

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.
Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies
Authors: Zhang H , Duan L X , Dornadula G , Pomerantz R J
Published: January 2020

Reverse transcription of retroviral genomic RNA in a target cell is influenced by cellular factors, including the concentration of deoxyribonucleoside triphosphates (dNTPs). In addition, recent data have demonstrated that reverse transcription can be driven within human immunodeficiency virus type 1 virions, prior to infection of a cell, by increasing extracellular concentrations of dNTPs. In attempts to increase the transduction efficiency of recombinant murine leukemia virus vectors, endogenous reverse transcription was initiated within cell-free, recombinant murine leukemia virus virions in the presence of relatively high concentrations of dNTPs. As a result, the expression of transduced genes via these retroviral vectors was increased approximately 10-fold by treatment of virions with dNTPs. Combined with our previous data, these observations suggest that virion-associated DNA synthesis can occur in diverse groups of retroviruses and positively alter retroviral infectivity. As such, these manipulations may be useful for increasing the efficiency of retrovirus-mediated gene delivery.
Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef
Authors: Goldsmith M A , Warmerdam M T , Atchison R E , Miller M D , Greene W C
Published: January 2020

Recent evidence indicates that the nef gene of human immunodeficiency virus type 1 augments rather than inhibits viral replication in both cell culture and in vivo models. In addition, nef alters various normal cellular processes, including the display of CD4 on the cell surface. However, it remains unknown whether the enhancement of infectivity and the downregulation of CD4 represent linked or independent biologic properties of this single protein. In the present studies, mutational analyses were performed to define structure-function relationships within the Nef protein that mediate these effects. To assess the functional consequences of these mutations, sensitive and reliable assays were developed to quantitate the viral infectivity enhancement and CD4 downregulation functions of Nef. The results indicate that membrane-targeting sequences at the N terminus of Nef are important for both functions of Nef, while certain other conserved regions are dispensable for both functions. A conserved proline-X-X repeat segment in the central core of the protein, which is reminiscent of an SH3-binding domain, is critical for the enhancement of infectivity function but is dispensable for CD4 downregulation. However, the downregulation of CD4 by Nef appears to involve a two-step process requiring the initial dissociation of p56lck from CD4 to permit engagement of the endocytic apparatus by CD4. Together, these findings demonstrate that the infectivity enhancement and CD4 downregulation activities of human immunodeficiency virus type 1 Nef can be dissociated. Thus, these processes may be independent of one another in the viral replication cycle.
Viral factors determine progression to AIDS in simian immunodeficiency virus-infected newborn rhesus macaques
Authors: Marthas M L , van Rompay K K , Otsyula M , Miller C J , Canfield D R , Pedersen N C , McChesney M B
Published: January 2020

To evaluate how viral variants may affect disease progression in human pediatric AIDS, we studied the potential of three simian immunodeficiency virus (SIV) isolates to induce simian AIDS in newborn rhesus macaques. The three virus isolates were previously shown to range from pathogenic (SIVmac251 and SIVmac239) to nonpathogenic (SIVmac1A11) when inoculated intravenously into juvenile and adult rhesus macaques. Six newborn macaques inoculated with pathogenic, uncloned SIVmac251 developed persistent, high levels of cell-associated and cell-free viremia, had no detectable antiviral antibodies, and had poor weight gain; these animals all exhibited severe clinical disease and pathologic lesions diagnostic for simian AIDS and were euthanatized 10 to 26 weeks after inoculation. Two newborns inoculated with pathogenic, molecularly cloned SIVmac239 developed persistent high virus load in peripheral blood, but both animals had normal weight gain and developed antiviral antibodies. One of the SIVmac239-infected neonates exhibited pathologic lesions diagnostic for SAIDS and was euthanatized at 34 weeks after inoculation; the other SIVmac239-infected neonate remained alive and exhibited no significant clinical disease for more than 1 year after inoculation. In contrast, three newborn rhesus macaques inoculated with the nonpathogenic molecular clone, SIVmac1A11, had transient, low-level viremia, seroconverted by 10 weeks after inoculation, had normal weight gain, and remained healthy for over 1 year. These results indicate that (i) newborn rhesus macaques infected with an uncloned, virulent SIVmac isolate have a more rapid, fulminant disease course than do adults inoculated with the same virus, (ii) the most rapid disease progression is associated with lack of a detectable humoral immune response in SIV-infected infant macaques, (iii) a molecularly cloned, attenuated SIV isolate is nonpathogenic in neonatal macaques, and (iv) SIV-infected neonatal macaques exhibit patterns of infection, virus load, and disease progression similar to those observed in human immunodeficiency virus-infected children. This SIV/neonatal rhesus model of pediatric AIDS provides a rapid, sensitive model with which to compare the virulence of SIV isolates and to study the mechanisms underlying the differences in disease progression in human immunodeficiency virus-infected infants.
Synergistic inhibition of human immunodeficiency virus type 1 envelope glycoprotein-mediated cell fusion and infection by an antibody to CD4 domain 2 in combination with anti-gp120 antibodies
Authors: Burkly L , Mulrey N , Blumenthal R , Dimitrov D S
Published: January 2020

Antibodies to several epitopes of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41) can synergize in inhibiting HIV-1 infection. In the present study we tested the ability of a monoclonal antibody (MAb), 5A8, which interacts with CD4 domain 2, and other CD4-specific MAbs to synergize with antibodies against gp120. We have previously found that 5A8 inhibits HIV-1 entry without interfering with gp120 binding to CD4, presumably by affecting a postbinding membrane fusion event. Because antibodies to the gp120 V3 loop also affect post-CD4-gp120-binding events, 5A8 was first tested in combination with anti-V3 loop antibodies for possible synergy. The anti-V3 loop antibodies 0.5 beta, NEA-9205, and 110.5 acted synergistically with 5A8 in inhibiting syncytium formation between gp120-gp41- and CD4-expressing cells. A human MAb to an epitope of gp120 involved in CD4 binding, IAM 120-1B1, and another anti-CD4 binding site antibody, PC39.13, also exerted synergistic effects in combination with 5A8. Similarly, an antibody against the gp120 binding site on CD4, 6H10, acted synergistically with an anti-V3 loop antibody, NEA-9205. However, a control anti-CD4 antibody, OKT4, which does not significantly inhibit syncytium formation alone, produced only an additive effect when combined with NEA-9205. Serum from HIV-1-infected individuals, which presumably contains antibodies to the V3 loop and the CD4 binding site, exhibited a strong synergistic effect with 5A8 in inhibiting infection by a patient HIV-1 isolate (0104B) and in blocking syncytium formation. These results indicate that therapeutics based on antibodies affecting both non-gp120 binding and gp120 binding epitopes of the target receptor molecule, CD4, could be efficient in patients who already contain anti-gp120 antibodies and could also be used to enhance passive immunization against HIV-1 in combination with anti-gp120 antibodies.
Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients
Authors: Scarselli E , Cerino A , Esposito G , Silini E , Mondelli M U , Traboni C
Published: January 2020

The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme-linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl-terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed.
Isolation of a fully infectious variant of parvovirus H-1 supplanting the standard strain in human cells
Authors: Faisst S , Faisst S R , Dupressoir T , Plaza S , Pujol A , Jauniaux J C , Rhode S L , Rommelaere J
Published: January 2020

A variant H-1 virus, designated H-1 dr virus, was isolated from stock of the standard H-1 virus strain propagated in the newborn human kidney cell line NB-E. Molecular cloning and sequence analysis revealed an in-frame deletion at map positions 39 to 41. This deletion affects the open reading frames encoding the nonstructural proteins NS-1 and NS-2 and the untranslated leader sequence of the R3 transcripts encoding the capsid proteins. In addition, H-1 dr virus harbors a 58-nucleotide duplication inboard from the right-hand terminal palindrome. Internal deletions and terminal reiterations are hallmarks of H-1 virus type I variants that typically are defective interfering particles. Indeed, H-1 dr virus was found to progressively supplant the standard strain in serially coinfected NB-E cell cultures. However, H-1 dr virus differed from previously described type I variants in its full infectivity, as was apparent from its ability to give yields of replication and progeny virus production that were similar to those of the standard virus strain in NB-E cells. Hence, the interference of H-1 dr virus in the propagation of standard H-1 virus in coinfected cells was not accompanied by a drop in the titer of infectious virus. Moreover, H-1 dr virus proved to induce the same pathogenic effects in newborn hamsters as the standard virus strain did.
Monocyte-derived cultured dendritic cells are susceptible to human immunodeficiency virus infection and transmit virus to resting T cells in the process of nominal antigen presentation
Authors: Tsunetsugu-Yokota Y , Akagawa K , Kimoto H , Suzuki K , Iwasaki M , Yasuda S , Häusser G , Hultgren C , Meyerhans A , Takemori T
Published: January 2020

The susceptibility of monocyte-derived cultured dendritic cells (DCs) to human immunodeficiency virus (HIV) infection and their role in viral transmission in the immune response were studied in detail. We observed that highly purified cultured DCs were infected with the T-tropic Lai strain of HIV type 1 (HIV-1Lai) via the CD4 receptor, and this was followed by formation of the complete provirus as detected by PCR. HIV mRNAs were transcribed at only low levels, and virus production was undectable; however, the addition of the purified protein derivative antigen of tuberculin and of autologous resting T cells to HIV-1Lai-infected DCs but not to HIV-1Lai-infected macrophages led to massive HIV transmission and production. These data suggest that the interaction of infected DCs with T cells during the normal immune response could play an important role in the activation and expansion of HIV.
Aberrant Gag protein composition of a human immunodeficiency virus type 1 vif mutant produced in primary lymphocytes
Authors: Simm M , Shahabuddin M , Chao W , Allan J S , Volsky D J
Published: January 2020

Productive, spreading infection of peripheral blood lymphocytes (PBL) with human immunodeficiency virus type 1 (HIV-1) requires the viral protein Vif. To study the requirement for vif in this system, we infected PBL with a phenotypically complemented HIV-1 clone mutated in vif. Progeny virus was produced which was noninfectious in PBL but replicated in SupT1 cells. Analysis of metabolically labeled proteins of sedimentable extracellular particles made in PBL by radioimmunoprecipitation with either serum from a patient with AIDS or a monoclonal antibody reactive with HIV-1 Gag proteins revealed that vif-negative but not wild-type particles carry higher levels of p55, p41, and p38 Gag-specific proteins compared with those of p24. Similar results were obtained with sucrose-purified virions. Our data indicate that vif plays a role in Gag protein processing or in incorporation of processed Gag products into mature virions. The presence of unprocessed precursor Gag polyprotein (Pr55gag) and other Gag processing intermediates in PBL-derived vif-negative extracellular particles may contribute to the reduced infectivity of this virus.
Arginine-rich regions succeeding the nuclear localization region of the herpes simplex virus type 1 regulatory protein ICP27 are required for efficient nuclear localization and late gene expression
Authors: Hibbard M K , Sandri-Goldin R M
Published: January 2020

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.
Vaccination with recombinant vaccinia viruses expressing ICP27 induces protective immunity against herpes simplex virus through CD4+ Th1+ T cells
Authors: Manickan E , Francotte M , Kuklin N , Dewerchin M , Molitor C , Gheysen D , Slaoui M , Rouse B T
Published: January 2020

This study was designed to evaluate the efficacy and mechanisms of protection mediated by recombinant vaccinia viruses encoding immediate-early (IE) proteins of herpes simplex virus type 2 (HSV-2). Three mouse strains were immunized against the IE proteins ICP27, ICP0, and ICP4, and mice were challenged intracutaneously in the zosteriform model with HSV-2 strain MS. Protection was observed only following immunization with the ICP27 construct and then only in the BALB/c mouse strain. Protection in BALB/c mice was ablated by CD4+ T-cell suppression but remained intact in animals depleted of CD8+ T cells. Moreover, protection could be afforded to SCID nude recipients with CD4+ but not CD8+ T cells from ICP27-immunized mice. Only BALB/c mice developed a delayed-type hypersensitivity reaction to HSV-2, and in vitro measurements of humoral and cell-mediated immunity revealed response patterns to ICP27 and HSV that differed between protected BALB/c and unprotected mouse strains. Accordingly, BALB/c responses showed antigen-induced cytokine profiles dominated by type 1 cytokines, whereas C57BL/6 and C3H/HeN mice generated cytokine responses mainly of the type 2 variety. Our results may indicate that protection against zosterification is mainly mediated by CD4+ T cells that express a type 1 cytokine profile and that protective vaccines against HSV which effectively induce such T-cell responses should be chosen.
Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4
Authors: Bachmann M F , Schorle H , Kühn R , Müller W , Hengartner H , Zinkernagel R M , Horak I
Published: January 2020

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.
Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase
Authors: Cooper J , Conner J , Clements J B
Published: January 2020

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.
Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis
Authors: Aiken C , Trono D
Published: January 2020

The Nef protein of human immunodeficiency virus type 1 (HIV-1) stimulates viral infectivity. The mechanism of this phenotype was investigated. Viruses containing disrupted nef genes were 4 to 40 times less infectious than wild-type HIV-1 in a single-round infection. The Nef-mediated stimulation HIV-1 infectivity was dependent on the association of Nef with the plasma membrane and could be observed when Nef was provided in trans in the virus producer but not target cells. The impaired infectiousness of nef-defective (delta Nef) virions was observed whether or not CD4 was present in either of these cells. Furthermore, it was independent of the mode of viral entry, since it was not rescued by pseudotyping Env- HIV-1 virions with the amphotropic murine leukemia virus envelope glycoproteins. As predicted from this result, wild-type and delta Nef virions entered cells with equal efficiencies. However, despite their normal content in viral genomic RNA and reverse transcriptase activity, delta Nef viruses were limited in their ability to perform reverse transcription once internalized in several cell types, including peripheral blood lymphocytes. Since Nef does not appear to be abundant in virions, these results suggest that Nef acts in producer cells to allow the generation of particles fully competent for completing steps that follow entry, leading to efficient reverse transcription of the HIV-1 genome. Using a trans complementation assay, we found that Nef proteins from a number of primary HIV-1 isolates as well as, to a milder degree, those from HIV-2ST and SIVMAC239 could enhance the infectivity of delta Nef HIV-1. This indicates that the Nef-mediated stimulation of proviral DNA synthesis is highly conserved and likely plays an important role in vivo.