bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutralizing Antibodies in Sera from Macaques Infected with Chimeric Simian-Human Immunodeficiency Virus Containing the Envelope Glycoproteins of either a Laboratory-Adapted Variant or a Primary Isolate of Human Immunodeficiency Virus Type 1
Authors: Montefiori David C. , Reimann Keith A. , Wyand Michael S. , Manson Kelledy , Lewis Mark G. , Collman Ronald G. , Sodroski Joseph G. , Bolognesi Dani P. , Letvin Norman L.
Published: December 2019

The magnitude and breadth of neutralizing antibodies raised in response to infection with chimeric simian-human immunodeficiency virus (SHIV) in rhesus macaques were evaluated. Infection with either SHIV-HXB2, SHIV-89.6, or SHIV-89.6PD raised high-titer neutralizing antibodies to the homologous SHIV (SHIV-89.6P in the case of SHIV-89.6PD-infected animals) and significant titers of neutralizing antibodies to human immunodeficiency virus type 1 (HIV-1) strains MN and SF-2. With few exceptions, however, titers of neutralizing antibodies to heterologous SHIV were low or undetectable. The antibodies occasionally neutralized heterologous primary isolates of HIV-1; these antibodies required >40 weeks of infection to reach detectable levels. Notable was the potent neutralization of the HIV-1 89.6 primary isolate by serum samples from SHIV-89.6-infected macaques. These results demonstrate that SHIV-HXB2, SHIV-89.6, and SHIV-89.6P possess highly divergent, strain-specific neutralization epitopes. The results also provide insights into the requirements for raising neutralizing antibodies to primary isolates of HIV-1.
Adenovirus Internalization and Infection Require Dynamin
Authors: Wang Kena , Huang Shuang , Kapoor-Munshi Archana , Nemerow Glen
Published: December 2019

The cell receptors that facilitate adenovirus internalization into cells have been identified; however, the infectious pathway of virus entry has not been established. Adenovirus entry and infection were examined in HeLa cells lacking or overexpressing mutant dynamin, a protein that specifically regulates clathrin-mediated endocytosis. Expression of mutant dynamin significantly reduced adenovirus internalization and gene delivery, indicating a functional requirement for this molecule. These findings are consistent with virus entry via the clathrin-coated pit pathway.
The CAR1 Gene Encoding a Cellular Receptor Specific for Subgroup B and D Avian Leukosis Viruses Maps to the Chicken
tvb
Locus
Authors: Smith Eugene J. , Brojatsch Jürgen , Naughton John , Young John A. T.
Published: December 2019

Host susceptibility to subgroup B, D, and E avian leukosis viruses (ALV) is determined by specific alleles of the chicken
tvb
locus. Recently, a chicken gene that encodes a cellular receptor, designated CAR1, specific for subgroups B and D ALV was cloned, and it was proposed that this gene was the
s3
allele of
tvb
(J. Brojatsch, J. Naughton, M. M. Rolls, K. Zingler, and J. A. T. Young, Cell 87:845–855, 1996). We now report that in a backcross derived from an F
1
(Jungle Fowl × White Leghorn [WL]) male mated with inbred WL females, the cloned ALV receptor gene cosegregated with two markers linked to
tvb
. The two markers used were a

tvb
s1

-specific antigen recognized by the chicken R2 alloantiserum and restriction fragment length polymorphisms associated with the expressed sequence tag com152e. With all three markers, no crossovers were observed among 52 backcross progeny tested and LOD linkage scores of 15.7 were obtained. These data demonstrate that
CAR1
is the subgroup B and D ALV susceptibility gene located at

tvb
s3
.
Origin and Rapid Evolution of a Novel Murine Erythroleukemia Virus of the Spleen Focus-Forming Virus Family
Authors: Hoatlin Maureen E. , Gomez-Lucia Esperanza , Lilly Frank , Beckstead Jay H. , Kabat David
Published: December 2019

The Friend spleen focus-forming virus (SFFV)
env
gene encodes a glycoprotein with apparent
M
r
of 55,000 that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. A retroviral vector that does not encode any Env glycoprotein was packaged into retroviral particles and was coinjected into mice in the presence of a nonpathogenic helper virus. Although most mice remained healthy, one mouse developed splenomegaly and polycythemia at 67 days; the virus from this mouse reproducibly caused the same symptoms in secondary recipients by 2 to 3 weeks postinfection. This disease, which was characterized by extramedullary erythropoietin-independent erythropoiesis in the spleens and livers, was also reproduced in long-term bone marrow cultures. Viruses from the diseased primary mouse and from secondary recipients converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives but had no effect on the interleukin-3-dependent parental BaF3 cells. Most of these factor-independent cell clones contained a major Env-related glycoprotein with an
M
r
of 60,000. During further in vivo passaging, a virus that encodes an
M
r
-55,000 glycoprotein became predominant. Sequence analysis indicated that the ultimate virus is a new SFFV that encodes a glycoprotein of 410 amino acids with the hallmark features of classical gp55s. Our results suggest that SFFV-related viruses can form in mice by recombination of retroviruses with genomic and helper virus sequences and that these novel viruses then evolve to become increasingly pathogenic.
The Roles of Pol and Env in the Assembly Pathway of Human Foamy Virus
Authors: Baldwin David N. , Linial Maxine L.
Published: December 2019

Human foamy virus (HFV) is the prototype of the
Spumavirus
genus of retroviruses. These viruses have a genomic organization close to that of other complex retroviruses but have similarities to hepadnaviruses such as human hepatitis B virus (HBV). Both HFV and HBV express their Pol protein independently of their structural proteins. Retroviruses and hepadnaviruses differ in their requirements for particle assembly and genome packaging. Assembly of retroviral particles containing RNA genomes requires only the Gag structural protein. The Pol protein is not required for capsid assembly, and the Env surface glycoprotein is not required for release of virions from the cell. In contrast, assembly of extracellular HBV particles containing DNA requires core structural protein and polymerase (P protein) for assembly of nucleocapsids and requires surface glycoproteins for release from the cell. We investigated the requirements for synthesis of extracellular HFV particles by constructing mutants with either the
pol
or
env
gene deleted. We found that the Pol protein is dispensable for production of extracellular particles containing viral nucleic acid. In the absence of Env, intracellular particles are synthesized but few or no extracellular particles could be detected. Thus, foamy virus assembly is distinct from that of other reverse transcriptase-encoding mammalian viruses.
Transgenic Mice Secreting Coronavirus Neutralizing Antibodies into the Milk
Authors: Sola Isabel , Castilla Joaquín , Pintado Belén , Sánchez-Morgado José M. , Whitelaw C. Bruce A. , Clark A. John , Enjuanes Luis
Published: December 2019

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 104by radioimmunoassay (RIA) and neutralized virus infectivity up to 104-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 106as determined by RIA, neutralized virus infectivity by 106-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic β-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and β-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of β-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.
Dendritic Cells Efficiently Induce Protective Antiviral Immunity
Authors: Ludewig Burkhard , Ehl Stephan , Karrer Urs , Odermatt Bernhard , Hengartner Hans , Zinkernagel Rolf M.
Published: December 2019

Cytotoxic T lymphocytes (CTL) are essential for effective immunity to various viral infections. Because of the high speed of viral replication, control of viral infections imposes demanding functional and qualitative requirements on protective T-cell responses. Dendritic cells (DC) have been shown to efficiently acquire, transport, and present antigens to naive CTL in vitro and in vivo. In this study, we assessed the potential of DC, either pulsed with the lymphocytic choriomeningitis virus (LCMV)-specific peptide GP33-41 or constitutively expressing the respective epitope, to induce LCMV-specific antiviral immunity in vivo. Comparing different application routes, we found that only 100 to 1,000 DC had to reach the spleen to achieve protective levels of CTL activation. The DC-induced antiviral immune response developed rapidly and was long lasting. Already at day 2 after a single intravenous immunization with high doses of DC (1 × 105to 5 × 105), mice were fully protected against LCMV challenge infection, and direct ex vivo cytotoxicity was detectable at day 4 after DC immunization. At day 60, mice were still protected against LCMV challenge infection. Importantly, priming with DC also conferred protection against infections in which the homing of CTL into peripheral organs is essential: DC-immunized mice rapidly cleared an infection with recombinant vaccinia virus-LCMV from the ovaries and eliminated LCMV from the brain, thereby avoiding lethal choriomeningitis. A comparison of DC constitutively expressing the GP33-41 epitope with exogenously peptide-pulsed DC showed that in vivo CTL priming with peptide-loaded DC is not limited by turnover of peptide-major histocompatibility complex class I complexes. We conclude that the priming of antiviral CTL responses with DC is highly efficient, rapid, and long lasting. Therefore, the use of DC should be considered as an efficient means of immunization for antiviral vaccination strategies.
Effect of Water-Based Microencapsulation on Protection against EDIM Rotavirus Challenge in Mice
Authors: Moser Charlotte A. , Speaker Tully J. , Offit Paul A.
Published: December 2019

We determined the capacity of microcapsules formed by the combination of sodium alginate, an aqueous anionic polymer, and spermine hydrochloride, an aqueous cationic amine, to enhance protection against rotavirus challenge in mice. Adult BALB/c mice were orally inoculated with either free or microencapsulated rotavirus (simian rotavirus strain RRV) and challenged 6 or 16 weeks later with murine rotavirus strain EDIM. Virus-specific humoral immune responses were determined at the time of challenge and 4 days after challenge by intestinal fragment culture. We found that spermine-alginate microcapsules enhanced protection against challenge 16 weeks after immunization but not 6 weeks after immunization. Quantities of virus-specific immunoglobulin A produced by small intestinal lamina propria lymphocytes were correlated with the degree of protection against challenge afforded by spermine-alginate microcapsules. Possible mechanisms by which microcapsules enhance protection against rotavirus challenge are discussed.
Detection of a Novel Bovine Lymphotropic Herpesvirus
Authors: Rovnak Joel , Quackenbush Sandra L. , Reyes Richard A. , Baines Joel D. , Parrish Colin R. , Casey James W.
Published: December 2019

Degenerate PCR primers which amplify a conserved region of the DNA polymerase genes of the herpesvirus family were used to provide sequence evidence for a new bovine herpesvirus in bovine B-lymphoma cells and peripheral blood mononuclear cells (PBMC). The sequence of the resultant amplicon was found to be distinct from those of known herpesvirus isolates. Alignment of amino acid sequences demonstrated 70% identity with ovine herpesvirus 2, 69% with alcelaphine herpesvirus 1, 65% with bovine herpesvirus 4, and 42% with bovine herpesvirus 1. Phylogenetic analysis placed this putative virus within the tumorigenic
Gammaherpesvirinae
subfamily, and it is tentatively identified as bovine lymphotropic herpesvirus. This novel agent was expressed in vitro from infected PBMC, and cell-free supernatants were used to transfer infection to a bovine B-cell line, BL3. Analysis, with specific PCR primers, of DNA from bovine PBMC and lymphoma cells identified infection in blood of 91% of adult animals (
n
= 101), 63% of lymphomas (
n
= 32), and 38% of juveniles (
n
= 13). Of the adults, herpesvirus infection was present in 94% of animals that were seropositive for bovine leukemia virus (BLV) (
n
= 63) and in 87% of BLV-seronegative animals (
n
= 38). Of the seropositive group, 17 animals exhibited persistent lymphocytosis, and 100% of these were herpesvirus positive by PCR. A role for bovine lymphotropic herpesvirus as a cofactor in BLV pathogenesis is considered.
Evolution of Hepatitis C Virus Quasispecies in Hypervariable Region 1 and the Putative Interferon Sensitivity-Determining Region during Interferon Therapy and Natural Infection
Authors: Polyak Stephen J. , McArdle Susan , Liu Shan-Lu , Sullivan Daniel G. , Chung Minjun , Hofgärtner Wolfgang T. , Carithers Robert L. , McMahon Brian J. , Mullins James I. , Corey Lawrence , Gretch David R.
Published: December 2019

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (
r
= 0.941,
P
< 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year;
P
= 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year;
P
= 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.
Identification of a Full-Length cDNA for an Endogenous Retrovirus of Miniature Swine
Authors: Akiyoshi Donna E. , Denaro Maria , Zhu Haihong , Greenstein Julia L. , Banerjee Papia , Fishman Jay A.
Published: December 2019

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames forgag,pol, andenvin PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attach- ment) region ofenv. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.
Mutational and Functional Analysis of the C-Terminal Region of the C3H Mouse Mammary Tumor Virus Superantigen
Authors: Wrona Thomas J. , Lozano Mary , Binhazim Awadh A. , Dudley Jaquelin P.
Published: December 2019

The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vβ) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vβ chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+Vβ14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of thesagmutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps thesaggene within the LTR, suggesting that loss of Sag function may be compensated, at least partially, by loss of transcriptional suppression in certain tissues. Together these results confirm the importance of Sag for efficient milk-borne transmission and indicate that the entire C-terminal region is needed for complete Sag function.
Herpes Simplex Virus Type 1 Glycoprotein B Requires a Cysteine Residue at Position 633 for Folding, Processing, and Incorporation into Mature Infectious Virus Particles
Authors: Laquerre Sylvie , Anderson Dina B. , Argnani Rafaela , Glorioso Joseph C.
Published: December 2019

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) resides in the virus envelope in an oligomeric form and plays an essential role in virus entry into susceptible host cells. The oligomerizing domain is a movable element consisting of amino acids 626 to 653 in the gB external domain. This domain contains a single cysteine residue at position 633 (Cys-633) that is predicted to form an intramolecular disulfide bridge with Cys-596. In this study, we examined gB oligomerization, processing, and incorporation into mature virus during infection by two mutant viruses in which either the gB Cys-633 [KgB(C633S)] or both Cys-633 and Cys-596 [KgB(C596S/C633S)] residues were mutated to serine. The result of immunofluorescence studies and analyses of released virus particles showed that the mutant gB molecules were not transported to the cell surface or incorporated into mature virus envelopes and thus infectious virus was not produced. Immunoprecipitation studies revealed that the mutant gB molecules were in an oligomeric configuration and that these mutants produced hetero-oligomers with a truncated form of gB consisting of residues 1 to 43 and 595 to 904, the latter containing the oligomerization domain. Pulse-chase experiments in combination with endoglycosidase H treatment determined that the mutant molecules were improperly processed, having been retained in the endoplasmic reticulum (ER). Coimmunoprecipitation experiments revealed that the cysteine mutations resulted in gB misfolding and retention by the molecular chaperones calnexin, calreticulin, and Grp78 in the ER. The altered conformation of the gB mutant glycoproteins was directly detected by a reduction in monoclonal antibody recognition of two previously defined distinct antigenic sites located within residues 381 to 441 and 595 to 737. The misfolded molecules were not transported to the cell surface as hetero-oligomers with wild-type gB, suggesting that the conformational change could not be corrected by intermolecular interactions with the wild-type molecule. Together, these experiments confirmed that a disulfide bridge involving Cys-633 and Cys-596 is not essential for oligomerization but rather is required for proper folding and maintenance of a gB domain essential to complete posttranslational modification, transport, and incorporation into mature virus particles.
In Vivo Distribution of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Coreceptors: CXCR4, CCR3, and CCR5
Authors: Zhang Linqi , He Tian , Talal Andrew , Wang Gloria , Frankel Sarah S. , Ho David D.
Published: December 2019

We have evaluated the in vivo distribution of the major human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) coreceptors, CXCR4, CCR3, and CCR5, in both rhesus macaques and humans. T lymphocytes and macrophages in both lymphoid and nonlymphoid tissues are the major cell populations expressing HIV/SIV coreceptors, reaffirming that these cells are the major targets of HIV/SIV infection in vivo. In lymphoid tissues such as the lymph node and the thymus, approximately 1 to 10% of the T lymphocytes and macrophages are coreceptor positive. However, coreceptor expression was not detected on follicular dendritic cells (FDC) in lymph nodes, suggesting that the ability of FDC to trap extracellular virions is unlikely to be mediated by a coreceptor-specific mechanism. In the thymus, a large number of immature and mature T lymphocytes express CXCR4, which may render these cells susceptible to infection by syncytium-inducing viral variants that use this coreceptor for entry. In addition, various degrees of coreceptor expression are found among different tissues and also among different cells within the same tissues. Coreceptor-positive cells are more frequently identified in the colon than in the rectum and more frequently identified in the cervix than in the vagina, suggesting that the expression levels of coreceptors are differentially regulated at different anatomic sites. Furthermore, extremely high levels of CXCR4 and CCR3 expression are found on the neurons from both the central and peripheral nervous systems. These findings may be helpful in understanding certain aspects of HIV and SIV pathogenesis and transmission.
Simian-Human Immunodeficiency Virus (SHIV) Containing thenef/Long Terminal Repeat Region of the Highly Virulent SIVsmmPBj14 Causes PBj-Like Activation of Cultured Resting Peripheral Blood Mononuclear Cells, but the Chimera Showed No Increase in Virulence
Authors: Stephens Edward B. , Mukherjee Sampa , Liu Zhen Qian , Sheffer Darlene , Lamb-Wharton Rebecca , Leung Kevin , Zhuge Wu , Joag Sanjay V. , Li Zhuang , Foresman Larry , Adany Istvan , Narayan Opendra
Published: December 2019

SIVsmmPBj14 is a highly pathogenic lentivirus which causes acute diarrhea, rash, massive lymphocyte proliferation predominantly in the gastrointestinal tract, and death within 7 to 14 days. In cell culture, the virus has mitogenic effects on resting macaque T lymphocytes. In contrast, SIVmac239 causes AIDS in rhesus macaques, generally within 2 years after inoculation. In a previous study, replacement of amino acid residues 17 and 18 of the Nef protein of SIVmac239 with the corresponding amino acid residues of the Nef protein of SIVsmmPBj14 yielded a PBj-like virus that caused extensive activation of resting T lymphocytes in cultures and acute PBj-like disease when inoculated into pig-tailed macaques. This study suggested thatnefplayed a major role in both processes. In this study, we replaced thenef/long terminal repeat (LTR) region of a nonpathogenic simian-human immunodeficiency virus (SHIV), SHIVPPc, with the corresponding region from SIVsmmPBj14 and examined the biological properties of the resultant virus. Like SIVsmmPBj14, SHIVPPcPBjnef caused massive stimulation of resting peripheral blood mononuclear cells (PBMC), which then produced virus in the absence of extraneous interleukin 2. However, when inoculated into macaques, the virus failed to replicate productively or cause disease. Thus, while these results confirmed that thenef/LTR region of SIVsmmPBj14 played a major role in the activation of resting PBMC, duplication of the cellular activation process in macaques may require a further interaction betweennefand the envelope glycoprotein of simian immunodeficiency virus because SHIV, containing the envelope of human immunodeficiency virus type 1, failed to cause activation in vivo.
Rabbit Genital Tissue Is Susceptible to Infection by Rabbit Oral Papillomavirus: an Animal Model for a Genital Tissue-Targeting Papillomavirus
Authors: Harvey Stephen B. , Cladel Nancy M. , Budgeon Lynn R. , Welsh Patricia A. , Griffith James W. , Lang C. Max , Christensen Neil D.
Published: December 2019

Rabbit oral papillomavirus (ROPV) is a mucosatropic papillomavirus which naturally infects oral mucosal sites of domestic rabbits. In this study, we tested the hypothesis that rabbit genital mucosa is also susceptible to ROPV infection by using the athymic mouse xenograft system and adult immunocompetent rabbits. Subrenal xenografts of ROPV-infected rabbit vulvar and penile sheath tissues were strongly positive for ROPV infection by histologic, in situ hybridization, and Southern analyses. Direct inoculation of adult rabbit penises with infectious ROPV produced small raised lesions of approximately 1 by 1 by 1 mm that were ROPV positive by both in situ hybridization and Southern analyses and were also viral capsid antigen positive by immunohistological staining. Infection of rabbit genital tissues with ROPV may be a useful animal model for the study of genital tissue-targeting papillomaviruses.
Recombination of Engineered Defective RNA Species Produces Infective Potyvirus In Planta
Authors: Gal-On Amit , Meiri Eti , Raccah Benjamin , Gaba Victor
Published: December 2019

Recombination occurred between viral genomes when squash plants were cobombarded with mixtures of engineered disabled constructs of a zucchini yellow mosaic potyvirus. Single and double recombinants were detected in the progeny. Genes involved in the recombination process and the mechanisms of recombination were studied in potyviruses for the first time.
Identification of Replication-Competent Strains of Simian Immunodeficiency Virus Lacking Multiple Attachment Sites for N-Linked Carbohydrates in Variable Regions 1 and 2 of the Surface Envelope Protein
Authors: Reitter Julie N. , Desrosiers Ronald C.
Published: December 2019

Carbohydrates comprise about 50% of the mass of gp120, the external envelope glycoprotein of simian immunodeficiency virus (SIV) and human immunodeficiency virus. We identified 11 replication-competent derivatives of SIVmac239 lacking two, three, four, or five potential sites for N-linked glycosylation. These sites were located within and around variable regions 1 and 2 of the surface envelope protein of the virus. Asn (AAT) of the canonical N-linked glycosylation recognition sequence (Asn X Ser/Thr) was changed in each case to the structurally similar Gln (CAG or CAA) such that two nucleotide changes in the codon would be required for reversion. Replication of one triple mutant (g456), however, was severely impaired. A revertant of the g456 mutant was recovered from CEMx174 cells with a Met-to-Val compensatory substitution at position 144, 2 amino acids upstream of attachment site 5. Thus, a debilitating loss of sites for N-linked glycosylation can be compensated for by amino acid changes not involving the Asn-X-Ser/Thr consensus motif. These results provide a framework to begin testing the hypothesis that carbohydrates form a barrier that can limit the humoral immune responses to the virus.
Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA
3
Lys
rather than tRNA
1,2
Lys
Authors: Zhang Zhijun , Yu Qin , Kang Sang-Moo , Buescher James , Morrow Casey D.
Published: December 2019

All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA
3
Lys
. However, within HIV-1 virions, there are approximately equal amounts of tRNA
1
Lys
, tRNA
2
Lys
, and tRNA
3
Lys
. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA
3
Lys
, tRNA
1
Lys
and tRNA
2
Lys
could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a
gpt
gene encoding xanthine-guanine phosphoribosyl transferase in place of the
env
gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA
3
Lys
, not tRNA
1
Lys
or tRNA
2
Lys
. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA
1,2
Lys
. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA
3
Lys
from proviral DNA isolated from
gpt
-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA
1,2
Lys
resulted in
gpt
- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA
1,2
Lys
. The results of these studies suggest that the selection of tRNA
3
Lys
for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.
Intracellular Signaling by the Chemokine Receptor US28 during Human Cytomegalovirus Infection
Authors: Billstrom Marcella A. , Johnson Gary L. , Avdi Natalie J. , Worthen G. Scott
Published: December 2019

In patients with impaired cell-mediated immune responses (e.g., lung transplant recipients and AIDS patients), cytomegalovirus (CMV) infection causes severe disease such as pneumonitis. However, although immunocompetency in the host can protect from CMV disease, the virus persists by evading the host immune defenses. A model of CMV infection of the endothelium has been developed in which inflammatory stimuli, such as the CC chemokine RANTES, bind to the endothelial cell surface, stimulating calcium flux during late times of CMV infection. At 96 h postinfection, CMV-infected cells express mRNA of the CMV-encoded CC chemokine receptor US28 but do not express mRNA of other CC chemokine receptors that bind RANTES (CCR1, CCR4, CCR5). Cloning and stable expression of the receptor CMV US28 in human kidney epithelial cells (293 cells) with and without the heterotrimeric G protein α16indicated that CMV US28 couples to both Gαiand Gα16proteins to activate calcium flux in response to the chemokines RANTES and MCP-3. Furthermore, cells that coexpress US28 and Gα16responded to RANTES stimulation with activation of extracellular signal-regulated kinase, which could be attributed, in part, to specific Gα16coupling. Thus, through expression of the CC chemokine receptor US28, CMV may utilize resident G proteins of the infected cell to manipulate cellular responses stimulated by chemokines.
Role of the Pre-S2 Domain of the Large Envelope Protein in Hepatitis B Virus Assembly and Infectivity
Authors: Le Seyec J. , Chouteau P. , Cannie I. , Guguen-Guillouzo C. , Gripon P.
Published: December 2019

Among the three viral proteins present in the hepatitis B virus (HBV) envelope, both the small and large polypeptides, but not the middle polypeptide, are necessary for the production of complete viral particles. Whereas it has been established that the C-terminal extremity of the pre-S1 region is required for HBV morphogenesis, whether the pre-S2 region of the large surface protein plays a critical role remains questionable. In the present study, we have analyzed the role of the large-polypeptide pre-S2 region in viral maturation and infectivity. For this purpose, mutants bearing contiguous deletions covering the entire pre-S2 domain were generated. First, the efficient expression of all the mutant large envelope proteins was verified and their ability to substitute for the wild-type form in virion secretion was tested. We found that distinct deletions covering the domain between amino acids 114 and 163 still allowed virion production. In contrast, the polypeptide lacking the first 5 amino acids of pre-S2 (amino acids 109 to 113) was unable to support viral secretion. This result shows that the domain of the large surface protein, required for this process, must be extended to the N-terminal extremity of pre-S2. We then demonstrated that all the mutants competent for virion release were able to infect normal human hepatocytes in primary culture. Taken together, these results indicate that only 10% of the large-protein pre-S2 region at its N-terminal extremity is essential for virion export and that the remaining part, dispensable for viral secretion, is also dispensable for infectivity.
Particle Polymorphism Caused by Deletion of a Peptide Molecular Switch in a Quasiequivalent Icosahedral Virus
Authors: Dong X. Fan , Natarajan Padmaja , Tihova Mariana , Johnson John E. , Schneemann Anette
Published: December 2019

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.
Structure and Phylogenetic Analysis of an Endogenous Retrovirus Inserted into the Human Growth Factor Gene Pleiotrophin
Authors: Schulte Anke M. , Wellstein Anton
Published: December 2019

A human endogenous retrovirus-like element (HERV), flanked by long terminal repeats of 502 and 495 nucleotides is inserted into the human pleiotrophin (PTN) gene upstream of the open reading frame. Based on its Glu-tRNA primer binding site specificity and the location within the PTN gene, we named this element HERV-E.PTN. HERV-E.PTN appears to be a recombined viral element based on its high homology (70 to 86%) in distinct areas to members of two distantly related HERV type C families, HERV-E and retrovirus-like element I (RTVL-I). Furthermore, its pseudogene region is organized from 5′ to 3′ intogag-,pol-,env-,pol-,env-similar sequences. Interestingly, full-length and partial HERV-E.PTN-homologous sequences were found in the human X chromosome, the human hereditary haemochromatosis region, and the BRCA1 pseudogene. Finally, Southern analyses indicate that the HERV-E.PTN element is present in the PTN gene of humans, chimpanzees, and gorillas but not of rhesus monkeys, suggesting that genomic insertion occurred after the separation of monkeys and apes about 25 million years ago.
Heparan Sulfate Proteoglycan Binding by Herpes Simplex Virus Type 1 Glycoproteins B and C, Which Differ in Their Contributions to Virus Attachment, Penetration, and Cell-to-Cell Spread
Authors: Laquerre Sylvie , Argnani Rafaela , Anderson Dina B. , Zucchini Silvia , Manservigi Roberto , Glorioso Joseph C.
Published: December 2019

Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB’s role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK
−
also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK
−
gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK
−
. Compared with wild-type virus, KgBpK
−
showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the
lacZ
gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK
−
gC
−
) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK
−
mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.
Role of E4 in Eliciting CD4 T-Cell and B-Cell Responses to Adenovirus Vectors Delivered to Murine and Nonhuman Primate Lungs
Authors: Chirmule Narendra , Hughes Joseph V. , Gao Guang-Ping , Raper Steven E. , Wilson James M.
Published: December 2019

Adenovirus vectors delivered to lung are being considered in the treatment of cystic fibrosis (CF). Vectors from which E1 has been deleted elicit T- and B-cell responses which confound their use in the treatment of chronic diseases such as CF. In this study, we directly compare the biology of an adenovirus vector from which E1 has been deleted to that of one from which E1 and E4 have been deleted, following intratracheal instillation into mouse and nonhuman primate lung. Evaluation of the E1 deletion vector in C57BL/6 mice demonstrated dose-dependent activation of both CD4 T cells (i.e., TH1 and TH2 subsets) and neutralizing antibodies to viral capsid proteins. Deletion of E4 and E1 had little impact on the CD4 T-cell proliferative response and cytolytic activity of CD8 T cells against target cells expressing viral antigens. Analysis of T-cell subsets from mice exposed to the vector from which E1 and E4 had been deleted demonstrated preservation of TH1 responses with markedly diminished TH2 responses compared to the vector with the deletion of E1. This effect was associated with reduced TH2-dependent immunoglobulin isotypes and markedly diminished neutralizing antibodies. Similar results were obtained in nonhuman primates. These studies indicate that the vector genotype can modify B-cell responses by differential activation of TH1 subsets. Diminished humoral immunity, as was observed with the E1 and E4 deletion vectors in lung, is indeed desired in applications of gene therapy where readministration of the vector is necessary.
Inhibition of Simian Immunodeficiency Virus (SIV) Replication by CD8+T Lymphocytes from Macaques Immunized with Live Attenuated SIV
Authors: Gauduin Marie-Claire , Glickman Rhona L. , Means Robert , Johnson R. Paul
Published: December 2019

Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8+T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Δnef or SIVmac239Δ3 to inhibit SIV replication. CD8+T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4+T cells. Suppression of SIV replication by unstimulated CD8+T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8+T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8+T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8+cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1α (MIP-1α), or MIP-1β from stimulated CD8+T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8+T cells of control animals. However, addition of antibodies that neutralize these β-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8+T cells. Our results indicate that inhibition of SIV replication by CD8+T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1α, and MIP-1β.
Viral Genetic Evolution in Macaques Infected with Molecularly Cloned Simian Immunodeficiency Virus Correlates with the Extent of Persistent Viremia
Authors: Hirsch Vanessa M. , Dapolito George , Hahn Anna , Lifson Jeff , Montefiori David , Brown Charles R. , Goeken Robert
Published: December 2019

Genetic evolution of the simian immunodeficiency virus (SIV) envelope glycoprotein was evaluated in a group of six macaques (Macaca nemestrina) infected with the molecularly cloned, moderately pathogenic SIVsm62d. The extent of envelope evolution was subsequently evaluated within the context of the individual pattern of viremia and disease outcome. Two macaques in this cohort developed AIDS by 1.5 years postinoculation (progressors), whereas the remaining four macaques remained asymptomatic (nonprogressors). Compared with the nonprogressor macaques, the two progressor macaques exhibited higher persistent plasma viremia, higher homologous neutralizing antibody titers, and more extensive mutation and evolution in the V1 region of envelope. Although clearly distinct in each of these parameters from the progressors, the four nonprogressors exhibited more individual variability with respect to the extent of persistent viremia and genetic evolution of the V1 region of envelope. The extent of V1 envelope varied from no apparent V1 evolution in a macaque with good viral containment to extensive evolution in one macaque with persistent viremia. This study underscores the critical role of persistent replication in the genetic evolution of SIV.
A Role for Herpesvirus Saimiri orf14 in Transformation and Persistent Infection
Authors: Duboise Monroe , Guo Jie , Czajak Sue , Lee Heuiran , Veazey Ronald , Desrosiers Ronald C. , Jung Jae U.
Published: December 2019

The product of open reading frame 14 (orf14) of herpesvirus saimiri (HVS) exhibits significant homology with mouse mammary tumor virus superantigen. orf14 encodes a 50-kDa secreted glycoprotein, as shown previously (Z. Yao, E. Maraskovsky, M. K. Spriggs, J. I. Cohen, R. J. Armitage, and M. R. Alderson, J. Immunol. 156:3260–3266, 1996). orf14 expressed from recombinant baculovirus powerfully induces proliferation of CD4-positive cells originating from several different species. To study the role of orf14 in transformation, a mutant form of HVS (HVS Δorf14) was constructed with a deletion in the orf14 gene. The transforming potential of HVS Δorf14 was tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, while the HVS Δorf14 mutant did not produce such a growth transformation. In addition, HVS Δorf14 was nononcogenic in common marmosets. In contrast to other nononcogenic HVS mutant viruses which were repeatedly isolated from peripheral blood mononuclear cells of infected marmosets for more than 5 months, HVS Δorf14 did not persist at a high level in vivo. These results demonstrate that orf14 of HVS is not required for replication but is required for transformation and for high-level persistence in vivo.