Cell and Developmental Biology Commons^™

A Subset Of Latency-Reversing Agents Expose Hiv-Infected Resting Cd4+ T-Cells To Recognition By Cytotoxic T-Lymphocytes., R. Brad Jones, Stefanie Mueller, Rachel O'Connor, Katherine Rimpel, Derek D Sloan, Allison S. Thomas, Sara Karandish, Szu-Han Huang, +13 More

Microbiology, Immunology, and Tropical Medicine Faculty Publications

Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing …

Lineage-Specific Differences In The Gp120 Inner Domain Layer 3 Of Human And Simian Immunodeficiency Viruses, Shilei Ding, Halima Medjahed, Jérémie Prévost, Mathieu Coutu, Shi-Hua Xiang, Andrés Finzi

Nebraska Center for Virology: Faculty Publications

Binding of HIV-1 and SIV gp120 exterior envelope glycoprotein to CD4 triggers conformational changes in gp120 that promote its interaction with one of the chemokine receptors, usually CCR5, ultimately leading to gp41-mediated virus-cell membrane fusion and entry. We previously described that topological Layers (Layer 1, Layer 2 and Layer 3) in the gp120 inner domain contribute to gp120-trimer association in the unliganded state but also help secure CD4 binding. Relative to Layer 1 of HIV-1 gp120, the SIVmac239 gp120 Layer 1 plays a more prominent role in maintaining gp120-trimer association but is minimally involved in promoting CD4 binding, which could …

Giant Chloroviruses: Five Easy Questions, James L. Van Etten, David Dunigan

Nebraska Center for Virology: Faculty Publications

Chloroviruses are large, icosahedral, dsDNA-containing viruses that replicate in certain unicellular, chlorella-like green algae [1,2]. They exist in freshwater throughout the world with titers as high as thousands of plaque-forming units (PFU) per ml of indigenous water although titers are typically 1–100 PFU/ml. Titers fluctuate during the year with the highest titers typically occurring in the spring and late fall. Known chlorovirus hosts, which are normally symbionts and are often referred to as zoochlorellae, are associated with either the protozoan Paramecium bursaria (Fig 1A), the coelenterate Hydra viridis, or the heliozoan Acanthocystis turfacea. Zoochlorellae are resistant to viruses …

Genetic Barrier To Direct Acting Antivirals In Hcv Sequences Deposited In The European Databank, Dimas Alexandre Kliemann, Cristiane Valle Tovo, Ana Beatriz Gorini Da Veiga, André Luiz Machado, John T. West

Nebraska Center for Virology: Faculty Publications

Background & Aims: Development of resistance results from mutations in the viral genome, and the presence of selective drug pressure leads to the emergence of a resistant virus population. The aim of this study was to analyze the impact of genetic variability on the genetic barrier to drug resistance to DAAs.

Methods: The genetic barrier was quantified based on the number and type of nucleotide mutations required to impart resistance, considering full-length HCV NS3, NS5A and NS5B regions segregated by genotype into subtypes 1a, 1b, 2a, 2b and 3a. This study analyzed 789 NS3 sequences, 708 sequences and 536 NS5B …

Kaposi’S Sarcoma-Associated Herpesvirus Reduces Cellular Myeloid Differentiation Primary-Response Gene 88 (Myd88) Expression Via Modulation Of Its Rna, Amy Lingel, Erica Ehlers, Qianli Wang, Mingxia Cao, Charles Wood, Rongtuan Lin, Luwen Zhang

Nebraska Center for Virology: Faculty Publications

Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Interleukin 1 (IL-1) is a major mediator for inflammation and plays an important role in both innate and adaptive immunity. Myeloid differentiation primary response gene 88 (MyD88) is an essential adaptor molecule for IL-1 as well as most Toll-like receptor signaling. In this study, we identified a novel mechanism by which KSHV interferes with host inflammation and immunity. KSHV RTA specifically reduces the steady-state protein levels of …

Nf45 And Nf90 Bind Hiv-1 Rna And Modulate Hiv Gene Expression, Yan Li, Michael Belshan

Nebraska Center for Virology: Faculty Publications

A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1) replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 …

Mucosal Vaccination By Adenoviruses Displaying Reovirus Sigma 1, Eric A. Weaver, Zenaido T. Camacho, Matthew L. Hillestad, Catherine M. Crosby, Mallory A. Turner, Adam J. Guenzel, Hind J. Fadel, George T. Mercier, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

We previously developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but had 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generated stronger T cell responses than Ad5 when used for mucosal immunization. New Ad5- fiber-sigma vectors were generated here by varying the number of fiber β-spiral shaft repeats (R) fused between fiber tail and the sigma. Ad5 virions encoding R3, R14, and R20 chimeras were rescued. Increasing chimera length led to their decreasing encapsidation of these proteins in the virions. …

Recapitulating Cross-Species Transmission Of Sivcpz To Humans Using Humanized-Blt Mice, Zhe Yuan, Guobin Kang, Fangrui Ma, Wuxun Lu, Wenjin Fan, Christine M. Fennessey, Brandon F. Keele, Qingsheng Li

Nebraska Center for Virology: Faculty Publications

The origins of HIV-1 have been widely accepted to be the consequence of simian immunodeficiency viruses from wild chimpanzees (SIVcpz) crossing over to humans. However, there has not been any in vivo study of SIVcpz infection of humans. Also, it remains largely unknown why only specific SIVcpz strains have achieved cross-species transmission and what transmission risk might exist for those SIVcpz strains that have not been found to infect humans. Closing this knowledge gap is essential for better understanding cross-species transmission and predicting the likelihood of additional cross-species transmissions of SIV into humans. Here we show hu-BLT mice are susceptible …

High Glucose Induces Reactivation Of Latent Kaposi’S Sarcoma-Associated Herpesvirus, Fengchun Ye, Yan Zeng, Jingfeng Sha, Tiffany Jones, Kurt Kuhne, Charles Wood, Shou-Jiang Gao

Nebraska Center for Virology: Faculty Publications

High prevalence of Kaposi’s sarcoma (KS) is seen in diabetic patients. It is unknown if the physiological condition of diabetes contributes to KS development. We found elevated levels of viral lytic gene expression when Kaposi’s sarcoma-associated herpesvirus (KSHV) infected cells were cultured in high glucose medium. To demonstrate the association between high glucose and KSHV replication, we xeno29

grafted telomerase-immortalized human umbilical vein endothelial cells that are infected with KSHV (TIVE-KSHV) into hyperglycemic and normal nude mice. The injected cells expressed significantly higher levels of KSHV lytic genes in hyperglycemic mice than in normal mice. We further demonstrated that high …

Persistent Low-Level Replication Of Sivδnef Drives Maturation Of Antibody And Cd8 T Cell Responses To Induce Protective Immunity Against Vaginal Siv Infection, Sama Adnan, R. Keith Reeves, Jacqueline Gillis, Fay E. Wong, Yi Yu, Jeremy V. Camp, Qingsheng Li, Michelle Connole, Yuan Li, Michael Piatak Jr., Jeffrey D. Lifson, Wenjun Li, Brandon F. Keele, Pamela A. Kozlowski, Ronald C. Desrosiers, Ashley T. Haase, R. Paul Johnson

Nebraska Center for Virology: Faculty Publications

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef …

Complete Genome Sequence Of Highly Virulent Porcine Reproductive And Respiratory Syndrome Virus Variants That Recently Emerged In The United States, Aspen M. Workman, Timothy P.L. Smith, Fernando A. Osorio, Hiep L.X. Vu

Nebraska Center for Virology: Faculty Publications

A recent outbreak of particularly virulent disease caused by porcine reproductive and respiratory syndrome virus has occurred in swine herds across the United States. We report here the complete genome sequence of eight viral isolates from four Nebraska herds experiencing an outbreak of severe disease in 2016.

Domain I Of The 5′ Non-Translated Genomic Region In Coxsackievirus B3 Rna Is Not Required For Productive Replication, L. Jaramillo, S. Smithee, S. Tracy, N. M. Chapman

Nebraska Center for Virology: Faculty Publications

Domain I is a cloverleaf-like secondary structure at the 5′ termini of all enterovirus genomes, comprising part of a cis-acting replication element essential for efficient enteroviral replication. 5′ genomic terminal deletions up to as much as 55% of domain I can occur without lethality following coxsackie B virus infections. We report here that the entire CVB structural domain I can be deleted without lethality.

Reversion To Wildtype Of A Mutated And Nonfunctional Coxsackievirus B3cre(2c), Shane Smithee, Steven Tracy, Nora M. Chapman

Nebraska Center for Virology: Faculty Publications

The cis-acting replication element (CRE) in the 2C protein coding region [CRE(2C)] of enteroviruses (EV) facilitates the addition of two uridine residues (uridylylation) onto the virus-encoded protein VPg inorder for it to serve as the RNA replication primer. We demonstrated that coxsackievirus B3 (CVB3) is replication competent in the absence of a native (uridylylating) CRE(2C) and also demonstrated that lackof a functional CRE(2C) led to generation of 5’ terminal genomic deletions in the CVB3 CRE-knock-out (CVB3-CKO) population. We asked whether reversion of the mutated CRE(2C) occurred, thus permitting sustained replication, and when were 5’ terminal deletions generated during replication. Virions …

Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses, Catherine M. Crosby, William E. Matchett, Stephanie S. Anguiano-Zarate, Christopher A. Parks, Eric A. Weaver, Larry R. Pease, Richard J. Webby, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Head-to-head comparisons of conventional influenza vaccines with ade- novirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we de- veloped “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than …

Role Of Stat3 In Human Nk Cell Functions, Prasad V. Phatarpekar

Dissertations and Theses (Open Access)

Natural Killer (NK) cells are cytotoxic lymphocytes, which play a critical role in the immune response against malignant cells and microbial infections. NK cells are equipped with activating receptors, which upon detecting ligands expressed on stressed cells induce cytolytic activity of NK cells. Stimulation of NK cell proliferation and priming of NK cytolytic capability are accomplished by cytokines, which mediate their signals mainly through JAK-STAT signaling pathway. Previously, we found that K562 cells genetically modified to express membrane bound IL-21 (mbIL-21), which predominantly activates STAT3, induce robust expansion and activation of human NK cells. Further investigations revealed role of STAT3 …

Early Initiation Of Antiretroviral Therapy Can Functionally Control Productive Hiv-1 Infection In Humanized-Blt Mice, Qingsheng Li, For Yue Tso, Guobin Kang, Wuxun Lu, Yue Li, Wenjin Fan, Zhe Yuan, Christopher J. Destache, Charles Wood

Nebraska Center for Virology: Faculty Publications

Background—Recent reports showed that functional control of HIV-1 infection for a prolonged time is possible by early anti-retroviral therapy (ART); however its underlying mechanism needs to be studied with a suitable animal model. Recently, humanized-BLT (bone marrow, liver and thymus) mouse (hu-BLT) was shown to be an excellent model for studying HIV-1 infection. We thus tested the feasibility of studying functional control of HIV-1 infection using hu-BLT mice.

Methods—Animals in three treatment groups (Rx-6h, Rx-24h, Rx-48h) and untreated group were infected with HIV-1, followed by ART initiation at 6, 24 or 48 hours post-infection and continued daily for two weeks. …

The Role Of Dendritic Cell Subsets And Innate Immunity In The Pathogenesis Of Type 1 Diabetes And Other Autoimmune Diseases, Jeffrey D. Price, Kristin V. Tarbell

Nebraska Center for Virology: Faculty Publications

Dendritic cells (DCs) are key antigen-presenting cells that have an important role in autoimmune pathogenesis. DCs control both steady-state T cell tolerance and activation of pathogenic responses. The balance between these two outcomes depends on several factors, including genetic susceptibility, environmental signals that stimulate varied innate responses, and which DC subset is presenting antigen. Although the specific DC phenotype can diverge depending on the tissue location and context, there are four main subsets identified in both mouse and human: conventional cDC1 and cDC2, plasmacytoid DCs, and monocyte-derived DCs. In this review, we will discuss the role of these subsets in …

A Nuclear Fraction Of Turnip Crinkle Virus Capsid Protein Is Important For Elicitation Of The Host Resistance Response, Sung-Hwan Kang, Feng Qu, Thomas J. Morris

Nebraska Center for Virology: Faculty Publications

The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. …

A Novel Codon-Optimized Siv Gag-Pol Immunogen For Genebased Vaccination, Catherine M. Crosby, Eric A. Weaver, Reeti Khare, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Simian immunodeficiency virus (SIV) is a robust pathogen used in non-human primates to model HIV vaccines. SIV encodes a number of potential vaccine targets. By far the largest and most conserved protein target in SIV is its gag-pol protein that bears many epitopes to drive multivalent immune T cell responses. While gag-pol is an attractive antigen, it is only translated after a frame shift between gag and pol with the effect that gag and pol are expressed at an approximate 10/1 ratio. The codon bias of native lentiviral genes are also mismatched with the abundance of tRNAs in mammalian cells …

Whole-Genome Sequencing Of Kaposi’S Sarcoma-Associated Herpesvirus From Zambian Kaposi’S Sarcoma Biopsy Specimens Reveals Unique Viral Diversity, Landon Olp, Adrien Jeanniard, Clemence Marimo, Charles Wood

Nebraska Center for Virology: Faculty Publications

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi’s sarcoma (KS). Both KSHV and KS are endemic in sub-Saharan Africa where approximately 84% of global KS cases occur. Nevertheless, whole-genome sequencing of KSHV has only been completed using isolates from Western countries—where KS is not endemic. The lack of whole-genome KSHV sequence data from the most clinically important geographical region, sub-Saharan Africa, represents an important gap since it remains unclear whether genomic diversity has a role on KSHV pathogenesis. We hypothesized that distinct KSHV genotypes might be present in sub-Saharan Africa compared to Western countries. Using a KSHV-targeted enrichment …

Observed And Expected Incidence Of Cervical Cancer In Lusaka And The Southern And Western Provinces Of Zambia, 2007 - 2012, Mulele Kalima, Kennedy Lishimpi, Jane L. Meza, Shinobu Watanabe-Galloway, Susan C. Msadabwe, Catherine K. Mwaba, Aaron L. Shibemba, Lewis Banda, Charles Wood, Robert M. Chamberlain, Amr S. Soliman

Nebraska Center for Virology: Faculty Publications

Objectives—Cervical cancer is increasing but underestimated in developing countries. We calculated the observed and expected incidence of cervical cancer in Lusaka and Southern and Western provinces of Zambia.

Methods/Materials—Data for 2007-2012 was obtained for the 3 provinces. Data included age, residence, year of diagnosis, marital status, occupation, HIV, stage, radiotherapy and chemotherapy. Expected incidence in Southern and Western provinces was calculated based on observed incidence for Lusaka province, adjusting for HIV.

Results—Crude and age-standardized incidence rates (ASR) in Lusaka were 2-4 times higher than incidence in the other 2 provinces. Lusaka had a rate of 54.1/10⁵ and ASR of …

Crf07_Bc Strain Dominates The Hiv-1 Epidemic In Injection Drug Users In Liangshan Prefecture Of Sichuan, China, Lin Li, Daying Wei, Wan-Ling Hsu, Tianyi Li, Tao Gui, Charles Wood, Yongjian Liu, Hanping Li, Zuoyi Bao, Siyang Liu, Xiaolin Wang, Jingyun Li

Nebraska Center for Virology: Faculty Publications

The Liangshan prefecture in Sichuan province is an area in China severely affected by the HIV epidemic, with intravenous drug use (IDU) as the main risk factor. No reports on HIV subtypes prevalent in IDUs in Liangshan prefecture could be found. In this study, we have characterized the genotypes of HIV-1 in the IDU population in Liangshan prefecture and further determined the phylogenetic relationship of the CRF07_BC strains to HIV-1 sequences from the other regions of China, including Xinjiang and Yunnan provinces, to explore the pattern and possible diffusion pathway of HIV-1 in these regions. HIV-1-seropositive drug-naive IDUs identified in …

Characterization Of Cd8+ T Cell Differentiation Following Sivδnef Vaccination By Transcription Factor Expression Profiling, James M. Billingsley, Premeela A. Rajakumar, Michelle A. Connole, Nadine C. Salisch, Sama Adnan, Yury V. Kuzmichev, Henoch S. Hong, R. Keith Reeves, Hyung-Joo Kang, Wenjun Li, Qingsheng Li, Ashley T. Haase, R. Paul Johnson

Nebraska Center for Virology: Faculty Publications

The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8⁺ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8+⁺ T cells, and in SIV-specific CD8⁺ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8⁺ T cells into groups concordant with cell surface phenotype. Transcription factor …

Escherichia Coli Surface Display Of Single-Chain Antibody Vrc01 Against Hiv-1 Infection, Lin-Xu Wang, Michael Mellon, Dane Bowder, Meghan Quinn, Danielle Shea, Charles Wood, Shi-Hua Xiang

Nebraska Center for Virology: Faculty Publications

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene from …

Evidence For Placental Hpv Infection In Both Hiv Positive And Negative Women, Chrispin Chisanga, Dawn Eggert, Charles D. Mitchell, Charles Wood, Peter C. Angeletti

Nebraska Center for Virology: Faculty Publications

Human papillomaviruses (HPVs) have previously been reported to infect epithelial trophoblast cells of the placenta. To investigate this possibility, 200 placental samples from Zambian women were separated into HIV+ and HIV− groups and tested for HPV by redundant primer PCR, using GP5+/GP6+ and CPI/CPII primer sets. Three HPV genotypes (HPV6, 16 and 90) were detected in placental samples. Whereas, 20 different HPV genotypes were detected in vaginal sampling of the same patients, suggesting that compartment specific sub-populations of HPV may exist. The incidence of HPV16 in placental samples was almost 2-fold greater in HIV+ women compared to HIV− (p = …