Browsing by Subject "Virus Replication"

Now showing 1 - 10 of 10
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    Biogenesis and molecular characteristics of serum hepatitis B virus RNA
    (Public Library of Science, 2020-10-20) Shen, Sheng; Xie, Zhanglian; Cai, Dawei; Yu, Xiaoyang; Zhang, Hu; Kim, Elena S.; Zhou, Bin; Hou, Jinlin; Zhang, Xiaoyong; Huang, Qi; Sun, Jian; Guo, Haitao; Medicine, School of Medicine
    HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
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    Characterization of the Termini of Cytoplasmic Hepatitis B Virus Deproteinated Relaxed Circular DNA
    (American Society for Microbiology, 2020-12-09) Cai, Dawei; Yan, Ran; Xu, Jerry Z.; Zhang, Hu; Shen, Sheng; Mitra, Bidisha; Marchetti, Alexander; Kim, Elena S.; Guo, Haitao; Microbiology and Immunology, School of Medicine
    The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5' end of the minus strand [(-)strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5'/3' rapid amplification of cDNA ends (RACE), 5' radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3' end of (-)strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3' end of plus strand [(+)strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5' terminus of (+)strand DNA with either a phosphate or hydroxyl group; and (iv) the 5' end of the DP-rcDNA (-)strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5' tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis.IMPORTANCE The covalently closed circular DNA (cccDNA) is the persistent form of the hepatitis B virus (HBV) genome in viral infection and an undisputed antiviral target for an HBV cure. HBV cccDNA is converted from viral genomic relaxed circular DNA (rcDNA) through a complex process that involves removing the covalently bound viral polymerase from rcDNA, which produces a deproteinated-rcDNA (DP-rcDNA) intermediate for cccDNA formation. In this study, we characterized the four termini of cytoplasmic DP-rcDNA and compared them to its rcDNA precursor. While rcDNA and DP-rcDNA have an identical 3' terminus of (-)strand DNA, the 3' terminus of (+)strand DNA on DP-rcDNA is further elongated. Furthermore, the peculiarities on rcDNA 5' termini, specifically the RNA primer on the (+)strand and the polymerase on the (-)strand, are absent from DP-rcDNA. Thus, our study provides new insights into a better understanding of HBV rcDNA deproteination and cccDNA biosynthesis.
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    The HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B
    (Public Library of Science, 2018-05-09) Carmichael, Jillian C.; Yokota, Hiroki; Craven, Rebecca C.; Schmitt, Anthony; Wills, John W.; Biomedical Engineering, School of Engineering and Technology
    All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1), direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor), we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B), and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4) blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease.
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    The Interferon-Inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion
    (American Society for Microbiology, 2015-09-15) Yan, Ran; Zhao, Xuesen; Cai, Dawei; Liu, Yuanjie; Block, Timothy M.; Guo, Ju-Tao; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Interferon alpha (IFN-α) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-α elicits a pleiotropic antiviral state in hepatitis B virus (HBV)-infected hepatocytes, but whether or not IFN-α impedes the late steps of the HBV life cycle, such as HBV secretion, remains elusive. Here we report that IFN-α treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon-stimulated gene(s) that is responsible for the reduction of HBV virion release, we found that tetherin, a broad-spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-α in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, GPI anchor-truncated tetherin exhibited a dominant-negative effect and was incorporated into the liberated virions. We also found colocalization of tetherin and HBV L protein at the intracellular multivesicular body, where the budding of HBV virions takes place. In line with this, electron microscopy demonstrated that HBV virions were tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knockdown of tetherin or overexpression of dominant negative tetherin attenuated the IFN-α-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-α inhibits HBV virion egress from hepatocytes through the induction of tetherin. IMPORTANCE: Tetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface with its membrane anchor domains. Here we report that interferon directly and selectively inhibits the secretion of HBV virions, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to liberated HBV virions. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virions occurs in the intracellular cisterna and that tetherin colocalizes with HBV virions on the multivesicular body, which is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.
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    Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA
    (PLOS, 2017-04-11) Liu, Yuanjie; Nie, Hui; Mao, Richeng; Mitra, Bidisha; Cai, Dawei; Yan, Ran; Guo, Ju-Tao; Block, Timothy M.; Mechti, Nadir; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.
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    Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication
    (Elsevier, 2015-04) Kanginakudru, Sriramana; DeSmet, Marsha; Thomas, Yanique; Morgan, Iain M.; Androphy, Elliot J.; Department of Dermatology, IU School of Medicine
    The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reduced viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number.
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    Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination
    (American Society for Microbiology (ASM), 2014-06) Byrd, Daniel; Shepherd, Nicole; Lan, Jie; Hu, Ningjie; Amet, Tohti; Yang, Kai; Desai, Mona; Yu, Qigui; Department of Microbiology & Immunology, IU School of Medicine
    Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serum-derived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-γ) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1β treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. IMPORTANCE Our results provide critical information to the burgeoning fields of cancer-killing (oncolytic) virus therapy with vaccinia virus (VACV). One type of macrophage (M2) is considered a common presence in tumors and is associated with poor prognosis. Our results demonstrate a preference for VACV replication in M2 macrophages and could assist in designing treatments and engineering poxviruses with special considerations for their effect on M2 macrophage-containing tumors. Additionally, this work highlights the importance of macrophages in the field of vaccine development using poxviruses as vectors. The understanding of the dynamics of poxvirus-infected foci is central in understanding the effectiveness of the immune response to poxvirus-mediated vaccine vectors. Monocytic cells have been found to be an important part of VACV skin lesions in mice in controlling the infection as well as mediating virus transport out of infected foci.
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