Viral mRNA Transcription Research Articles

PTD-fused p53 as a potential antiviral agent directly suppresses HBV transcription and expression

In Hepatitis B virus (HBV) infection, the virus generates numerous viral mRNAs/proteins and viral loads, which plays a major role in driving T cell tolerance, viral persistence, and hepatocellular carcinoma. However, currently available anti-HBV agents have no direct effect on viral mRNA transcription and protein expression. In this study, we designed a recombinant fusion of p53 protein with the cell-penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p53 internalization into hepatocytes. PTD-p53 effectively suppressed HBV transcription and antigen expression by interaction with viral enhancers. We further provide evidence that PTD-p53 counteracts the viral transcription feedback loop and effectively suppressed HBV production of viral mRNAs, as well as HBsAg, HBeAg, and HBcAg, both in vitro and in vivo. Our results thereby provide a basis for developing a new therapeutic approach against HBV infection.

Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

Inhibition of PA endonuclease activity of influenza virus RNA polymerase by Kampo medicines.

To find a novel influenza inhibitor targeting the endonuclease activity of influenza A virus polymerase acidic protein (PA), which is essential for the acquisition of primers for viral mRNA transcription, seven Kampo extracts were tested in vitro for their ability to inhibit endonuclease activity of the recombinant PA protein that was expressed and purified from Escherichia coli. The Kampo medicines Kakkonto, Shosaikoto, Saikokeishito, Keishito, Maobushisaishinto, and Maoto, but not Chikujountanto, inhibited PA endonuclease activity in a dose-dependent manner. Our results indicate that Kampo medicines are good sources providing a structural lead for optimization of an influenza endonuclease inhibitor.

The C-type Lectin Langerin Functions as a Receptor for Attachment and Infectious Entry of Influenza A Virus.

It is well established that influenza A virus (IAV) attachment to and infection of epithelial cells is dependent on sialic acid (SIA) at the cell surface, although the specific receptors that mediate IAV entry have not been defined and multiple receptors may exist. Lec2 Chinese hamster ovary (CHO) cells are SIA deficient and resistant to IAV infection. Here we demonstrate that the expression of the C-type lectin receptor langerin in Lec2 cells (Lec2-Lg) rendered them permissive to IAV infection, as measured by replication of the viral genome, transcription of viral mRNA, and synthesis of viral proteins. Unlike SIA-dependent infection of parental CHO cells, IAV attachment and infection of Lec2-Lg cells was mediated via lectin-mediated recognition of mannose-rich glycans expressed by the viral hemagglutinin glycoprotein. Lec2 cells expressing endocytosis-defective langerin bound IAV efficiently but remained resistant to IAV infection, confirming that internalization via langerin was essential for infectious entry. Langerin-mediated infection of Lec2-Lg cells was pH and dynamin dependent, occurred via clathrin- and caveolin-mediated endocytic pathways, and utilized early (Rab5(+)) but not late (Rab7(+)) endosomes. This study is the first to demonstrate that langerin represents an authentic receptor that binds and internalizes IAV to facilitate infection. Moreover, it describes a unique experimental system to probe specific pathways and compartments involved in infectious entry following recognition of IAV by a single cell surface receptor. On the surface of host cells, sialic acid (SIA) functions as the major attachment factor for influenza A viruses (IAV). However, few studies have identified specific transmembrane receptors that bind and internalize IAV to facilitate infection. Here we identify human langerin as a transmembrane glycoprotein that can act as an attachment factor and a bone fide endocytic receptor for IAV infection. Expression of langerin by an SIA-deficient cell line resistant to IAV rendered cells permissive to infection. As langerin represented the sole receptor for IAV infection in this system, we have defined the pathways and compartments involved in infectious entry of IAV into cells following recognition by langerin.

Exosomes derived from HTLV-1 infected cells contain viral proteins and mRNA

The HTLV-1 transactivator protein Tax controls many critical cellular pathways including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Recently, exosomes have been shown to play critical roles during pathogenic viral infections as delivery vehicles for host and viral components including proteins, mRNA and miRNA. We hypothesized that exosomes derived from HTLV-1 infected cells contain unique host and viral proteins that may contribute to pathogenesis. We have characterized exosomes released from uninfected and HTLV-1 infected cell lines, as well as ATL and HAM/TSP material. The functional impact of exosomes derived from HTLV-1 infected cells on naive recipient cells was evaluated by utilizing transcription and reactive oxygen species (ROS) assays. Exosomes from HTLV-1 infected cells displayed unique proteomic profiles distinct from exosomes derived from uninfected cells. For instance, proinflammatory mediators are contained within the exosomes, as well as viral mRNA transcripts including Tax, HBZ, and Env. We found that exosomes from infected cells deliver functional Tax to naA¯ve recipient cells as well as cytokines. The release of factors was through Calcium and Calcium channels. We observed that exosomes released from HTLV-1 infected, Tax-expressing cells contributed to enhanced survival of target cells treated with α-FAS. Two other critical proteins were found in these exosomes that could contribute to overall activation of neighboring cells including Tax1BP1 and PrP. Both proteins are critical for activation of NFkB patway in recipient cells. Collectively, our results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. Furthermore, exosomes derived from infected cells are capable of inducing an ROS response in naA¯ve cells and contribute to an anti-apoptotic phenotype in cells treated with α-FAS.

Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5′-3′ mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial viral endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases. In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1. Collectively, these results indicate that mRNA decay rates impact transcription and that gamma-herpesviruses use this feedback mechanism to facilitate viral gene expression.

Molecular characterization of full-length Tat in HIV-1 subtypes B and C.

HIV-1Tat (trans-acting activator of transcription) plays essential roles in the replication through viral mRNA and genome transcription from the HIV-1 LTR promoter. However, Tat undergoes continuous amino acid substitutions. As a consequence, the virus escapes from host immunity indicating that genetic diversity of Tat protein in major HIV-1 subtypes is required to be continuously monitored. We analyzed available full-length HIV-1 sequences of subtypes B (n=493) and C (n=280) strains circulating worldwide. We observed 81% and 84% nucleotide sequence identities of HIV-1 Tat for subtypes B and C, respectively. Based on phylogenetic and mutation analyses, global diversity of subtype B was apparently higher compared to that of subtype C. Positively selected sites, such as positions Ser68 and Ser70 in both subtypes, were located in the Tat-transactivation responsive RNA (TAR) interaction domain. We also found positively selected sites in exon 2, such as positions Ser75, Pro77, Asp80, Pro81 and Ser87 for both subtypes. Our study provides useful information on the full-length HIV-1 Tat sequences in globally circulating strains.

Human papillomavirus does not have a causal role in colorectal carcinogenesis.

To investigate the presence of human papillomavirus (HPV) DNA along with the integration, the quantification and the expression of the HPV16 in colorectal cancers. A prospective series of colorectal tumors were genotyped for HPV DNA. The clinical and pathological variables of the HPV-positive tumors were compared to those of HPV-negative samples. The integration status of HPV16 was evaluated by calculating E2/E6 ng ratios. HPV16-positive tumors were also evaluated for (1) E2, E4, E5, E6 and E7 viral gene ng quantification; (2) relative quantification compared to W12 cells; and (3) viral E2, E4, E5, E6 and E7 mRNA transcripts by real-time polymerase chain reaction. HPV infection was detected in 16.9% of all tumors examined, and HPV16 was the most frequent type detected (63.6% of positive tissues). Notably, the clinical and pathological features of HPV-positive colorectal cancers were not significantly different than those of HPV-negative cancers (χ (2) and t-test for all clinical and pathological features of HPV-positive vs HPV-negative colorectal cancers: p ns). HPV16 DNA was present exclusively in episomal form, and the HPV16 E2, E4, E5, E6 and E7 genes were detected in trace nanogram quantities. Furthermore, the HPV16 genes ranged from 10(-3) to 10(-9) compared to W12 cells at an episomal stage. Although the extractions were validated by housekeeping gene expression, all the HPV16 positive tissues were transcriptionally inactive for the E2, E4, E5, E6 and E7 mRNAs. Based on our results, HPV is unlikely involved in colorectal carcinogenesis.

Promoter Targeting RNAs: Unexpected Contributors to the Control of HIV-1 Transcription

In spite of prolonged and intensive treatment with combined antiretroviral therapy (cART), which efficiently suppresses plasma viremia, the integrated provirus of HIV-1 persists in resting memory CD4+ T cells as latent infection. Treatment with cART does not substantially reduce the burden of latent infection. Once cART is ceased, HIV-1 replication recrudesces from these reservoirs in the overwhelming majority of patients. There is increasing evidence supporting a role for noncoding RNAs (ncRNA), including microRNAs (miRNAs), antisense (as)RNAs, and short interfering (si)RNA in the regulation of HIV-1 transcription. This appears to be mediated by interaction with the HIV-1 promoter region. Viral miRNAs have the potential to act as positive or negative regulators of HIV transcription. Moreover, inhibition of virally encoded long-asRNA can induce positive transcriptional regulation, while antisense strands of siRNA targeting the NF-κB region suppress viral transcription. An in-depth understanding of the interaction between ncRNAs and the HIV-1 U3 promoter region may lead to new approaches for the control of HIV reservoirs. This review focuses on promoter associated ncRNAs, with particular emphasis on their role in determining whether HIV-1 establishes active or latent infection.

The N terminus of the influenza B virus nucleoprotein is essential for virus viability, nuclear localization, and optimal transcription and replication of the viral genome.

The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However, NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50-amino-acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension; however, this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. The truncation of more than three amino acids prevented virus recovery, suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in the nuclear localization of BNP, with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first 10 residues of BNP demonstrated few differences in nuclear localization; however, the viruses exhibited significant reductions in viral mRNA transcription and genome replication, resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall, our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP but also for regulating viral mRNA transcription and genome replication. The multifunctional NP of influenza viruses has roles throughout the viral replication cycle; therefore, it is essential for virus viability. Despite high levels of homology between the NP of influenza A and B viruses, the NP of influenza B virus contains an evolutionarily conserved 50-amino-acid N-terminal extension that is absent from the NP of influenza A viruses. In this study, we show that this N-terminal extension is essential for virus viability, and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore, we demonstrate for the first time that the N terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension, these viruses may have evolved separate mechanisms to regulate these processes.

Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells.

Inhibition of Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication by HIV-1 Nef and Cellular MicroRNA hsa-miR-1258

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to several AIDS-related malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. The interaction of human immunodeficiency virus type 1 (HIV-1) and KSHV has a central role in promoting the aggressive manifestations of AIDS-KS. We have previously shown that negative factor (Nef), a secreted HIV-1 protein, synergizes with KSHV viral interleukin-6 (vIL-6) to promote angiogenesis and tumorigenesis by activating the AKT pathway (X. Zhu, et al., Oncogene, 22 April 2013, http://dx.doi.org/10.1038/onc.2013.136). Here, we further demonstrated the role of soluble and ectopic Nef in the regulation of KSHV latency. We found that both soluble Nef protein and ectopic expression of Nef by transfection suppressed the expression of KSHV viral lytic mRNA transcripts and proteins and the production of infectious viral particles. MicroRNA (miRNA) microarray analysis identified a number of Nef-regulated miRNAs. Bioinformatics and luciferase reporter analyses showed that one of the Nef-upregulated miRNAs, cellular miRNA 1258 (hsa-miR-1258), directly targeted a seed sequence in the 3' untranslated region (UTR) of the mRNA encoding the major lytic switch protein (RTA), which controls KSHV reactivation from latency. Ectopic expression of hsa-miR-1258 impaired RTA synthesis and enhanced Nef-mediated inhibition of KSHV replication, whereas repression of hsa-miR-1258 has the opposite effect. Mutation of the seed sequence in the RTA 3'UTR abolished downregulation of RTA by hsa-miR-1258. Collectively, these novel findings demonstrate that, by regulating cellular miRNA, Nef may inhibit KSHV replication to promote viral latency and contribute to the pathogenesis of AIDS-related malignancies. This study found that Nef, a secreted HIV-1 protein, suppressed KSHV lytic replication to promote KSHV latency. Mechanistic studies indicated that a Nef-upregulated cellular miRNA, hsa-miR-1258, inhibits KSHV replication by directly targeting a seed sequence in the KSHV RTA 3'UTR. These results illustrate that, in addition to viral miRNAs, cellular miRNAs also play an important role in regulating the life cycle of KSHV. Overall, this is the first study to report the involvement of Nef in KSHV latency, implying its likely important role in the pathogenesis of AIDS-related malignancies.

Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions.

Comparative Structural and Functional Analysis of Orthomyxovirus Polymerase Cap-Snatching Domains

Orthomyxovirus Influenza A virus (IAV) heterotrimeric polymerase performs transcription of viral mRNAs by cap-snatching, which involves generation of capped primers by host pre-mRNA binding via the PB2 subunit cap-binding site and cleavage 10–13 nucleotides from the 5′ cap by the PA subunit endonuclease. Thogotoviruses, tick-borne orthomyxoviruses that includes Thogoto (THOV), Dhori (DHOV) and Jos (JOSV) viruses, are thought to perform cap-snatching by cleaving directly after the cap and thus have no heterogeneous, host-derived sequences at the 5′ extremity of their mRNAs. Based on recent work identifying the cap-binding and endonuclease domains in IAV polymerase, we determined the crystal structures of two THOV PB2 domains, the putative cap-binding and the so-called ‘627-domain’, and the structures of the putative endonuclease domains (PA-Nter) of THOV and DHOV. Despite low sequence similarity, corresponding domains have the same fold confirming the overall architectural similarity of orthomyxovirus polymerases. However the putative Thogotovirus cap-snatching domains in PA and PB2 have non-conservative substitutions of key active site residues. Biochemical analysis confirms that, unlike the IAV domains, the THOV and DHOV PA-Nter domains do not bind divalent cations and have no endonuclease activity and the THOV central PB2 domain does not bind cap analogues. On the other hand, sequence analysis suggests that other, non-influenza, orthomyxoviruses, such as salmon anemia virus (isavirus) and Quaranfil virus likely conserve active cap-snatching domains correlating with the reported occurrence of heterogeneous, host-derived sequences at the 5′ end of the mRNAs of these viruses. These results highlight the unusual nature of transcription initiation by Thogotoviruses.

Improved dual promotor-driven reverse genetics system for influenza viruses

Reverse genetic systems for influenza A virus (IAV) allow the generation of genetically manipulated infectious virus from a set of transfected plasmid DNAs encoding the eight genomic viral RNA segments (vRNA). For this purpose, cDNAs representing these eight vRNA segments are cloned into specific plasmid vectors that allow the generation of vRNA-like transcripts using polymerase I (Pol I). In addition, these plasmids support the transcription of viral mRNA by polymerase II (Pol II), leading to the expression of viral protein(s) encoded by the respective transcripts. In an effort to develop this system further, we constructed the bi-directional vector pMPccdB. It is based on pHW2000 (Hoffmann et al., 2000b) but contains additionally (i) the ccdB gene whose expression is lethal for most Escherichia coli strains and therefore used as a negative selection marker and (ii) more efficient AarI cloning sites that flank the ccdB gene on either side. Furthermore, we used a modified one-step restriction/ligation protocol to insert the desired cDNA into the respective pMPccdB vector DNA. Both the use of a negative selection marker and an improved cloning protocol were shown to facilitate the generation of genetically engineered IAV as illustrated in this study by the cloning and rescue of the 2009 pandemic isolate A/Giessen/6/2009 (Gi-H1N1).

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

The human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene is unusual as it contains an editing site where nontemplated ribonucleotide residues can be inserted. This RNA editing can lead to the expression of the viral P, PD, putative W, and theoretical V protein from a single gene. Although the HPIV3 PD protein has been detected, its function and those of the W and V proteins are poorly understood. Therefore, we first used reverse genetics techniques to construct and rescue a recombinant (r)HPIV3 clone with a polyhistidine sequence at the 5' end of the P gene for tagged protein detection. Western blot analysis demonstrated the presence of the P, PD, and W proteins, but no V protein was detected. Then, we functionally studied the D domain of the PD protein by constructing two rHPIV3 knockout clones that are deficient in the expression of the D domain. Results from growth kinetic studies with infected MA-104 and A596 cells showed that viral replication of the two knockout viruses (rHPIV3-ΔES and rHPIV3-ΔD) was comparable to that of the parental virus in both cell lines. However, viral mRNA transcription and genomic replication was significantly reduced. Furthermore, cytokine/chemokine profiles of A549 cells infected with either knockout virus were unchanged or showed lower levels compared to those from cells infected with the parental virus. These data suggest that the D domain of the PD protein may play a luxury role in HPIV3 RNA synthesis and may also be involved in disrupting the expression of beta interferon.

ZASC1 knockout mice exhibit an early bone marrow-specific defect in murine leukemia virus replication

BackgroundZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010).MethodsTo determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies.ResultsMice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection.ConclusionsThese studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.

Distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev function

RNA helicase plays an important role in host mRNA and viral mRNA transcription, transport, and translation. Many viruses utilize RNA helicases in their life cycle, while human immunodeficiency virus type 1 (HIV-1) does not encode an RNA helicase. Thus, host RNA helicase has been involved in HIV-1 replication. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether distinct DDX RNA helicases cross-talk and cooperate to modulate the HIV-1 Rev function. In this study, we noticed that distinct DDX RNA helicases, including DDX1, DDX3, DDX5, DDX17, DDX21, DDX56, except DDX6, bound to the Rev protein and they colocalized with Rev in nucleolus or nucleus. In this context, these DEAD-box RNA helicases except DDX6 markedly enhanced the HIV-1 Rev-dependent RNA export. Furthermore, DDX3 interacted with DDX5 and synergistically enhanced the Rev function. As well, combination of other distinct DDX RNA helicases cooperated to stimulate the Rev function. Altogether, these results suggest that distinct DDX DEAD-box RNA helicases cooperate to modulate the HIV-1 Rev function.

Interaction of cellular poly(C)-binding protein 2 with nonstructural protein 1β is beneficial to Chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication

Non-structural protein1β (Nsp1β) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be involved in suppressing the host innate immune response and mediating viral subgenomic mRNA transcription. In the present study, we have analyzed the interaction of Nsp1β of Chinese highly pathogenic PRRSV (HP-PRRSV) with cellular poly(C)-binding 2 (PCBP2) by means of the yeast two-hybrid screening in a pulmonary alveolar macrophages (PAMs) cDNA library and co-immunoprecipitation (Co-IP) assay. Our results indicated that the Nsp1β of the HP-PRRSV is able to bind and interact with cellular PCBP2 strongly in both the infected cells and plasmid transfected cells. Their minimal binding regions were identified to be the residues 85–203 aa (PCPβ and CTE domains) for the Nsp1β and the residues 96–168 aa (KH2 domain) for PCBP2, respectively. Next, we used confocal immunofluorescence analysis and discovered that, during PRRSV infection in MARC-145 cells and/or pasmid-transfected cells, the Nsp1β and PCBP2 mainly colocalized in the cytoplasm and perinuclear pattern. Moreover, the siRNA-mediated silencing of PCBP2 gene in the MARC-145 cells resulted in significant reduction of the virus titer in supernatants as well as viral proteins, while no significant effects on the expression of the type I interferon α and interferon β, suggesting that the interaction of the Nsp1β with cellular PCBP2 is beneficial to Chinese HP-PRRSV replication in MARC-145 cells.

Antiviral activity of salivary microRNAs for ophthalmic herpes zoster

Ophthalmic herpes zoster is a common ocular infection caused by the varicella-zoster virus (VZV). Viral mRNA transcripts play a major role in the replicative cycle of the virus and current antiviral agents have little effect in preventing and treating the complications. Therapeutic use of saliva for certain painful ocular diseases such as ophthalmic herpes zoster is a well-known public practice in our region. We thought that antiviral activity of saliva may stem from salivary microvesicles and we aimed to look for molecules with antiviral activity in these vesicles. As a possible candidate for antiviral activity, salivary microvesicles contain at least 20 microRNAs (miRNAs), small noncoding RNAs, which suppress the translation of target mRNAs. miRNAs not only participate in maintenance of normal cell functions, but are also involved in host–virus interactions and limit the replication of certain virus types. Thus, miRNA gene therapy by targeting mRNAs required for VZV survival may find a niche in the treatment of ophthalmic herpes zoster. But, how could salivary microvesicles reach into the corneal cells to demonstrate their antiviral activity. We suggest that human salivary microvesicles can be effective carriers of miRNA for corneal cells, because they contain a molecular machinery for vesicle trafficking and fusion allowing them to be endocytosed by target cells. After binding to the plasma membrane, microvesicles seem to enter into the corneal cells through the clathrin-mediated endocytosis. In the cytosol, human salivary miRNAs base-pair with specific viral mRNAs and inhibit their translation, thus limiting the replication of the virus.