Morphology-Dependent HIV-Enhancing Effect of Semen-Derived Enhancer of Viral Infection

PAP248–286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248–286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248–286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors

The properties of constitutive promoters within adeno-associated viral (AAV) vectors have not yet been fully characterized. In this study, AAV vectors, in which enhanced GFP expression was directed by one of the six constitutive promoters (human β-actin, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40, and herpes simplex virus thymidine kinase), were constructed and introduced into the HCT116, DLD-1, HT-1080, and MCF-10A cell lines. Quantification of GFP signals in infected cells demonstrated that the CMV promoter produced the highest GFP expression in the six promoters and maintained relatively high GFP expression for up to eight weeks after infection of HCT116, DLD-1, and HT-1080. Exogenous human CDKN2A gene expression was also introduced into DLD-1 and MCF-10A in a similar pattern by using AAV vectors bearing the human β-actin and the CMV promoters. The six constitutive promoters were subsequently placed upstream of the neomycin resistance gene within AAV vectors, and HCT116, DLD-1, and HT-1080 were infected with the resulting vectors. Of the six promoters, the CMV promoter produced the largest number of G418-resistant colonies in all three cell lines. Because AAV vectors have been frequently used as a platform to construct targeting vectors that permit gene editing in human cell lines, we lastly infected the three cell lines with AAV-based targeting vectors against the human PIGA gene in which one of the six promoters regulate the neomycin resistance gene. This assay revealed that the CMV promoter led to the lowest PIGA gene targeting efficiency in the investigated promoters. These results provide a clue to the identification of constitutive promoters suitable to express exogenous genes with AAV vectors, as well as those helpful to conduct efficient gene targeting using AAV-based targeting vectors in human cell lines.     

Inferring Host Gene Subnetworks Involved in Viral Replication

Systematic, genome-wide loss-of-function experiments can be used to identify host factors that directly or indirectly facilitate or inhibit the replication of a virus in a host cell. We present an approach that combines an integer linear program and a diffusion kernel method to infer the pathways through which those host factors modulate viral replication. The inputs to the method are a set of viral phenotypes observed in single-host-gene mutants and a background network consisting of a variety of host intracellular interactions. The output is an ensemble of subnetworks that provides a consistent explanation for the measured phenotypes, predicts which unassayed host factors modulate the virus, and predicts which host factors are the most direct interfaces with the virus. We infer host-virus interaction subnetworks using data from experiments screening the yeast genome for genes modulating the replication of two RNA viruses. Because a gold-standard network is unavailable, we assess the predicted subnetworks using both computational and qualitative analyses. We conduct a cross-validation experiment in which we predict whether held-aside test genes have an effect on viral replication. Our approach is able to make high-confidence predictions more accurately than several baselines, and about as well as the best baseline, which does not infer mechanistic pathways. We also examine two kinds of predictions made by our method: which host factors are nearest to a direct interaction with a viral component, and which unassayed host genes are likely to be involved in viral replication. Multiple predictions are supported by recent independent experimental data, or are components or functional partners of confirmed relevant complexes or pathways. Integer program code, background network data, and inferred host-virus subnetworks are available at http://www.biostat.wisc.edu/~craven/chasman_host_virus/.     

病毒RNA如何逃避宿主细胞的降解

细胞RNA的降解机制不仅在基因表达调节方面具有重要作用,而且也是一种重要的病毒防御机制. 作为一种必须在细胞内增殖的微生物,病毒已经进化出了多种机制,以保护它们的RNA免被宿主细胞降解,如病毒RNA模拟宿主细胞mRNA的结构、形成磷脂包膜、形成局部二级结构、结合自己或宿主细胞编码的蛋白质和编码核酸酶增强宿主细胞mRNA降解等. 本文主要论述了病毒RNA逃避宿主细胞降解的方式,并对其应用前景进行了展望,尤其是在研发抗病毒药物方面的应用前景.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Aminoquinoline Surfen Inhibits the Action of SEVI (Semen-derived Enhancer of Viral Infection)

In semen, proteolytic peptide fragments from prostatic acid phosphatase can form amyloid fibrils termed SEVI (semen-derived enhancer of viral infection). These fibrils greatly enhance human immunodeficiency virus (HIV) infectivity by increasing the attachment of virions to target cells. Therefore, SEVI may have a significant impact on whether HIV is successfully transmitted during sexual contact. Here, we demonstrate that surfen, a small molecule heparan sulfate proteoglycan antagonist, inhibits both SEVI- and semen-mediated enhancement of HIV type 1 infection. Surfen interferes with the binding of SEVI to both target cells and HIV type 1 virions but does not deaggregate SEVI fibrils. Because SEVI can increase HIV infectivity by several orders of magnitude, supplementing current HIV microbicide candidates with SEVI inhibitors, such as surfen, might greatly increase their potency.     

病毒的侵染策略和植物的防卫反策略

病毒对植物的侵染及植物对病毒侵染的抵抗实际上是病毒与寄主植物之间相互作用的结果,病毒和寄主植物共同参与调控植物的亲和反应及防卫反应。     

Viral Host Jumps: Moving toward a Predictive Framework

In order to predict pathogen emergence, we must distinguish between emergence phenomena that occur via different processes. Focusing on the appearance of viral pathogens in new host species, I outline a framework that uses specific molecular characteristics to rank virus families by their expected a priori ability to complete each of three steps in the emergence process (encounter, infection, and propagation). I then discuss the degree to which the patterns expected, based solely on molecular-level structural characteristics, agree with observations regarding the ability of animal viruses to infect humans. This approach yields predictions consistent with empirical observations regarding the ability of specific viral families to infect novel host species but highlights the need for consideration of other factors, such as the ecology of host interactions and the determinants of cellular susceptibility and permissivity to specific virus groups, when trying to predict the frequency with which a virus will encounter a novel host species or the probability of propagation within a novel host species once infection has occurred.     

Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2α phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2α by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2α phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2α phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5′ untranslated region (5′UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5′UTR and IGR IRES.For productive viral protein expression, viruses have to compete for and hijack the host translational machinery (45). Some viruses such as poliovirus, vesicular stomatitis virus (VSV), and influenza virus selectively antagonize the translation apparatus to shut off host translation, resulting in the release of ribosomes from host mRNAs and the inhibition of antiviral responses. On the other hand, the host cell can counteract through antiviral mechanisms to shutdown viral translation. For instance, viral RNA replication intermediates can trigger PKR, leading to an inhibition of overall translation. To bypass the block in translation, viruses have evolved unique mechanisms to preferentially recruit the ribosome for viral protein synthesis. Thus, the control of the translational machinery during infection is a major focal point in the battle between the host and the virus and often, elucidation of these viral translational shutoff strategies reveals key targets of translational regulation.The majority of cellular mRNAs initiate translation through the recruitment of the cap-binding complex, eukaryotic translation initiation factor 4F (eIF4F), to the 5′ cap of the mRNA (56). eIF4F consists of the cap-binding protein eIF4E, the RNA helicase, eIF4A, and the adaptor protein eIF4G. eIF4G acts as a bridge to join eIF4E and the 40S subunit via eIF3. With the ternary eIF2-Met-tRNA_i-GTP complex bound, the 40S subunit scans in a 5′-to-3′ direction until an AUG start codon is encountered. Here, eIF5 mediates GTP hydrolysis on the ternary complex, releasing the eIFs and subsequently leading to 60S subunit joining to assemble an elongation-competent 80S ribosome. The ternary eIF2-Met-tRNA_i-GTP complex is reactivated for another round of translation by exchange of GDP for GTP, which is mediated by the guanine nucleotide exchange factor, eIF2B. The 3′ poly(A) tail of the mRNA also stimulates translational initiation by binding to the poly(A) binding protein (PABP), which in turn interacts with eIF4G at the 5′end, resulting in a circularized mRNA. PABP has been proposed to enhance eIF4E affinity for the 5′cap and promote 60S joining, indicating that PABP functions at multiple steps of translational initiation (33).A common tactic viruses use to inhibit host translation is to selectively target eIFs. One of the best studied is the cleavage of eIF4G by viral proteases during picornavirus infection. In humans, two isoforms, eIF4GI and eIF4GII, are cleaved early in poliovirus infection by the viral protease 2A, where cleavage of eIFGII correlates more precisely with host translation shutoff (20). Cleavage of eIF4G produces an amino-terminal fragment that binds to eIF4E and a C-terminal fragment that binds to eIF4A and eIF3 (26, 39, 42). PABP is also cleaved by the viral protease 3C during poliovirus infection, thus contributing to shutoff of both host and viral translation and thereby enabling the switch from viral translation to replication (3, 31, 38). Another major target is the availability of the cap-binding protein eIF4E, which is regulated by binding to the repressor protein 4E-BP (21, 41). 4E-BP and eIF4G compete for an overlapping site on eIF4E (42). In its hypophosphorylated state, 4E-BP binds to and sequesters eIF4E, preventing eIF4G recruitment. Dephosphorylation and activation of 4E-BP has been observed during poliovirus, encephalomyocarditis (EMCV), and VSV infections (7, 18).During virus infection, host antiviral responses are triggered that also inhibit translation to counteract viral protein synthesis. An integral antiviral response is phosphorylation at Ser⁵¹ of eIF2α, which reduces the pool of the ternary complex by blocking the eIF2B-dependent exchange of GDP to GTP. In mammals, four known eIF2α kinases exist including the endoplasmic reticulum (ER)-stress-inducible PERK, GCN2, which senses the accumulation of deacylated tRNAs during amino acid starvation conditions; the heme-regulated kinase HRI; and the interferon-inducible double-stranded RNA-binding PKR (64). In mammalian cells, PKR is activated by binding to double-stranded viral RNA replication intermediates, leading to eIF2α phosphorylation and inhibition of overall host and viral translation. PERK and GCN2 have also been shown to be activated during virus infections by VSV and members of the alphavirus family (2, 6, 43, 65, 79). Often, viruses rely on the ER for synthesis and proper folding of viral proteins. The large burden on the ER activates PERK to phosphorylate eIF2α, thereby inhibiting global protein synthesis to reduce the load on the ER (23). Some viruses such as HCV and herpes simplex viruses have adapted to responses that induce eIF2α phosphorylation by producing viral proteins that counteract PKR or modulate the ER stress response (27, 76). Thus, virus infection can trigger several eIF2α kinases that lead to translational shutoff to counteract viral protein synthesis.To circumvent these translation blocks, viruses such as poliovirus and hepatitis C virus utilize internal ribosome entry sites (IRES), which are RNA elements that directly recruit ribosomes in a cap-independent manner and require only a subset of canonical eIFs (15, 25). It is generally thought that IRES-containing viral mRNAs can be translated under conditions when specific eIFs are compromised during infection. Except for a few cases, the specific mechanisms and factors that lead to IRES stimulation is poorly understood. For example, poliovirus and the related EMCV possess an IRES that allows viral translation despite cleavage of eIF4G during infection or inhibiting eIF4E by 4E-BP binding. This type of IRES can still bind to the central domain of eIF4G and mediate 40S subunit recruitment (11, 37, 57).One of the most unique and simplest IRES is found within the intergenic region (IGR) of the Dicistroviridae family (for extensive reviews, see references 28, 36, and 49). Members of this family include the cricket paralysis virus (CrPV), drosophila C virus (DCV), taura syndrome virus, the Plautia stali intestine virus (PSIV), the Rhopalosiphum padi virus (RhPV), and several bee viruses such as the black queen cell virus and the Israeli acute paralysis virus, which has been recently linked to colony collapse disorder (10). The dicistroviruses encode a positive-strand 8- to 10-kb single-stranded RNA genome, which contains two main open reading frames, ORF1 and ORF2, encoding the nonstructural and structural proteins, respectively, separated by an IGR (see Fig. ​Fig.1A).1A). The 5′ end of the CrPV RNA is linked to the viral protein VpG and the 3′ end contains a poly(A) tail (16). Radiolabeling of intracellular RNA in infected cells reveals no subgenomic RNA species smaller than the full-length genomic RNA, and this has been supported by Northern blot analysis (16, 81). Translation of ORF2 is directed by the IGR IRES, whereas ORF1 expression is mediated by an IRES within the 5′ untranslated region (5′UTR) (35, 67, 81, 82). Remarkably, the IGR IRES element can directly recruit the ribosome independently of eIFs or the initiator Met-tRNA_i (29, 30, 54, 80). Furthermore, the IRES occupies the P-site of the ribosome to initiate translation from the ribosomal A-site encoding non-AUG codon (35, 81). Extensive biochemical and structural analyses from several groups have revealed that the IGR IRES mimics a tRNA that occupies the mRNA cleft of the ribosome and sets the ribosome into an elongation state (9, 29, 30, 34, 51, 55, 58, 68, 72, 83). Using reporter constructs, it has also been demonstrated that CrPV IGR IRES-mediated translation is active under a number of cellular conditions when the activity of the ternary complex eIF2-Met-tRNA_i-GTP is compromised (17, 63, 78, 80). Because IGR IRES-mediated translation does not require initiation factors, the IRES can direct translation under a number of cellular conditions when the activity of multiple eIFs is compromised (12). Although the majority of studies have focused on the IGR IRES of CrPV, PSIV, and TSV, it is predicted that the IGRs within this viral family all function similarly based on the predicted conserved RNA structures (28, 36, 49). In contrast, only the 5′UTR IRES mechanism of RhPV has been studied in detail (77). Despite the wealth of studies on the mechanics of these IRES, the mechanisms that lead to translational shutoff during dicistrovirus infection and the interaction of dicistrovirus with the host machinery to allow virus production have been relatively unexplored.Open in a separate windowFIG. 1.Kinetics of host protein synthesis and viral protein expression in CrPV-infected Drosophila S2 cells. (A) Genomic arrangement of the CrPV RNA. The viral open reading frames, ORF1 and ORF2, that encode nonstructural (NS) and structural (S) proteins, respectively, are shown, which are separated by the intergenic internal ribosome entry site (IGR IRES). Translation of ORF1 and ORF2 is directed by the 5′UTR IRES and the IGR IRES, respectively. The first amino acid of ORF2 directed by the IGR IRES is encoded by a GCU alanine codon. (B) Autoradiography of protein lysates resolved on a SDS-12% PAGE gel. The protein lysates were collected from S2 cells that were untreated (U), mock infected (M), CrPV infected (5 FFU/cell), or thapsigargin treated (Tg; 0.4 μM) for the indicated times (h p.i.) and metabolically labeled with [³⁵S]methionine for 30 min at each time point. The migration of proteins with known molecular masses is shown on the left. The expression of detectable nonstructural (NS) and structural (S) proteins is denoted. (C) Quantitation of host protein synthesis during CrPV infection. To calculate the host translation at each time point, the amount of radioactivity of the bands between 55 and 70 kDa in panel A was quantitated by using ImageQuant, and the percent translation was calculated at each time point of virus infection or thapsigargin treatment compared to the mock infection. Shown are averages (± the standard deviation) from at least three independent experiments. (D) Immunoblots of viral ORF1 and ORF2 during CrPV infection at various times postinfection (h p.i.). Antibodies were raised against peptides within ORF1 and ORF2. The expression of ORF1 and ORF2 was quantitated by a LI-COR Odyssey system, plotted against time of infection, and normalized to the amount of ORF1 or ORF2 expression at 6 h p.i. (100%). As a comparison, viral RNA synthesis as detected by Northern blot analysis (see Fig. ​Fig.2B)2B) is plotted on the same graph.Previous studies have shown that the CrPV and the related DCV can infect a wide range of insect hosts, including the Drosophila melanogaster S2 cell line (60, 69). In the present study, we have explored how CrPV infection leads to host translational shutoff in S2 cells. Two steps of translational initiation are targeted during CrPV infection. First, the interaction of deIF4G with deIF4E is disrupted early in infection and remains dissociated during the course of infection. Second, deIF2α is phosphorylated at a time that lags after the initial host translational shutoff during infection. Premature phosphorylation of deIF2α early in infection inhibited translation directed by the 5′UTR IRES, but IGR IRES-mediated translation remained relatively resistant. These results support the model that multiple mechanisms, including impairment of deIF4F complex formation and induction of deIF2α phosphorylation, contribute to the host translational shutoff during CrPV infection. The inhibition of host translation and the release of ribosomes from host mRNAs ensures that translation mediated by the 5′UTR and IGR IRES is optimal to produce sufficient viral nonstructural and structural proteins for proper CrPV maturation and assembly.     

Patterns of Oligonucleotide Sequences in Viral and Host Cell RNA Identify Mediators of the Host Innate Immune System

The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context - which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans - is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.