Viral Messages Research Articles

Tpl2/Cot Signals Activate ERK, JNK, and NF-κB in a Cell-type and Stimulus-specific Manner

Macrophages and B-cells from Tpl2 knock-out mice exhibit a restricted defect in lipopolysaccharide and death receptor signaling that is limited to the activation of ERK. Here we show that Tpl2-/- MEFs exhibit defects in ERK, JNK, and NF-kappaB activation, or ERK activation only when stimulated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, respectively. In addition, we show that the activation of Tpl2 by TNF-alpha depends on signals transduced by both TRAF2 and RIP1. Activated Tpl2 phosphorylates MKK4/SEK1 upstream of JNK and stimulates NF-kappaB DNA binding and transcriptional activity by mechanisms that are independent of the nuclear translocation of p50 and p65. Tpl2-transduced TNF-alpha signals instead promote the phosphorylation of p65 at Ser276 and modulate the spectrum of proteins associated with p65. Phosphorylation stimulates the transcriptional activity of NF-kappaB but does not affect its ability to bind DNA, which may be affected by the composition of the nuclear NF-kappaB complexes. These data confirm that defects caused by a single mutation may be cell-type and signal-specific and delineate the role of Tpl2 in the transduction of TNF-alpha signals that activate JNK and NF-kappaB in MEFs.

Inhibition of Host and Viral Translation during Vesicular Stomatitis Virus Infection: eIF2 IS RESPONSIBLE FOR THE INHIBITION OF VIRAL BUT NOT HOST TRANSLATION

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.

Processing Internet Communications: A Motivation, Opportunity and Ability Framework

Despite the rapid growth of the Internet as a vehicle for communication and commerce, substantive theory to guide Web-based marketing communications is still in its infancy. Combining the distinctive characteristics of the Internet with recent models and research findings regarding information processing, this paper proposes a framework for understanding consumer response to Web-based communications. Consideration of Internet communication options (advertisements, Web sites, viral messaging), message characteristics (attention devices, encoding variability, framing, mood tone) and individual-difference moderators (involvement, cognitive/affective motivations, gender, context) leads to propositions regarding consumer motivation, opportunity and ability to process.

SR proteins and hnRNP H regulate the splicing of the HIV-1 tev-specific exon 6D.

A naturally arising point mutation in the env gene of HIV-1 activates the aberrant inclusion of the cryptic exon 6D into most viral messages, leading to inefficient viral replication. We set out to understand how a single nucleotide substitution could cause such a dramatic change in splicing. We have determined that the exon 6D mutation promotes binding of the SR protein SC35 to the exon. Mutant exon 6D sequences function as a splicing enhancer when inserted into an enhancer-dependent splicing construct. hnRNP H family proteins bind to the enhancer as well; their binding is dependent on the sequence GGGA located just downstream of the point mutation and depletion-- reconstitution studies show that hnRNP H is essential for enhancer activity. A polypurine sequence located further downstream in exon 6D binds SR proteins but acts as an exonic splicing silencer. hnRNP H is required for interaction of U1 snRNP with the enhancer, independent of the point mutation. We propose that SC35 binding to the point mutation region may convert the hnRNP H-U1 snRNP complex into a splicing enhancer.

Advances in HIV molecular biology

Advances in HIV molecular biology

Cellular changes induced by low-risk human papillomavirus type 11 in keratinocytes that stably maintain viral episomes.

Infections by low-risk papillomavirus types, such as human papillomavirus (HPV) type 6 (HPV-6) and HPV-11, induce benign genital warts that rarely progress to malignancy. In contrast, lesions induced by high-risk HPV types have the potential to progress to cancer. Considerable information is available concerning the pathogenesis of high-risk HPV types, but little is known about the life cycle of low-risk HPV types. Although functionally distinct, both high- and low-risk virus types infect keratinocytes and induce virion production upon differentiation. This information suggests that they may share common mechanisms for regulating their productive life cycles. Using tissue culture methods developed to study high-risk HPV types, we examined the ability of HPV-11 to be stably maintained as episomes following transfection of normal human keratinocytes with cloned viral DNA. HPV-11 genomes were found to be maintained in keratinocytes for extended passages in cultures in 14 independent experiments involving transfection of cloned HPV-11 DNA. Interestingly, the HPV-11-positive cells exhibited an extended life span that averaged approximately twofold longer than that of control neomycin-transfected cells. In organotypic cultures, HPV-11-positive cells exhibited altered differentiation patterns, but the extent of disruption was less severe than that seen with high-risk HPV types. In addition, the amplification of HPV-11 DNA, as well as the induction of several viral messages, was observed following differentiation of transfected cells in semisolid media. To determine whether global changes in cellular gene expression induced by HPV-11 were similar to those observed with high-risk HPV-31 (Y. E. Chang and L. A. Laimins, J. Virol. 74:4174-4182, 2000), microarray analysis of 7,075 expressed sequences was performed. A spectrum of cellular genes different from that previously reported for HPV-31 was found to be activated or repressed by HPV-11. The expression of only a small set of genes was similarly altered by both high- and low-risk HPV types. This result suggests that different classes of HPVs have distinct effects on global cellular transcription patterns during infection. The methods described allow for a genetic analysis of HPV-11 in the context of its differentiation-dependent life cycle.

Generation of a Ribozyme-Adenoviral Vector Against K-ras Mutant Human Lung Cancer Cells

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.

An Mpsi-containing heterologous RNA, but not env mRNA, is efficiently packaged into avian retroviral particles.

Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MPsi, is located upstream of the 5' splice site and therefore is present on both genomic and spliced RNAs. We now show that an MPsi-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MPsi. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MPsi is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3' end of MPsi was found to reduce the packaging efficiency of heterologous RNAs.

Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) evolved to stimulate the expression of intronless viral messages. To determine whether this ability to enhance expression could be useful in nonviral and heterologous viral gene delivery systems, we analyzed the ability of the WPRE to elevate the expression of a cDNA encoding the green fluorescent protein (GFP) in these contexts. We find that the WPRE can stimulate the expression of GFP when the gene is delivered by transfection or transduction with recombinant adeno-associated virus (AAV). Enhancement occurred both during transient expression and when the gene is stably incorporated into the genome of target cells. This enhancement required that the WPRE be located in cis within the GFP message, and was observed in both transformed cell lines and primary human fibroblasts. These results demonstrate that the WPRE will be an effective tool for increasing the long-term expression of transgenes in gene therapy.

The Hepatitis C Virus Internal Ribosome Entry Site Adopts an Ion-dependent Tertiary Fold

Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5′ untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.

The Ins and Outs of HIV Rev

The Rev protein of the human immunodeficiency virus mediates the nuclear export of the intron-containing viral messages. This export is a consequence of the continuous shuttling of HIV Rev between the nucleus and cytoplasm. This shuttling is mediated by a nuclear localization signal and a nuclear export signal contained within Rev. Recently, several factors which are required for the movement of Rev through the nuclear pore have been identified. This review will focus on these factors and their role the nucleocytoplasmic shuttling of HIV Rev.

Human immunodeficiency virus infection of dorsal root ganglion neurons detected by polymerase chain reaction in situ hybridization.

A predominantly sensory peripheral neuropathy is common with human immunodeficiency virus (HIV) infection, but the cause is unknown. Formalin-fixed dorsal root ganglia (DRG), obtained at postmortem from patients with neuropathy and HIV infection and from control subjects, were examined for the presence of HIV DNA by using polymerase chain reaction (PCR)-amplified in situ hybridization. Viral message RNA was detected using reverse transcription in situ PCR with gag-specific primers. HIV DNA and RNA sequences were detected in many satellite cells, mononuclear cells, and occasional neurons in 5 of 5 patients with HIV and neuropathy. HIV DNA was detected only in rare interstitial and satellite cells from 3 of 4 patients with HIV infection without neuropathy and was not detected in 6 patients without HIV infection. HIV infection of DRG neurons and supporting cells may contribute to the HIV-associated sensory neuropathy.

Role of Mason-Pfizer Monkey Virus (MPMV) Constitutive Transport Element (CTE) in the Propagation of MPMV Vectors by Genetic Complementation Using Homologous/HeterologousenvGenes

To study Mason-Pfizer monkey virus (MPMV) replication over a single round, virus particles were generated that contain a replication-defective vector encoding a dominant selectable marker, thehygromycin B phosphotransferase (hygr)gene. Genetic complementation with a homologous MPMV envelope glycoprotein (Env-gp) or pseudotyping by several heterologous Env-gps from a variety of viruses resulted in infectious MPMV particles containing the replication-defective RNA. Recently, it has been shown that human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) Rev and Rev-responsive element (RRE) functions can be substitutedin vitroby acis-acting sequence, the constitutive transport element (CTE), from simian type D retroviruses like MPMV and simian retrovirus type 1 (SRV-1). To determine whether CTE of MPMV is necessary for MPMV nucleic acid propagation, an MPMV vector that lacked the terminally located CTE was generated. Propagation of this vector was completely abrogated in the absence of CTE, showing the importance of CTE in MPMV replication. Insertion of CTE back into the MPMV genome in the sense orientation rescued replication to wild-type levels. Slot-blot analysis of nuclear versus cytoplasmic RNA fractions revealed that most of the messages were sequestered in the nucleus of cells transfected with the CTE(−) vectors and very little was transported to the cytoplasm. To test whether HIV-1 or SIV RREs could complement CTE function, the HIV-1 or SIV RREs were inserted in the CTE(−) vectors.transcomplementation of CTE(−)RRE(+) vectors with Env- and Rev- expression plasmids rescued propagation of the CTE(−) vectors. Computer analysis predicted an RNA secondary structure in MPMV CTE analogous to the HIV-1 and SIV RREs that could form three stable stem loops, the first of which contains a site similar to the Rev-binding domain in the HIV-1 RRE. The presence of a higher-order CTE structure was analyzed by mutational analysis. We conclude that CTE is important in the replication of MPMV and affects the nucleocytoplasmic transport and/or stability of viral messages similar to the Rev/RRE regulatory system of HIV-1 and SIV.

Rapid automated combined in situ hybridization and immunohistochemistry for sensitive detection of cytomegalovirus in paraffin-embedded tissue biopsies.

The authors questioned whether an automated kinetic mode assay of combined cytomegalovirus (CMV) late viral message and immediate and early antigens might result in a more sensitive and timely CMV diagnosis relevant to speedy treatment in the transplant setting. Toward this end, two cohorts were studied using automated in situ hybridization (ISH) for CMV as well as immunohistochemistry (IHC). The first cohort of patients consisted of 19 cases that were histologically positive (CMV-associated cytopathic change). A second cohort consisted of 10 cases that were histologically negative, yet culture positive. From the first cohort of histologically positive cases, 100% were positive by both ISH and IHC run on separate slides. In the second cohort, CMV was detected overall in 70% of cases (50% by ISH alone and 30% by IHC alone). These results indicate that a combined assay of ISH and IHC can detect more cases than routine hematoxylin and eosin staining or either assay alone. In two illustrative cases, used to demonstrate the feasibility of combining ISH and IHC, the authors used a combined two-color assay (ISH and IHC) performed sequentially on the same slide. The combined assays resulted in colocalized single cell message and protein in some cells and demonstrated more positive cells overall (some positive by IHC alone, some by ISH alone, and some by both) than either assay alone. The combined dual color assay can be completed within 4 to 5 hours giving the prospect of a same day result, which is faster than shell vial technique with immunofluorescence (24 to 48 hours) or culture (7 to 14 days). This study demonstrates that combining CMV message and protein assays results in a more sensitive assay and, when carried out in the kinetic mode, allows a speedy result relevant to early anti-CMV therapy.

High susceptibility of U937-derived subclones to human immunodeficiency virus type 1 infection correlates with accumulation of unintegrated circular viral DNA.

Our previous report showed that U937-derived subclones were differentiated into at least three types (high, middle, and low types), even in the subclones expressing similar levels of surface CD4, in terms of the kinetics of the appearance of viral antigens and virus production after infection with human immunodeficiency virus type 1 (HIV-1). Here we showed the evidence that high susceptibility to HIV-1 infection, which was confirmed by the profound expression of viral messages and antigens, was exclusively associated with a high number of the unintegrated extrachromosomal form of viral DNA, but not with the amounts of adsorbed virus RNA nor those of integrated DNA form. The difference in the amounts of extrachromosomal form of viral DNA was also observed in the culture with 3'-azido-3'-deoxythymidine (AZT), indicating that the susceptibility is essentially unrelated to multiple infection events. Thus, the susceptibility of U937-derived subclones to HIV-1 infection seems to be affected by the occurrence of specific events involved in the accumulation of unintegrated viral DNA after viral adsorption.

Unique insertion sequence and pattern of CD4 expression in variants selected with immunotoxins from human immunodeficiency virus type 1-infected T cells.

To study the variability of human immunodeficiency virus type 1 (HIV-1), we used immunotoxins to select for variants within a population of H9 cells persistently infected with a molecular clone of HIV-1 designated NL4-3. Chimeric immunotoxin CD4-PE40 (a chimeric fusion protein consisting of the amino-terminal two domains of CD4 and the carboxy-terminal domains of Pseudomonas exotoxin A) was used to select for cells lacking cell surface expression of HIV Env (envelope proteins gp160, gp120, and gp41). The cells described here (A1, A7, C9, and E9) fail to express HIV proteins because they have markedly diminished transcription of the integrated provirus (A1, A7, and E9) or no HIV provirus (C9). Analysis demonstrated that two different cloned variants, A1 and E9, contain the complementary sequence of tRNA(3Lys) (45 bp) inserted 3' to the primer-binding site, following by a 169-bp deletion through the start of the gag gene. No HIV mRNA was detected by Northern (RNA) blotting, but PCR demonstrated the presence of the viral message. These variants were found very infrequently in the unselected H9/NL4-3 cell population, and they contained proviruses distinct from that found in the dominant population. In addition, all of these variants had similar patterns of CD4 surface expression that allowed them to escape reinfection within the tissue culture. The data are discussed with regard to mechanisms and errors of HIV reverse transcription, as well as the evolution of mutants within a population of persistently infected cells.

Monocyte maturation controls expression of equine infectious anemia virus

In vivo, equine infectious anemia virus (EIAV) replicates in tissues rich in macrophages, and it is widely believed that the tissue macrophage is the principal, if not sole, cell within the host that replicates virus. No viral replication has been detected in circulating peripheral blood monocytes. However, proviral DNA can be detected in these cells, and monocytes may serve as a reservoir for the virus. In this study, an in vitro model was developed to clarify the role of monocyte maturation in regulating EIAV expression. Freshly isolated, nonadherent equine peripheral blood monocytes were infected with a macrophage-tropic strain of EIAV, and expression of EIAV was monitored in cells held as nonadherent monocytes and cells allowed to adhere and differentiate into macrophages. A 2- to 3-day delay in viral antigen expression was observed in the nonadherent cells. This restriction of viral expression in monocytes was supported by nuclear run-on studies demonstrating that on day 5 postinfection, the level of actively transcribed viral messages was 4.7-fold lower in monocyte cultures than in macrophage cultures. Electrophoretic mobility shift assays identified three regions of the U3 enhancer that interacted with nuclear extracts from normal equine macrophages. Each region contained the core binding motif of a family of transcription factors that includes the product of the proto-oncogene ets. Antibodies to the Ets family member PU.1 caused a supershifting of retarded bands in an electrophoretic mobility shift assay. Transfection studies of ets motif mutants demonstrated that the U3 ets sites were important in the regulation of EIAV transcription in macrophages. Interactions between the ets motif and nuclear extracts from freshly isolated, nonadherent monocytes, macrophages adherent for 1 or 2 days, or macrophages adherent for 5 days gave different patterns of retarded bands, although the binding specificities were similar with all three extracts. The different complexes formed by monocyte and macrophage nuclear extracts may explain the enhanced ability of mature macrophages to support EIAV expression.

Instability of Frog Virus 3 mRNA in Productively Infected Cells

Cloned DNA restriction fragments encoding representative frog virus 3 messages were used as probes to assess the stability of viral transcripts in infected fathead minnow cells. Analysis of Northern blot hybridization profiles confirmed earlier findings and revealed that in infected cells the steady-state level of representative frog virus 3 (FV3) messages increased throughout the replication cycle. However, when actinomycin D was added at 4 hr after infection to block the synthesis of new transcripts, viral messages were observed to turn over rapidly, with half-lives of approximately 2 hr. These results indicate that viral transcripts were not preferentially stabilized in FV3-infected cells and suggest that the high steady-state level of viral messages present at late times after infection was due to viral transcription outpacing message degradation. Moreover, the instability of viral messages challenges the suggestion that the terminal dyed symmetry (hairpin structure) observed in all frog Virus 3 messages sequenced to date plays a role in transcript stability.

Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex

N6-Methyladenosine is found at internal positions of mRNA in higher eukaryotes. This post-transcriptional modification occurs at a frequency of one to three methylation/average mRNA molecule in mammalian cell lines and is sequence-specific. A highly conserved consensus recognition site for the methyltransferase has been determined from both viral and cellular messages, consisting of the sequence Pu(G/A)AC(U/A) (with A being methylated). Despite the ubiquity and the specificity of this modification, little is known about the mechanism of formation of N6-methyladenosine. Utilizing an in vitro methylation system from HeLa cell nuclear extracts, and a substrate RNA derived from the mRNA coding for bovine prolactin, the mRNA N6-adenosine methyltransferase has been characterized and partially purified. Unique among other characterized nucleic acid methyltransferases, the enzyme is composed of three components which are separable under non-denaturing conditions. The molecular masses of the components are 30, 200, and 875 kDa as determined by gel filtration and glycerol gradient sedimentation. The 200-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit. The 875-kDa component has affinity for single-stranded DNA-agarose, suggesting that it may contain the mRNA-binding site. N6-Adenosine methyltransferase is not sensitive to treatment with micrococcal nuclease, nor to immunodepletion using an anti-trimethylguanosine antibody, suggesting that it does not contain an essential RNA component.

5′ sequence of vesicular stomatitis virus N-gene confers selective translation of mRNA

The infection of cells by vesicular stomatitis virus results in the rapid inhibition of host-cell protein synthesis, but not of viral protein synthesis. To determine if this translational selectivity might be conferred by the viral mRNA, we constructed a plasmid (pUCLNβ-4) containing the 5′ end of the viral nucleocapsid (N)-gene, including the ribosome binding site, fused in frame with the gene encoding β-galactosidase, and compared it to a control plasmid (pMC1924) containing the cellular rabbit beta-globin gene 5′ end fused with the β-galactosidase encoding gene. Both plasmids contained identical promoter and 3′ nontranslated regions and expressed similar levels of β-galactosidase in the indicator cell line 293. In cells transfected with either plasmid, viral infection resulted in a ∼70% decrease in protein synthesis by five hours. The level of β-galactosidase from cells transfected with pMC1924 also decreased concomitantly with the decrease in total protein synthesis. However, the level of β-galactosidase from cells transfected with pUCLNβ-4 was not affected by viral infection. Our data suggest that sequences in the 5′ end of the viral mRNA allow for the selective translation of the viral message in the presence of an inhibited translational machinery.