Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5' Caps.

Eukaryotic RNAs are heavily processed, including co- and post-transcriptional formation of various 5' caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7m G cap is hypermethylated at the N2 -position, whereas in higher eukaryotes and viruses 2'-O-methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid-phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site-specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di-and trimethylated caps, as well as RNAs with caps methylated at the 2'-O-position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.

Involvement of the cellular phosphatase DUSP1 in vaccinia virus infection.

Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection.

Integrin ß1 mediates vaccinia virus entry through activation of PI3K/Akt signaling.

Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin ß1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin ß1 in lipid rafts on the cell surface, and the knockdown of integrin ß1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin ß1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin ß1-dependent manner, suggesting that integrin ß1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin ß1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.

The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence.

Sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) pathway in infected cells has been shown to be crucial for full replication efficiency of orthopoxviruses in cell culture. In infected cells, this pathway is mainly activated by the vaccinia virus growth factor (VGF), an epidermal growth factor (EGF)-like protein. We show here that chorioallantois vaccinia virus Ankara (CVA), but not modified vaccinia virus Ankara (MVA), induced sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in infected human 293 cells, although both viruses direct secretion of functional VGF. A CVA mutant lacking the O1L gene (CVA-ΔO1L) demonstrated that the O1 protein was required for sustained upregulation of the ERK1/2 pathway in 293 cells as well as in other mammalian cell lines. The highly conserved orthopoxvirus O1L gene encodes a predicted 78-kDa protein with a hitherto-unknown function. CVA-ΔO1L showed reduced plaque size and an attenuated cytopathic effect (CPE) in infected cell cultures and reduced virulence and spread from lungs to ovaries in intranasally infected BALB/c mice. Reinsertion of an intact O1L gene into MVA, which in its original form harbors a fragmented O1L open reading frame (ORF), restored ERK1/2 activation in 293 cells but did not increase replication and spread of MVA in human or other mammalian cell lines. Thus, the O1 protein was crucial for sustained ERK1/2 activation in CVA- and MVA-infected human cells, complementing the autocrine function of VGF, and enhanced virulence in vivo.

A vaccinia virus-driven interplay between the MKK4/7-JNK1/2 pathway and cytoskeleton reorganization.

Viral manipulation of transduction pathways associated with key cellular functions such as survival, response to microbial infection, and cytoskeleton reorganization can provide the supportive milieu for a productive infection. Here, we demonstrate that vaccinia virus (VACV) infection leads to activation of the stress-activated protein kinase (SAPK)/extracellular signal-regulated kinase (ERK) 4/7 (MKK4/7)-c-Jun N-terminal protein kinase 1/2 (JNK1/2) pathway; further, the stimulation of this pathway requires postpenetration, prereplicative events in the viral replication cycle. Although the formation of intracellular mature virus (IMV) was not affected in MKK4/7- or JNK1/2-knockout (KO) cells, we did note an accentuated deregulation of microtubule and actin network organization in infected JNK1/2-KO cells. This was followed by deregulated viral trafficking to the periphery and enhanced enveloped particle release. Furthermore, VACV infection induced alterations in the cell contractility and morphology, and cell migration was reduced in the JNK-KO cells. In addition, phosphorylation of proteins implicated with early cell contractility and cell migration, such as microtubule-associated protein 1B and paxillin, respectively, was not detected in the VACV-infected KO cells. In sum, our findings uncover a regulatory role played by the MKK4/7-JNK1/2 pathway in cytoskeleton reorganization during VACV infection.

The interplay between Araçatuba virus and host signaling pathways: role of PI3K/Akt in viral replication.

In this study, we describe the interaction between Araçatuba virus (ARAV), a naturally occurring Brazilian vaccinia virus isolated from an outbreak at a dairy farm, and the host cell's signal transduction pathways. Even though ARAV infection led to phosphorylation of MAPKs MEK/ERK, JNK, and p38MAPK, genetic or pharmacological inhibition of these pathways had no impact on viral replication. We also provide evidence that ARAV stimulated the phosphorylation of Akt (PKB) at serine 473 (S473-P), a signaling event that is required for full activation of Akt during the infectious cycle. Furthermore, pharmacological inhibition of PI3K (LY294002) abrogated ARAV-induced Akt activation (S473-P) and affected early and late viral gene expression, which was followed by a decrease in virus yield (~1 log). Taken together, our data shed some light onto the biological differences between ARAV and vaccinia virus strain WR (VACV-WR), which could contribute, at least in part, to the low-virulence phenotype displayed by ARAV. Thus, while the requirement for the PI3K/Akt pathway for successful ARAV replication is also shared with VACV-WR and cowpox virus strain BR (CPXV-BR), ARAV showed a lower replicative capacity, as well as a smaller plaque-size phenotype after infection of A31 cells when compared to VACV-WR.

Mechanism and specificity of DNA strand exchange catalyzed by vaccinia DNA topoisomerase type I.

The type I DNA topoisomerase from vaccinia virus (vTopo) forms a reversible covalent 3'-phosphotyrosyl linkage with a single strand of duplex DNA at the preferred sequence 5'-(C/T)CCTTp downward arrowN(-1)N(-2)N(-3)-3'. The enzyme-DNA covalent adduct is recombinogenic in cells, because the nicked strand downstream of the cleavage site can dissociate and be replaced by another DNA strand, potentially resulting in genome rearrangements if the enzyme executes strand ligation. Topo I could play an active role in strand exchange, either by altering the kinetics or thermodynamics of DNA strand binding or by serving as a proofreading gate to prevent ligation of incoming DNA strands containing mismatches. To address these questions, we have measured the kinetic and thermodynamic parameters for strand annealing to a purified vaccinia Topo I-DNA (vTopo-DNA) covalent complex containing a single-strand overhang and then compared them with the same overhang duplex in the absence of vTopo. We found that vTopo accelerates the strand association rate by 2-fold but has no effect on the rate of strand dissociation. vTopo has a similar small effect on the annealing parameters of a series of DNA strands containing single mismatches. In contrast, single base mismatches at the -1, -2, or -3 positions decreased the forward rate and equilibrium constant for reversible strand ligation by 10-fold. These data establish that while vTopo is a bystander during the annealing step of strand exchange, the enzyme strongly discriminates against mismatches close to the cleavage site during the subsequent events leading to strand ligation. A mechanism emerges where vTopo oscillates between an open state where the downstream DNA segment does not interact with the enzyme and a closed state where catalytically important contacts are formed with this region. This oscillation between an open and closed state of the covalently bound enzyme is likely important for regulating the number of DNA superhelical turns that are removed during the lifetime of the covalent complex with supercoiled substrates.

Viral host-range factor C7 or K1 is essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase R-mediated phosphorylation of eukaryotic translation initiation factor 2alpha.

Vaccinia virus (VACV) infection induces phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha), which inhibits cellular and viral protein synthesis. In turn, VACV has evolved the capacity to antagonize this antiviral response by expressing the viral host-range proteins K3 and E3. This study revealed that the host-range genes K1L and C7L also prevent eIF2alpha phosphorylation in modified VACV Ankara (MVA) infection of several human and murine cell lines. Moreover, C7L-deleted MVA (MVA-DeltaC7L) lacked late gene expression, which could be rescued by the function of host-range factor K1 or C7. It was demonstrated that viral gene expression was blocked after viral DNA replication and that it was independent of apoptosis induction. Furthermore, it was found that eIF2alpha phosphorylation in MVA-DeltaC7L-infected cells is mediated by protein kinase R (PKR) as shown in murine embryonic fibroblasts lacking PKR function, and it was shown that this was not due to reduced E3L gene expression. The block of eIF2alpha phosphorylation by C7 could be complemented by K1 in cells infected with MVA-DeltaC7L encoding a reinserted K1L gene (MVA-DeltaC7L-K1L). Importantly, these data illustrated that eIF2alpha phosphorylation by PKR is not responsible for the block of late viral gene expression. This suggests that other mechanisms targeted by C7 and K1 are essential for completing the MVA gene expression cycle and probably also for VACV replication in a diverse set of cell types.

Viral exploitation of the MEK/ERK pathway - A tale of vaccinia virus and other viruses.

The VACV replication cycle is remarkable in the sense that it is performed entirely in the cytoplasmic compartment of vertebrate cells, due to its capability to encode enzymes required either for regulating the macromolecular precursor pool or the biosynthetic processes. Although remarkable, this gene repertoire is not sufficient to confer the status of a free-living microorganism to the virus, and, consequently, the virus relies heavily on the host to successfully generate its progeny. During the complex virus-host interaction, viruses must deal not only with the host pathways to accomplish their temporal demands but also with pathways that counteract viral infection, including the inflammatory, innate and acquired immune responses. This review focuses on VACV and other DNA or RNA viruses that stimulate the MEK (MAPK - Mitogen Activated Protein Kinase)/ERK- Extracellular signal-Regulated Kinase) pathway as part of their replication cycle.

Differential induction of topoisomerase I-DNA cleavage complexes by the indenoisoquinoline MJ-III-65 (NSC 706744) and camptothecin: base sequence analysis and activity against camptothecin-resistant topoisomerases I.

Camptothecin (CPT) and its derivatives target mammalian DNA topoisomerase I (top1) and are among the most effective novel anticancer drugs. However, the activity of CPTs is limited by several factors, including drug inactivation by lactone ring opening, tumor drug resistance, and toxicity in patients. Novel top1 inhibitors are being searched to overcome such limitations and expand the anticancer spectrum of camptothecins. MJ-III-65 (NSC 706744) is among the most promising indenoisoquinolines to date. In this study, we show that MJ-III-65 enhances top1 cleavage complexes by both inhibiting their reversal (religation) more efficiently than CPT and by enhancing their formation. The top1 DNA cleavage complexes induced by MJ-III-65 exhibit a different distribution pattern compared with CPT and exhibit different base sequence preferences immediately around the top1 cleavage sites. Although CPTs have a preference for thymine at the (-1) position and guanine at the (+1) position of the top1-mediated DNA cleavage sites, MJ-III-65 can accommodate different base pairs at the (-1), (+1), or (+2) position, with a preference for a cytosine at the (-1) position on the scissile strand. Another difference with CPTs is the activity of MJ-III-65 against CPT-resistant top1 enzymes, implying that the amino acid residue interactions with top1 are different for MJ-III-65 and CPTs. As with CPT, MJ-III-65 is inactive against vaccinia top1. This study shows the specific molecular interactions of MJ-III-65 with top1 and demonstrates that MJ-III-65 is a potentially useful top1 inhibitor that enhances and traps top1 cleavage sites not sensitive to CPTs.

Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.

The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.

Identification of induced protein kinase activities specific for the ribosomal proteins uniquely phosphorylated during infection of HeLa cells with vaccinia virus.

We have examined the ribosomal protein kinase activities in partially purified cytoplasmic extracts from HeLa cells infected with vaccinia virus. We found an activity or activities, absent from mock-infected cells, that was capable of phosphorylating the proteins S2 and S13 in vitro. The ribosomes phosphorylated in vitro exhibited the same multiple phosphorylation of S2 found in vivo, at least 3 phosphoryl residues being seen, and the same mono-phosphorylation of S13. Also as in vivo, ribosomal protein S2 contained phosphothreonine as well as phosphoserine, whereas S13 contained only phosphoserine. This strongly suggests that these new protein kinase activities are responsible for the ribosomal protein phosphorylations that occur during infection with vaccinia virus.

Recombinant mouse cytochromes P1-450 and P3-450: enzymatic characterization of the hemoprotein expressed in human cells infected with recombinant vaccinia virus.

We expressed mouse cytochrome P1-450 and P3-450 using recombinant vaccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the recombinant vaccinia virus containing either mouse cytochrome P1-450 or P3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were measured as an expression of the cytochrome P-450 enzyme activities. Both cell lines began to express these enzyme activities as early as 12h after infection. The activities increased linearly up to the 24 h time point, and were kept for 36 h. Western immunoblot analysis showed that these cytochrome P-450 proteins were detected at 16 h and reached maximum quantity at 24 h after infection. These data showed a good correlation between cytochrome P-450 enzyme activity and protein concentration throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzyme from the early stage of the protein expression.

Vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector VH1 from lateral bodies.

Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-γ-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors.

Vaccinia virus F11 promotes viral spread by acting as a PDZ-containing scaffolding protein to bind myosin-9A and inhibit RhoA signaling.

The vaccinia F11 protein promotes viral spread by modulating the cortical actin cytoskeleton by inhibiting RhoA signaling via an unknown mechanism. PDZ domains are widely conserved protein interaction modules whose occurrence in viral proteins is unprecedented. We found that F11 contains a central PDZ-like domain that is required to downregulate RhoA signaling and enhance viral spread. The PDZ-like domain interacts with the PDZ binding motif of the Rho GTPase-activating protein (GAP) Myosin-9A. In the absence of Myosin-9A, RhoA signaling is not inhibited, resulting in fewer actin tails and reduced virus release concomitant with less viral spread. The loss of Myosin-9A GAP activity or its ability to bind F11 also reduces actin tail formation. Furthermore, the ability of Myosin-9A to promote viral spread depends on F11 binding RhoA. Thus, F11 acts as a functional PDZ-containing scaffolding protein to inhibit RhoA signaling by binding Myosin-9A.

In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc). This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage. Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks), fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO(4) blunt-ended break (DNase I-type breaks), if T4 DNA ligase is present in the solution. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO(4) blunt-ended breaks, the ligase reacts with 5'PO(4) DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO(4) blunt-ended DNA breaks. This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

A selective Seoul-Fluor-based bioprobe, SfBP, for vaccinia H1-related phosphatase--a dual-specific protein tyrosine phosphatase.

We report a Seoul-Fluor-based bioprobe, SfBP, for selective monitoring of protein tyrosine phosphatases (PTPs). A rational design based on the structures at the active site of dual-specific PTPs can enable SfBP to selectively monitor the activity of these PTPs with a 93-fold change in brightness. Moreover, screening results of SfBP against 30 classical PTPs and 35 dual-specific PTPs show that it is selective toward vaccinia H1-related (VHR) phosphatase, a dual-specific PTP (DUSP-3).

Production of prostaglandin E2 in response to infection with modified vaccinia Ankara virus.

Prostaglandin E2 (PGE2) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE2 production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE2 to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A2 activity. The accumulation of AA and PGE2 was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE2 required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines.

Casein kinase 2 regulates vaccinia virus actin tail formation.

Casein kinase 2 (CK2) is a pleiotropic serine/threonine kinase that regulates numerous cellular processes and is essential to the infectious cycle of several viruses. Here we investigated the potential role of CK2 in vaccinia virus (VACV) infection. We used the CK2 inhibitor TBB and found that CK2 inactivation impaired VACV dissemination and actin tail formation. We used RNAi and confirmed that CK2 depletion impaired VACV actin tail formation. Furthermore, we designed a recombinant virus that allowed us to specifically detect cell-associated enveloped viruses (CEVs) at the plasma membrane and demonstrated that CK2 inactivation does not affect CEV formation. Finally, we showed that CK2 depletion impaired the recruitment of Src to CEVs. We discuss the possibility that CK2 may stimulate the A36-dependent recruitment of Src through A36 phosphorylation.