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Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells

September 2015
Journal of Virology 89(20)

DOI:10.1128/JVI.01252-15

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PubMed

Authors:

Augusta Jamin

Nouhou Ibrahim

National Institutes of Health

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Importance: The most well characterized role for the vaccinia B1 kinase is to facilitate viral DNA replication by phosphorylating and inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral lifecycle have been postulated for decades, but are difficult to examine directly due to the importance of B1 during DNA replication. Herein, we demonstrate that in U2OS cells, a B1-mutant virus escapes the block in DNA replication observed in other cell types and instead this mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral lifecycle, both dependent and independent of BAF.

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Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the

Viral Life Cycle in a BAF-Independent Manner in U2OS Cells

Augusta Jamin,

a,b

Nouhou Ibrahim,

a,b

April Wicklund,

a,b

Kaitlin Weskamp,

Matthew S. Wiebe

a,b

Nebraska Center for Virology

and the School of Veterinary Medicine and Biomedical Sciences,

University of Nebraska—Lincoln, Lincoln, Nebraska, USA; Department of

Biology, Nebraska Wesleyan University, Lincoln, Nebraska, USA

ABSTRACT

The vaccinia virus B1R gene encodes a highly conserved protein kinase that is essential for the poxviral life cycle. As demon-

strated in many cell types, B1 plays a critical role during viral DNA replication when it inactivates the cellular host defense effec-

tor barrier to autointegration factor (BAF or BANF1). To better understand the role of B1 during infection, we have character-

ized the growth of a B1-deﬁcient temperature-sensitive mutant virus (Cts2 virus) in U2OS osteosarcoma cells. In contrast to all

other cell lines tested to date, we found that in U2OS cells, Cts2 viral DNA replication is unimpaired at the nonpermissive tem-

perature. However, the Cts2 viral yield in these cells was reduced more than 10-fold, thus indicating that B1 is required at an-

other stage of the vaccinia virus life cycle. Our results further suggest that the host defense function of endogenous BAF may be

absent in U2OS cells but can be recovered through either overexpression of BAF or fusion of U2OS cells with mouse cells in

which the antiviral function of BAF is active. Interestingly, examination of late viral proteins during Cts2 virus infection demon-

strated that B1 is required for optimal processing of the L4 protein. Finally, execution point analyses as well as electron micros-

copy studies uncovered a role for B1 during maturation of poxviral virions. Overall, this work demonstrates that U2OS cells are

a novel model system for studying the cell type-speciﬁc regulation of BAF and reveals a role for B1 beyond DNA replication dur-

ing the late stages of the viral life cycle.

IMPORTANCE

The most well characterized role for the vaccinia virus B1 kinase is to facilitate viral DNA replication by phosphorylating and

inactivating BAF, a cellular host defense responsive to foreign DNA. Additional roles for B1 later in the viral life cycle have been

postulated for decades but are difﬁcult to examine directly due to the importance of B1 during DNA replication. Here, we dem-

onstrate that in U2OS cells, a B1 mutant virus escapes the block in DNA replication observed in other cell types and, instead, this

mutant virus exhibits impaired late protein accumulation and incomplete maturation of new virions. These data provide the

clearest evidence to date that B1 is needed for multiple critical junctures in the poxviral life cycle in a manner that is both depen-

dent on and independent of BAF.

Poxviruses are complex viruses containing linear double-

stranded DNA genomes with the unique characteristic of un-

dergoing viral replication in the cytoplasm of host cells. Vaccinia

virus, the most well studied poxvirus, has a genome that is 192 kb

in size and encodes approximately 200 proteins. The vaccinia vi-

rus life cycle includes a temporally regulated cascade of early gene

expression, DNA replication, and intermediate and late stages of

gene expression (1). This cascade culminates in the production

of the structural proteins needed for the assembly and maturation

of new virions in a process referred to as morphogenesis (2).

Viral DNA replication is orchestrated by a number of early

proteins, including the catalytic subunit of the viral DNA poly-

merase (the product of the viral E9 gene) (3–6), a heterodimeric

processivity factor (A20/D4) (7–9), a single-stranded DNA

(ssDNA)-binding protein (I3) (10,11), a DNA-independent nu-

cleotide triphosphatase (D5) (12–14), a putative scaffolding pro-

tein (H5) (15), and a serine/threonine protein kinase (B1) (6,

16–18). B1 is highly conserved within the members of the Poxviri-

dae family that infect mammals, with the only exceptions being

the Molluscipox and Parapox genera (19). It is well established that

the vaccinia virus B1 protein kinase is essential for productive

infection. This conclusion is drawn from studies of temperature-

sensitive mutant viruses with lesions in the B1 locus (Cts2 and

Cts25 viruses), the progeny of which are severely reduced in num-

ber during infection at nonpermissive temperatures, due to criti-

cal defects in viral DNA replication (16,20). Interestingly, there is

evidence that the severity of the Cts2 virus phenotype is cell type

dependent. For example, in L929 murine ﬁbroblasts, Cts2 virus

production at the nonpermissive temperature is reduced by 95%,

with a correlative decrease in the amount of viral DNA accumu-

lation to ⬍5% of the amount of viral DNA produced during a

permissive infection being found (16). In contrast, in BSC40 pri-

mate epithelial cells, the Cts2 viral yield is also reduced to ⬃15% of

wild-type (WT) viral titers, but viral DNA replication is less re-

stricted, with the virus producing 67% of the amount of viral DNA

relative to the amount produced during permissive infection (16).

Received 22 May 2015 Accepted 22 July 2015

Accepted manuscript posted online 29 July 2015

Citation Jamin A, Ibrahim N, Wicklund A, Weskamp K, Wiebe MS. 2015. Vaccinia

virus B1 kinase is required for postreplicative stages of the viral life cycle in a BAF-

independent manner in U2OS cells. J Virol 89:10247–10259.

doi:10.1128/JVI.01252-15.

Editor: G. McFadden

Address correspondence to Matthew S. Wiebe, mwiebe@unl.edu.

doi:10.1128/JVI.01252-15

October 2015 Volume 89 Number 20 jvi.asm.org 10247Journal of Virology

The Vaccinia Virus (VACV) B1 and Cellular VRK2 Kinases Promote VACV Replication Factory Formation through Phosphorylation-Dependent Inhibition of VACV B12

Article

Full-text available

Jan 2019

Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and vi- ral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a com- mon pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vac- cinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral repli- cation in the absence of B1, unlike other DNA viruses. Employing loss-of-function analy- sis, we demonstrate that vaccinia virus’s dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. More- over, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replica- tion factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-depen- dent manner.

The Vaccinia B1 and Cellular VRK2 Kinases Promote Vaccinia Replication Factory Formation through Phosphorylation Dependent Inhibition of Vaccinia B12

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Comparative examination of viral and host protein homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia virus B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called vaccinia virus-related kinases (VRKs). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia virus protein B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1, unlike other DNA viruses. Employing loss-of-function analysis, we demonstrate that vaccinia virus’s dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining coprecipitation of B12 by B1 and VRK2. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analyses of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling coregulation and support a model in which these enzymes modulate B12 in a phosphorylation-dependent manner.

Deletion of the Vaccinia B1 Kinase Reveals Essential Functions of this Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2

Article

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Jul 2017
J VIROL

The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses. IMPORTANCE Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62

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Full-text available

May 2024
J CELL BIOL

Autophagy is an essential degradation program required for cell homeostasis. Among its functions is the engulfment and destruction of cytosolic pathogens, termed xenophagy. Not surprisingly, many pathogens use various strategies to circumvent or co-opt autophagic degradation. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Even though these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been extensively explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that overexpression of the xenophagy receptors p62, NDP52, and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 initially targeted cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 was dependent upon p62 NLS2 and correlated with VACV kinase mediated phosphorylation of p62 T269/S272. This suggests that VACV targets p62 during the early stages of infection to avoid destruction and further implies that poxviruses exhibit multi-layered control of autophagy to facilitate cytoplasmic replication.

Vaccinia virus subverts xenophagy through phosphorylation and nuclear targeting of p62

Preprint

Full-text available

Apr 2021

Autophagy is an essential degradation program required to maintain cell homeostasis. Amongst its many functions is the engulfment and destruction of cytosolic pathogens, termed Xenophagy. Not surprisingly, many pathogens have adapted various strategies to circumvent or co-opt autophagic degradation during infection. For poxviruses, it is known that infection activates autophagy, which however is not required for successful replication. Despite the fact that these complex viruses replicate exclusively in the cytoplasm, autophagy-mediated control of poxvirus infection has not been explored. Using the prototypic poxvirus, vaccinia virus (VACV), we show that over-expression of the xenophagy receptors p62, NDP52 and Tax1Bp1 restricts poxvirus infection. While NDP52 and Tax1Bp1 were degraded, p62 was found to initially target cytoplasmic virions before being shunted to the nucleus. Nuclear translocation of p62 during infection was dependent upon p62 NLS2, and VACV kinase-mediated phosphorylation of associated p62 residues T269/S272. These results indicate that VACV actively targets the xenophagy receptor p62 during the early stages of infection to avoid destruction, and further suggest that poxviruses exhibit a unique multi-layered control of autophagy in order to facilitate cytoplasmic replication.

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by iPOND Coupled with Mass Spectrometry

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Full-text available

Sep 2017
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Poxviruses replicate within the cytoplasm and encode proteins for DNA and mRNA synthesis. To investigate poxvirus replication and transcription from a new perspective, we incorporated 5-ethynyl-2′-deoxyuridine (EdU) into nascent DNA in cells infected with vaccinia virus (VACV). The EdU-labeled DNA was conjugated to fluor- or biotin-azide and visualized by confocal, superresolution, and transmission electron microscopy. Nuclear labeling decreased dramatically after infection, accompanied by intense labeling of cytoplasmic foci. The nascent DNA colocalized with the VACV single-stranded DNA binding protein I3 in multiple puncta throughout the interior of factories, which were surrounded by endoplasmic reticulum. Complexes containing EdU-biotin-labeled DNA cross-linked to proteins were captured on streptavidin beads. After elution and proteolysis, the peptides were analyzed by mass spectrometry to identify proteins associated with nascent DNA. The known viral replication proteins, a telomere binding protein, and a protein kinase were associated with nascent DNA, as were the DNA-dependent RNA polymerase and intermediate- and late-stage transcription initiation and elongation factors, plus the capping and methylating enzymes. These results suggested that the replicating pool of DNA is transcribed and that few if any additional viral proteins directly engaged in replication and transcription remain to be discovered. Among the host proteins identified by mass spectrometry, topoisomerases IIα and IIβ and PCNA were noteworthy. The association of the topoisomerases with nascent DNA was dependent on expression of the viral DNA ligase, in accord with previous proteomic studies. Further investigations are needed to determine possible roles for PCNA and other host proteins detected. IMPORTANCE Poxviruses, unlike many well-characterized animal DNA viruses, replicate entirely within the cytoplasm of animal cells, raising questions regarding the relative roles of viral and host proteins. We adapted newly developed procedures for click chemistry and iPOND (Isolation of proteins on nascent DNA) to investigate vaccinia virus (VACV), the prototype poxvirus. Nuclear DNA synthesis ceased almost immediately following VACV infection, followed swiftly by the synthesis of viral DNA within discrete cytoplasmic foci. All viral proteins known from genetic and proteomic studies to be required for poxvirus DNA replication were identified in the complexes containing nascent DNA. The additional detection of the viral DNA-dependent RNA polymerase and intermediate and late transcription factors provided evidence for a temporal coupling of replication and transcription. Further studies are needed to assess the potential roles of host proteins, including topoisomerases IIα and IIβ and PCNA, which were found associated with nascent DNA.

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Osteosarcoma (OS) is the most common primary malignancy of the bone and has a high propensity for local invasion and metastasis. Although combining surgery with chemotherapy has immensely improved the outcomes of osteosarcoma patients, the prognosis of metastatic or recurrent osteosarcomas is still unsatisfactory. Immunotherapy has proven to be a promising therapeutic strategy against human malignancies and improved understanding of the immune response to OS, and biomarker development has increased the number of patients who benefit from immunotherapies in recent years. Here, we review recent advances in immunotherapy in osteosarcoma and discuss the mechanisms and status of immunotherapies in both preclinical and clinical trials as well as future therapies on the horizon. These advances may pave the way for novel treatments requisite for patients with osteosarcoma in need of new therapies.

The VACV B12 Pseudokinase Represses Viral Replication via Interaction with the Cellular Kinase VRK1 and Activation of the Antiviral Effector BAF

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The poxviral B1 and B12 proteins are a homologous kinase-pseudokinase pair, which modulate a shared host pathway governing viral DNA replication and antiviral defense. While the molecular mechanisms involved are incompletely understood, B1 and B12 seem to intersect with signaling processes mediated by their cellular homologs termed the vaccinia related kinases (VRKs). In this study, we expand upon our previous characterization of the B1-B12 signaling axis to gain insights into B12 function. We begin our studies by demonstrating that modulation of B12 repressive activity is a conserved function of B1 orthologs from divergent poxviruses. Next, we characterize the protein interactome of B12 using multiple cell lines and expression systems and discover that the cellular kinase VRK1 is a highly enriched B12 interactor. Using complementary VRK1 knockdown and overexpression assays, we first demonstrate that VRK1 is required for the rescue of a B1-deleted virus upon mutation of B12. Second, we find that VRK1 overexpression is sufficient to overcome repressive B12 activity during B1-deleted virus replication. Interestingly, we also evince that B12 interferes with the ability of VRK1 to phospho-inactivate the host defense protein BAF. Thus, B12 restricts vaccinia virus DNA accumulation in part by repressing the ability of VRK1 to inactivate BAF. Finally, these data establish that a B12-VRK1-BAF signaling axis forms during vaccinia infection and is modulated via kinases B1 and/or VRK2. These studies provide novel insights into the complex mechanisms poxviruses use to hijack homologous cellular signaling pathways during infection. IMPORTANCE Viruses from diverse families encode for both positive and negative regulators of viral replication. While their functions can sometimes be enigmatic, investigation of virus-encoded, negative regulators of viral replication has revealed fascinating aspects of virology. Studies of poxvirus-encoded genes have largely concentrated on positive regulators of their replication; however, examples of fitness gains attributed to poxvirus gene loss suggests that negative regulators of poxvirus replication also impact infection dynamics. This study focuses on the vaccinia B12 pseudokinase, a protein capable of inhibiting vaccinia DNA replication. Herein, we elucidate the mechanisms by which B12 inhibits vaccinia DNA replication, demonstrating that B12 activates the antiviral protein BAF by inhibiting the activity of VRK1, a cellular modulator of BAF. Combined with previous data, these studies provide evidence that poxviruses govern their replication by employing both positive and negative regulators of viral replication.

Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

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Unlabelled: The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1-H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vΔH5). Analysis of vΔH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis. Importance: Variola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.

Vaccinia Virus Mutations in the L4R Gene Encoding a Virion Structural Protein Produce Abnormal Mature Particles Lacking a Nucleocapsid

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Unlabelled: Electron micrographs from the 1960s revealed the presence of an S-shaped tubular structure in the center of the vaccinia virion core. Recently, we showed that packaging of virus transcription enzymes is necessary for the formation of the tubular structure, suggesting that the structure is equivalent to a nucleocapsid. Based on this study and on what is known about nucleocapsids of other viruses, we hypothesized that in addition to transcription enzymes, the tubular structure also contains the viral DNA and a structural protein as a scaffold. The vaccinia virion structural protein L4 stands out as the best candidate for the role of a nucleocapsid structural protein because it is abundant, it is localized in the center of the virion core, and it binds DNA. In order to gain more insight into the structure and relevance of the nucleocapsid, we analyzed thermosensitive and inducible mutants in the L4R gene. Using a cryo-fixation method for electron microscopy (high-pressure freezing followed by freeze-substitution) to preserve labile structures like the nucleocapsid, we were able to demonstrate that in the absence of functional L4, mature particles with defective internal structures are produced under nonpermissive conditions. These particles do not contain a nucleocapsid. In addition, the core wall of these virions is abnormal. This suggests that the nucleocapsid interacts with the core wall and that the nucleocapsid structure might be more complex than originally assumed. Importance: The vaccinia virus nucleocapsid has been neglected since the 1960s due to a lack of electron microscopy techniques to preserve this labile structure. With the advent of cryo-fixation techniques, like high-pressure freezing/freeze-substitution, we are now able to consistently preserve and visualize the nucleocapsid. Because vaccinia virus early transcription is coupled to the viral core structure, detailing the structure of the nucleocapsid is indispensable for determining the mechanisms of vaccinia virus core-directed transcription. The present study represents our second attempt to understand the structure and biological significance of the nucleocapsid. We demonstrate the importance of the protein L4 for the formation of the nucleocapsid and reveal in addition that the nucleocapsid and the core wall may be associated, suggesting a higher level of complexity of the nucleocapsid than predicted. In addition, we prove the utility of high-pressure freezing in preserving the vaccinia virus nucleocapsid.

Cell- and Virus-Mediated Regulation of the Barrier-to-Autointegration Factor's Phosphorylation State Controls Its DNA Binding, Dimerization, Subcellular Localization, and Antipoxviral Activity

Article

Full-text available

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Unlabelled: Barrier-to-autointegration factor (BAF) is a DNA binding protein with multiple cellular functions, including the ability to act as a potent defense against vaccinia virus infection. This antiviral function involves BAF's ability to condense double-stranded DNA and subsequently prevent viral DNA replication. In recent years, it has become increasingly evident that dynamic phosphorylation involving the vaccinia virus B1 kinase and cellular enzymes is likely a key regulator of multiple BAF functions; however, the precise mechanisms are poorly understood. Here we analyzed how phosphorylation impacts BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity through the characterization of BAF phosphomimetic and unphosphorylatable mutants. Our studies demonstrate that increased phosphorylation enhances BAF's mobilization from the nucleus to the cytosol, while dephosphorylation restricts BAF to the nucleus. Phosphorylation also impairs both BAF's dimerization and its DNA binding activity. Furthermore, our studies of BAF's antiviral activity revealed that hyperphosphorylated BAF is unable to suppress viral DNA replication or virus production. Interestingly, the unphosphorylatable BAF mutant, which is capable of binding DNA but localizes predominantly to the nucleus, was also incapable of suppressing viral replication. Thus, both DNA binding and localization are important determinants of BAF's antiviral function. Finally, our examination of how phosphatases are involved in regulating BAF revealed that PP2A dephosphorylates BAF during vaccinia infection, thus counterbalancing the activity of the B1 kinase. Altogether, these data demonstrate that phosphoregulation of BAF by viral and cellular enzymes modulates this protein at multiple molecular levels, thus determining its effectiveness as an antiviral factor and likely other functions as well. Importance: The barrier-to-autointegration factor (BAF) contributes to cellular genomic integrity in multiple ways, the best characterized of which are as a host defense against cytoplasmic DNA and as a regulator of mitotic nuclear reassembly. Although dynamic phosphorylation involving both viral and cellular enzymes is likely a key regulator of multiple BAF functions, the precise mechanisms involved are poorly understood. Here we demonstrate that phosphorylation coordinately regulates BAF's DNA binding, subcellular localization, dimerization, and antipoxviral activity. Overall, our findings provide new insights into how phosphoregulation of BAF modulates this protein at multiple levels and governs its effectiveness as an antiviral factor against foreign DNA.

Association of the Vaccinia Virus A11 Protein with the Endoplasmic Reticulum and Crescent Precursors of Immature Virions

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The apparent de novo formation of viral membranes within cytoplasmic factories is a mysterious, poorly understood, first step in poxvirus morphogenesis. Genetic studies identified several viral proteins essential for membrane formation and assembly of immature virus particles. Their repression results in abortive replication with accumulation of dense masses of viroplasm. In the present study we further characterized one of these proteins, A11, and investigated its association with cellular and viral membranes under normal and abortive replication conditions. We discovered that A11 colocalized in cytoplasmic factories with endoplasmic reticulum (ER) and L2, another viral protein required for morphogenesis. Confocal microscopy and subcellular fractionation indicated that A11 was not membrane-associated in uninfected cells, whereas L2 still co-localized with ER. Cell-free transcription and translation experiments indicated that both A11 and L2 are tail-anchored proteins that post-translationally associate with membranes and likely require specific cytoplasmic targeting chaperones. Transmission electron microscopy indicated that A11, like L2, associated with crescent membranes and immature virions during normal infection and with vesicles and tubules near masses of dense viroplasm during abortive infection in the absence of the A17 or A14 protein components of viral membranes. When synthesis of A11 was repressed, "empty" immature virion-like structures formed in addition to masses of viroplasm. The immature virion-like structures were labeled with antibodies to A17 and to the D13 scaffold protein and were closely associated with calnexin-labeled ER. These studies revealed similarities and differences between A11 and L2, both of which may be involved with recruitment of ER for virus assembly.

Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein, A6

Article

Full-text available

Sep 2012
J VIROL

A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

Molecular Genetic and Biochemical Characterization of the Vaccinia Virus I3 Protein, the Replicative Single-Stranded DNA Binding Protein

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May 2012
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Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its ∼200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus.

Vaccinia Virus L2 Protein Associates with the Endoplasmic Reticulum near the Growing Edge of Crescent Precursors of Immature Virions and Stabilizes a Subset of Viral Membrane Proteins

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Nov 2011
J VIROL

The initial step in poxvirus morphogenesis, the formation of crescent membranes, occurs within cytoplasmic factories. L2 is one of several vaccinia virus proteins known to be necessary for formation of crescents and the only one synthesized early in infection. Virus replication was unaffected when the L2R open reading frame was replaced by L2R containing an N-terminal epitope tag while retaining the original promoter. L2 colocalized with the endoplasmic reticulum (ER) protein calnexin throughout the cytoplasm of infected and transfected cells. Topological studies indicated that the N terminus of L2 is exposed to the cytoplasm with the hydrophobic C terminus anchored in the ER. Using immunogold labeling and electron microscopy, L2 was detected in tubular membranes outside factories and inside factories near crescents and close to the edge or rim of crescents; a similar labeling pattern was found for the ER luminal protein disulfide isomerase (PDI). The phenotype of L2 conditional lethal mutants and the localization of L2 suggest that it participates in elongation of crescents by the addition of ER membrane to the growing edge. Small amounts of L2 and PDI were detected within immature and mature virions, perhaps trapped during assembly. The repression of L2, as well as A11 and A17, two other proteins that are required for viral crescent formation, profoundly decreased the stability of a subset of viral membrane proteins including those comprising the entry-fusion complex. To avoid degradation, these unstable membrane proteins may need to directly insert into the viral membrane or be rapidly shunted there from the ER.

Molecular Characterization of the Host Defense Activity of the Barrier to Autointegration Factor against Vaccinia Virus

Article

Full-text available

Oct 2011
J VIROL

The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAF's activity as a host defense factor is not limited to poxviral infection. BAF's relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAF's molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.

Barrier to Autointegration Factor (BAF) Inhibits Vaccinia Virus Intermediate Transcription in the Absence of the Viral B1 Kinase

Article

Jul 2013
VIROLOGY

Barrier to autointegration factor (BAF/BANF1) is a cellular DNA-binding protein found in the nucleus and cytoplasm. Cytoplasmic BAF binds to foreign DNA and can act as a defense against vaccinia DNA replication. To evade BAF, vaccinia expresses the B1 kinase, which phosphorylates BAF and blocks its ability to bind DNA. Interestingly, B1 is also needed for viral intermediate gene expression via an unknown mechanism. Therefore, we evaluated the impact of B1-BAF signaling on vaccinia transcription. Strikingly, the decrease in vaccinia transcription caused by loss of B1 can be rescued by depletion of BAF. The repressive action of BAF is greatest on a viral promoter, and is more modest when non-vaccinia promoters are employed, which suggests BAF acts in a gene specific manner. These studies expand our understanding of the role of the B1 kinase during infection and provide the first evidence that BAF is a defense against viral gene expression.

The Genome of Molluscum Contagiosum Virus: Analysis and Comparison with Other Poxviruses

Article

Jul 1997

Analysis of the molluscum contagiosum virus (MCV) genome revealed that it encodes approximately 182 proteins, 105 of which have direct counterparts in orthopoxviruses (OPV). The corresponding OPV proteins comprise those known to be essential for replication as well as many that are still uncharacterized, including 2 of less than 60 amino acids that had not been previously noted. The OPV proteins most highly conserved in MCV are involved in transcription; the least conserved include membrane glycoproteins. Twenty of the MCV proteins with OPV counterparts also have cellular homologs and additional MCV proteins have conserved functional motifs. Of the 77 predicted MCV proteins without OPV counterparts, 10 have similarity to other MCV proteins and/or distant similarity to proteins of other poxviruses and 16 have cellular homologs including some predicted to antagonize host defenses. Clustering poxvirus proteins by sequence similarity revealed 3 unique MCV gene families and 8 families that are conserved in MCV and OPV. Two unique families contain putative membrane receptors; the third includes 2 proteins, each containing 2 DED apoptosis signal transduction domains. Additional families with conserved patterns of cysteines and putative redox active centers were identified. Promoters, transcription termination signals, and DNA concatemer resolution sequences are highly conserved in MCV and OPV. Phylogenetic analysis suggested that MCV, OPV, and leporipoxviruses radiated from a common poxvirus ancestor after the divergence of avipoxviruses. Despite the acquisition of unique genes for host interactions and changes in GC content, the physical order and regulation of essential ancestral poxvirus genes have been largely conserved in MCV and OPV.

Vaccinia Virus B1 Kinase Is Required for Postreplicative Stages of the Viral Life Cycle in a BAF-Independent Manner in U2OS Cells

Abstract

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