Closely related reovirus lab strains induce opposite expression of RIG-I/IFN-dependent versus -independent host genes, via mechanisms of slow replication versus polymorphisms in dsRNA binding σ3 respectively.

The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3DPL than T3DTD. Polymorphisms in reovirus σ3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.

Polymorphisms in the Most Oncolytic Reovirus Strain Confer Enhanced Cell Attachment, Transcription, and Single-Step Replication Kinetics.

Reovirus serotype 3 Dearing (T3D) replicates preferentially in transformed cells and is in clinical trials as a cancer therapy. Laboratory strains of T3D, however, exhibit differences in plaque size on cancer cells and differences in oncolytic activity in vivo This study aimed to determine why the most oncolytic T3D reovirus lab strain, the Patrick Lee laboratory strain (T3DPL), replicates more efficiently in cancer cells than other commonly used laboratory strains, the Kevin Coombs laboratory strain (T3DKC) and Terence Dermody laboratory (T3DTD) strain. In single-step growth curves, T3DPL titers increased at higher rates and produced ∼9-fold higher burst size. Furthermore, the number of reovirus antigen-positive cells increased more rapidly for T3DPL than for T3DTD In conclusion, the most oncolytic T3DPL possesses replication advantages in a single round of infection. Two specific mechanisms for enhanced infection by T3DPL were identified. First, T3DPL exhibited higher cell attachment, which was attributed to a higher proportion of virus particles with insufficient (≤3) σ1 cell attachment proteins. Second, T3DPL transcribed RNA at rates superior to those of the less oncolytic T3D strains, which is attributed to polymorphisms in M1-encoding µ2 protein, as confirmed in an in vitro transcription assay, and which thus demonstrates that T3DPL has an inherent transcription advantage that is cell type independent. Accordingly, T3DPL established rapid onset of viral RNA and protein synthesis, leading to more rapid kinetics of progeny virus production, larger virus burst size, and higher levels of cell death. Together, these results emphasize the importance of paying close attention to genomic divergence between virus laboratory strains and, mechanistically, reveal the importance of the rapid onset of infection for reovirus oncolysis.IMPORTANCE Reovirus serotype 3 Dearing (T3D) is in clinical trials for cancer therapy. Recently, it was discovered that highly related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a murine model of melanoma. The current study reveals two mechanisms for the enhanced efficiency of T3DPL in cancer cells. Due to polymorphisms in two viral genes, within the first round of reovirus infection, T3DPL binds to cells more efficiency and more rapidly produces viral RNAs; this increased rate of infection relative to that of the less oncolytic strains gives T3DPL a strong inherent advantage that culminates in higher virus production, more cell death, and higher virus spread.

Single Amino Acid Differences between Closely Related Reovirus T3D Lab Strains Alter Oncolytic Potency In Vitro and In Vivo.

Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

The herpes simplex virus host shutoff (vhs) RNase limits accumulation of double stranded RNA in infected cells: Evidence for accelerated decay of duplex RNA.

The herpes simplex virus virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs and blunts host innate antiviral responses. Previous work demonstrated that cells infected with vhs mutants display enhanced activation of the host double-stranded RNA (dsRNA)-activated protein kinase R (PKR), implying that vhs limits dsRNA accumulation in infected cells. Confirming this hypothesis, we show that partially complementary transcripts of the UL23/UL24 and UL30/31 regions of the viral genome increase in abundance when vhs is inactivated, giving rise to greatly increased levels of intracellular dsRNA formed by annealing of the overlapping portions of these RNAs. Thus, vhs limits accumulation of dsRNA at least in part by reducing the levels of complementary viral transcripts. We then asked if vhs also destabilizes dsRNA after its initial formation. Here, we used a reporter system employing two mCherry expression plasmids bearing complementary 3' UTRs to produce defined dsRNA species in uninfected cells. The dsRNAs are unstable, but are markedly stabilized by co-expressing the HSV dsRNA-binding protein US11. Strikingly, vhs delivered by super-infecting HSV virions accelerates the decay of these pre-formed dsRNAs in both the presence and absence of US11, a novel and unanticipated activity of vhs. Vhs binds the host RNA helicase eIF4A, and we find that vhs-induced dsRNA decay is attenuated by the eIF4A inhibitor hippuristanol, providing evidence that eIF4A participates in the process. Our results show that a herpesvirus host shutoff RNase destabilizes dsRNA in addition to targeting partially complementary viral mRNAs, raising the possibility that the mRNA destabilizing proteins of other viral pathogens dampen the host response to dsRNA through similar mechanisms.

Remodeling mTORC1 Responsiveness to Amino Acids by the Herpes Simplex Virus UL46 and Us3 Gene Products Supports Replication during Nutrient Insufficiency.

By sensing fundamental parameters, including nutrient availability, activated mechanistic target of rapamycin complex 1 (mTORC1) suppresses catabolic outcomes and promotes anabolic processes needed for herpes simplex virus 1 (HSV-1) productive growth. While the virus-encoded Us3 Ser/Thr kinase is required to activate mTORC1, whether stress associated with amino acid insufficiency impacts mTORC1 activation in infected cells and virus reproduction was unknown. In contrast to uninfected cells, where amino acid withdrawal inhibits mTORC1 activation, we demonstrate that mTORC1 activity is sustained in HSV-1-infected cells during amino acid insufficiency. We show that in the absence of Us3, the insensitivity of mTORC1 to amino acid withdrawal in infected cells was dependent on the host kinase Akt and establish a role for the HSV-1 UL46 gene product, which stimulates phosphatidylinositol (PI) 3-kinase signaling. Significantly, virus reproduction during amino acid insufficiency was stimulated by the viral UL46 gene product. By synergizing with Us3, UL46 reprograms mTORC1 such that it is insensitive to amino acid withdrawal and supports sustained mTORC1 activation and virus reproduction during amino acid insufficiency. This identifies an unexpected function for UL46 in supporting virus reproduction during physiological stress and identifies a new class of virus-encoded mTORC1 regulators that selectively uncouple mTORC1 activation from amino acid sufficiency.IMPORTANCE Mechanistic target of rapamycin complex 1 (mTORC1) is a multisubunit cellular kinase that coordinates protein synthesis with changing amino acid levels. During amino acid insufficiency, mTORC1 is repressed in uninfected cells, dampening protein synthesis and potentially restricting virus reproduction. Here, we establish that HSV-1 alters the responsiveness of mTORC1 to metabolic stress resulting from amino acid insufficiency. Unlike in uninfected cells, mTORC1 remains activated in HSV-1-infected cells deprived of amino acids. Synergistic action of the HSV-1 UL46 gene product, which stimulates PI 3-kinase, and the Us3 kinase supports virus reproduction during amino acid withdrawal. These results define how HSV-1, a medically important human pathogen associated with a range of diseases, uncouples mTORC1 activation from amino acid availability. Furthermore, they help explain how the virus reproduces during physiological stress. Reproduction triggered by physiological stress is characteristic of herpesvirus infections, where lifelong latency is punctuated by episodic reactivation events.

Expression of the Vaccinia Virus Antiapoptotic F1 Protein Is Blocked by Protein Kinase R in the Absence of the Viral E3 Protein.

Poxviruses encode many proteins with the ability to regulate cellular signaling pathways. One such protein is the vaccinia virus innate immunity modulator E3. Multiple functions have been ascribed to E3, including modulating the cellular response to double-stranded RNA, inhibiting the NF-κB and IRF3 pathways, and dampening apoptosis. Apoptosis serves as a powerful defense against damaged and unwanted cells and is an effective defense against viral infection; many viruses therefore encode proteins that prevent or delay apoptosis. Here, we present data indicating that E3 does not directly inhibit the intrinsic apoptotic pathway; instead, it suppresses apoptosis indirectly by stimulating expression of the viral F1 apoptotic inhibitor. Our data demonstrate that E3 promotes F1 expression by blocking activation of the double-stranded RNA-activated protein kinase R (PKR). F1 mRNA is present in cells infected with E3-null virus, but the protein product does not detectably accumulate, suggesting a block at the translational level. We also show that two 3' coterminal transcripts span the F1 open reading frame (ORF), a situation previously described for the vaccinia virus mRNAs encoding the J3 and J4 proteins. One of these is a conventional monocistronic transcript of the F1L gene, while the other arises by read-through transcription from the upstream F2L gene and does not give rise to appreciable levels of F1 protein.IMPORTANCE Previous studies have shown that E3-deficient vaccinia virus triggers apoptosis of infected cells. Our study demonstrates that this proapoptotic phenotype stems, at least in part, from the failure of the mutant virus to produce adequate quantities of the viral F1 protein, which acts at the mitochondria to directly block apoptosis. Our data establish a regulatory link between the vaccinia virus proteins that suppress the innate response to double-stranded RNA and those that block the intrinsic apoptotic pathway.

Herpes simplex virus 1 infection of T cells causes VP11/12-dependent phosphorylation and degradation of the cellular protein Dok-2.

Previous studies have shown that HSV-1 infection of lymphocytes induces the tyrosine phosphorylation of several proteins that might correspond to viral or host proteins. VP11/12, a viral tegument protein, is the major HSV-induced tyrosine phosphorylated protein identified thus far. In this report, we demonstrated that the cellular adaptor proteins Dok-2 and Dok-1 are tyrosine phosphorylated upon HSV-1 infection. In addition, HSV-1 induced the selective degradation of Dok-2. Finally, we provide evidence that Dok-2 interacts with VP11/12, and that HSV-induced tyrosine phosphorylation and degradation of Dok-2 require VP11/12. Inactivation of either the Src Family Kinases binding motifs or the SHC binding motif of VP11/12 eliminated the interaction of Dok-2 with VP11/12. Elimination of the binding of Dok-2 to VP11/12 prevented Dok-2 phosphorylation and degradation. We propose that HSV-induced Dok phosphorylation and Dok-2 degradation is an immune evasion mechanism to inactivate T cells that might play an important role in HSV pathogenesis.

Subversion of Host Responses to Energy Insufficiency by Us3 Supports Herpes Simplex Virus 1 Replication during Stress.

Cellular stress responses to energy insufficiency can impact virus reproduction. In particular, activation of the host AMP-activated protein kinase (AMPK) by low energy could limit protein synthesis by inhibiting mTORC1. Although many herpesviruses, including herpes simplex virus 1 (HSV-1), stimulate mTORC1, how HSV-1-infected cells respond to energy availability, a physiological indicator regulating mTORC1, has not been investigated. In addition, the impact of low-energy stress on productive HSV-1 growth and viral genetic determinants potentially enabling replication under physiological stress remains undefined. Here, we demonstrate that mTORC1 activity in HSV-1-infected cells is largely insensitive to stress induced by simulated energy insufficiency. Furthermore, resistance of mTORC1 activity to low-energy-induced stress, while not significantly influenced by the HSV-1 UL46-encoded phosphatidylinositol 3-kinase (PI3K)-Akt activator, was dependent upon the Ser/Thr kinase activity of Us3. A Us3-deficient virus was hypersensitive to low-energy-induced stress as infected cell protein synthesis and productive replication were reduced compared to levels in cells infected with a Us3-expressing virus. Although Us3 did not detectably prevent energy stress-induced AMPK activation, it enforced mTORC1 activation despite the presence of activated AMPK. In the absence of applied low-energy stress, AMPK activity in infected cells was restricted in a Us3-dependent manner. This establishes that the Us3 kinase not only activated mTORC1 but also enabled sustained mTORC1 signaling during simulated energy insufficiency that would otherwise restrict protein synthesis and virus replication. Moreover, it identifies the alphaherpesvirus-specific Us3 kinase as an mTORC1 activator that subverts the host cell energy-sensing program to support viral productive growth irrespective of physiological stress.IMPORTANCE Like all viruses, herpes simplex virus type 1 (HSV-1) reproduction relies upon numerous host energy-intensive processes, the most demanding of which is protein synthesis. In response to low energy, the cellular AMP-activated protein kinase (AMPK) triggers a physiological stress response that antagonizes mTORC1, a multisubunit host kinase that controls protein synthesis. This could restrict virus protein production and growth. Here, we establish that the HSV-1 Us3 protein kinase subverts the normal response to low-energy-induced stress. While Us3 does not prevent AMPK activation by low energy, it enforces mTORC1 activation and overrides a physiological response that couples energy availability and protein synthesis. These results help explain how reproduction of HSV-1, a ubiquitous, medically significant human pathogen causing a spectrum of diseases ranging from the benign to the life threatening, occurs during physiological stress. This is important because HSV-1 reproduction triggered by physiological stress is characteristic of reactivation of lifelong latent infections.

Role of Herpes simplex virus 1 VP11/12 tyrosine-based binding motifs for Src family kinases, p85, Grb2 and Shc in activation of the phosphoinositide 3-kinase-Akt pathway.

The abundant HSV-1 tegument protein VP11/12 encoded by gene UL46 is essential for induction of the PI3K/Akt-signaling pathway during infection. VP11/12 utilizes tyrosine-based motifs within its C-terminal region to bind the SH2 domains of Src family kinases, the p85 subunit of PI3 Kinase and Grb2, and the PTB domain of Shc. We previously proposed that the interaction with SFKs and p85 is used to gain control over the PI3K/Akt signaling pathway. We tested this model by evaluating the effects of mutations that eliminate each of these interactions on the ability of HSV-1 to activate Akt. Inhibiting the interaction of VP11/12 with SFKs, p85 and Grb2 reduced Akt activation, while inhibiting the interaction with Shc had little effect. Overall these data support the suggestion that VP11/12 stimulates the PI3K/Akt pathway by mimicking an activated growth factor receptor.

The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation.

UNLABELLED: The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. IMPORTANCE: The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects of RNA metabolism.

Mitochondrial DNA stress primes the antiviral innate immune response.

Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids. The abundant mtDNA-binding protein TFAM (transcription factor A, mitochondrial) regulates nucleoid architecture, abundance and segregation. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)-IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signalling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.

The herpes simplex virus 2 virion-associated ribonuclease vhs interferes with stress granule formation.

UNLABELLED: In a previous study, it was observed that cells infected with herpes simplex virus 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow-up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we reexamined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild-type UL41 following low-multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in the regulation of translation. IMPORTANCE: Eukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow-up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. The identification of a specific viral protein involved in disrupting SG formation is a key step toward understanding how HSV-2 interacts with these antiviral structures. Additionally, this understanding may provide insights into the biology of SGs that may find application in studies on human motor neuron degenerative diseases, like amyotrophic lateral sclerosis (ALS), which may arise as a result of dysregulation of SG formation.

The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

UNLABELLED: We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. IMPORTANCE: The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during virus infection, the overall amount of mRNA may increase drastically, possibly overloading the capacity of the translation apparatus. Our results suggest that the HSV-1 vhs protein, an mRNA-specific endoribonuclease, prevents mRNA overload during infection, thereby allowing translation of late viral mRNAs. The requirement for vhs varies between cell types. Further studies of the basis for this difference likely will offer insights into how cells regulate overall mRNA levels and access to the translational apparatus.

Herpes simplex virus protein kinases US3 and UL13 modulate VP11/12 phosphorylation, virion packaging, and phosphatidylinositol 3-kinase/Akt signaling activity.

UNLABELLED: The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays key roles in diverse cellular activities and promotes cell growth and survival. It is therefore unsurprising that most viruses modify this pathway in order to facilitate their replication and spread. Previous work has suggested that the herpes simplex virus 1 (HSV-1) tegument proteins VP11/12 and US3 protein kinase modulate the PI3K/Akt pathway, albeit in opposing ways: VP11/12 binds and activates Src family kinases (SFKs), is tyrosine phosphorylated, recruits PI3K in an SFK-dependent fashion, and is required for HSV-induced phosphorylation of Akt on its activating residues; in contrast, US3 inhibits Akt activation and directly phosphorylates downstream Akt targets. We examined if US3 negatively regulates Akt by dampening the signaling activity of VP11/12. Consistent with this hypothesis, the enhanced Akt activation that occurs during US3-null infection requires VP11/12 and correlates with an increase in SFK-dependent VP11/12 tyrosine phosphorylation. In addition, deleting US3 leads to a striking increase in the relative abundances of several VP11/12 species that migrate with reduced mobility during SDS-PAGE. These forms arise through phosphorylation, strictly require the viral UL13 protein kinase, and are excluded from virions. Taken in combination, these data indicate that US3 dampens SFK-dependent tyrosine and UL13-dependent serine/threonine phosphorylation of VP11/12, thereby inhibiting VP11/12 signaling and promoting virion packaging of VP11/12. These results illustrate that protein phosphorylation events mediated by viral protein kinases serve to coordinate the roles of VP11/12 as a virion component and intracellular signaling molecule. IMPORTANCE: Herpesvirus tegument proteins play dual roles during the viral life cycle, serving both as structural components of the virus particle and as modulators of cellular and viral functions in infected cells. How these two roles are coordinated during infection and virion assembly is a fundamental and largely unanswered question. Here we addressed this issue with herpes simplex virus VP11/12, a tegument protein that activates the cellular PI3K/Akt signaling pathway. We showed that protein phosphorylation mediated by the viral US3 and UL13 kinases serves to orchestrate its functions: UL13 appears to inhibit VP11/12 virion packaging, while US3 antagonizes UL13 action and independently dampens VP11/12 signaling activity.

Elimination of mitochondrial DNA is not required for herpes simplex virus 1 replication.

UNLABELLED: Infection with herpes simplex virus type 1 (HSV-1) results in the rapid elimination of mitochondrial DNA (mtDNA) from host cells. It is known that a mitochondrial isoform of the viral alkaline nuclease (UL12) called UL12.5 triggers this process. However, very little is known about the impact of mtDNA depletion on viral replication or the biology of HSV-1 infections. These questions have been difficult to address because UL12.5 and UL12 are encoded by overlapping transcripts that share the same open reading frame. As a result, mutations that alter UL12.5 also affect UL12, and UL12 null mutations severely impair viral growth by interfering with the intranuclear processing of progeny viral genomes. Therefore, to specifically assess the impact of mtDNA depletion on viral replication, it is necessary to eliminate the activity of UL12.5 while preserving the nuclear functions of UL12. Previous work has shown that the human cytomegalovirus alkaline nuclease UL98 can functionally substitute for UL12 during HSV-1 replication. We found that UL98 is unable to deplete mtDNA in transfected cells and therefore generated an HSV-1 variant in which UL98 coding sequences replace the UL12/UL12.5 open reading frame. The resulting virus was severely impaired in its ability to trigger mtDNA loss but reached titers comparable to those of wild-type HSV-1 in one-step and multistep growth experiments. Together, these observations demonstrate that the elimination of mtDNA is not required for HSV-1 replication in cell culture. IMPORTANCE: Herpes simplex virus types 1 and 2 destroy the DNA of host cell mitochondria, the powerhouses of cells. Epstein-Barr virus, a distantly related herpesvirus, has a similar effect, indicating that mitochondrial DNA destruction is under positive selection and thus confers a benefit to the virus. The present work shows that mitochondrial DNA destruction is not required for efficient replication of herpes simplex virus type 1 in cultured Vero kidney epithelial cells, suggesting that this activity likely benefits the virus in other cell types or in the intact human host.

Mitochondrial nucleases ENDOG and EXOG participate in mitochondrial DNA depletion initiated by herpes simplex virus 1 UL12.5.

Herpes simplex virus 1 (HSV-1) rapidly eliminates mitochondrial DNA (mtDNA) from infected cells, an effect that is mediated by UL12.5, a mitochondrial isoform of the viral alkaline nuclease UL12. Our initial hypothesis was that UL12.5 directly degrades mtDNA via its nuclease activity. However, we show here that the nuclease activities of UL12.5 are not required for mtDNA loss. This observation led us to examine whether cellular nucleases mediate the mtDNA loss provoked by UL12.5. We provide evidence that the mitochondrial nucleases endonuclease G (ENDOG) and endonuclease G-like 1 (EXOG) play key redundant roles in UL12.5-mediated mtDNA depletion. Overall, our data indicate that UL12.5 deploys cellular proteins, including ENDOG and EXOG, to destroy mtDNA and contribute to a growing body of literature highlighting roles for ENDOG and EXOG in mtDNA maintenance.

Role of herpes simplex virus VP11/12 tyrosine-based motifs in binding and activation of the Src family kinase Lck and recruitment of p85, Grb2, and Shc.

Previous studies have shown that the abundant herpes simplex virus 1 (HSV-1) tegument protein VP11/12, encoded by gene UL46, stimulates phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling: it binds the Src family kinase (SFK) Lck, is tyrosine phosphorylated, recruits the p85 subunit of PI3-kinase, and is essential for the activation of Akt during HSV-1 infection. The C-terminal region of VP11/12 contains tyrosine-based motifs predicted to bind the SH2 domains of SFKs (YETV and YEEI), p85 (YTHM), and Grb2 (YENV) and the phosphotyrosine-binding (PTB) domain of Shc (NPLY). We inactivated each of these motifs in the context of the intact viral genome and examined effects on binding and activation of Lck and recruitment of p85, Grb2, and Shc. Inactivating the p85, Grb2, or Shc motif reduced (p85) or eliminated (Grb2 and Shc) the interaction with the cognate signaling molecule without greatly affecting the other interactions or activation of Lck. Inactivating either SFK motif had only a minor effect on Lck binding and little or no effect on recruitment of p85, Grb2, or Shc. In contrast, inactivation of both SFK motifs severely reduced Lck binding and activation and tyrosine phosphorylation of VP11/12 and reduced (p85) or eliminated (Grb2 and Shc) binding of other signaling proteins. Overall, these data demonstrate the key redundant roles of the VP11/12 SFK-binding motifs in the recruitment and activation of SFKs and indicate that activated SFKs then lead (directly or indirectly) to phosphorylation of the additional motifs involved in recruiting p85, Grb2, and Shc. Thus, VP11/12 appears to mimic an activated growth factor receptor.

Human HERC5 restricts an early stage of HIV-1 assembly by a mechanism correlating with the ISGylation of Gag.

BACKGROUND: The identification and characterization of several interferon (IFN)-induced cellular HIV-1 restriction factors, defined as host cellular proteins or factors that restrict or inhibit the HIV-1 life cycle, have provided insight into the IFN response towards HIV-1 infection and identified new therapeutic targets for HIV-1 infection. To further characterize the mechanism underlying restriction of the late stages of HIV-1 replication, we assessed the ability of IFNbeta-induced genes to restrict HIV-1 Gag particle production and have identified a potentially novel host factor called HECT domain and RCC1-like domain-containing protein 5 (HERC5) that blocks a unique late stage of the HIV-1 life cycle. RESULTS: HERC5 inhibited the replication of HIV-1 over multiple rounds of infection and was found to target a late stage of HIV-1 particle production. The E3 ligase activity of HERC5 was required for blocking HIV-1 Gag particle production and correlated with the post-translational modification of Gag with ISG15. HERC5 interacted with HIV-1 Gag and did not alter trafficking of HIV-1 Gag to the plasma membrane. Electron microscopy revealed that the assembly of HIV-1 Gag particles was arrested at the plasma membrane, at an early stage of assembly. The mechanism of HERC5-induced restriction of HIV-1 particle production is distinct from the mechanism underlying HIV-1 restriction by the expression of ISG15 alone, which acts at a later step in particle release. Moreover, HERC5 restricted murine leukemia virus (MLV) Gag particle production, showing that HERC5 is effective in restricting Gag particle production of an evolutionarily divergent retrovirus. CONCLUSIONS: HERC5 represents a potential new host factor that blocks an early stage of retroviral Gag particle assembly. With no apparent HIV-1 protein that directly counteracts it, HERC5 may represent a new candidate for HIV/AIDS therapy.

The herpes simplex virus 1 vhs protein enhances translation of viral true late mRNAs and virus production in a cell type-dependent manner.

The herpes simplex virus 1 (HSV-1) virion host shutoff protein (vhs) degrades viral and cellular mRNAs. Here, we demonstrate for the first time that vhs also boosts translation of viral true late mRNAs in a cell type-dependent manner and that this effect determines the viral growth phenotype in the respective cell type. Our study was prompted by the detection of stress granules, indicators of stalled translation initiation, in cells infected with vhs mutants but not in wild-type-virus-infected cells. Accumulation of true late-gene products gC and US11 was strongly reduced in the absence of vhs in HeLa cells and several other restrictive cell lines but not in Vero and other permissive cells and was independent of phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Polysome analysis showed that gC and US11 transcripts were poorly translated in vhs-null-virus-infected HeLa cells, while translation of a cellular mRNA was not affected. Interestingly, hippuristanol, an eIF4A inhibitor, produced a similar phenotype in HeLa cells infected with wild-type HSV-1, while Vero cells were much more resistant to the inhibitor. These results suggest that translation of true late-gene transcripts is particularly sensitive to conditions of limited access to translation factors and that vhs is able either to prevent the limiting conditions or to facilitate translation initiation under these conditions. The varied permissivity of cell lines to vhs-null infection may stem from differences in the resilience of the translation machinery or the ability to control the accumulation of mRNAs.