Intracellular Sorting Signals for Sequential Trafficking of Human Cytomegalovirus UL37 Proteins to the Endoplasmic Reticulum and Mitochondria

Human cytomegalovirus UL37 antiapoptotic proteins, including the predominant UL37 exon 1 protein (pUL37x1), traffic sequentially from the endoplasmic reticulum (ER) through the mitochondrion-associated membrane compartment to the mitochondrial outer membrane (OMM), where they inactivate the proapoptotic activity of Bax. We found that widespread mitochondrial distribution occurs within 1 h of pUL37x1 synthesis. The pUL37x1 mitochondrial targeting signal (MTS) spans its first antiapoptotic domain (residues 5 to 34) and consists of a weak hydrophobicity leader (MTSα) and proximal downstream residues (MTSβ). This MTS arrangement of a hydrophobic leader and downstream proximal basic residues is similar to that of the translocase of the OMM 20, Tom20. We examined whether the UL37 MTS functions analogously to Tom20 leader. Surprisingly, lowered hydropathy of the UL37x1 MTSα, predicted to block ER translocation, still allowed dual targeting of mutant to the ER and OMM. However, increased hydropathy of the MTS leader caused exclusion of the UL37x1 high-hydropathy mutant from mitochondrial import. Conversely, UL37 MTSα replacement with the Tom20 leader did not retarget pUL37x1 exclusively to the OMM; rather, the UL37x1-Tom20 chimera retained dual trafficking. Moreover, replacement of the UL37 MTSβ basic residues did not reduce OMM import. Ablation of the MTSα posttranslational modification site or of the downstream MTS proline-rich domain (PRD) increased mitochondrial import. Our results suggest that pUL37x1 sequential ER to mitochondrial trafficking requires a weakly hydrophobic leader and is regulated by MTSβ sequences. Thus, HCMV pUL37x1 uses a mitochondrial importation pathway that is genetically distinguishable from that of known OMM proteins.During infection of permissive cells, the human cytomegalovirus (HCMV) UL37 immediate-early locus encodes multiple UL37 isoforms (4, 11, 16, 22, 24, 25) (Fig. ​(Fig.1A).1A). The predominant isoform, the UL37 exon 1 protein (pUL37x1), or the viral mitochondrial inhibitor of apoptosis (vMIA), is an essential HCMV gene product required for its growth in humans (17) and in cell culture (14, 20, 36, 47). pUL37x1 induces calcium efflux from the endoplasmic reticulum (ER) (40), regulates viral early gene expression (6, 12), disrupts the F-actin cytoskeleton (35, 40), binds and inactivates Bax at the mitochondrial outer membrane (OMM) (5, 32-34), and inhibits mitochondrial serine protease at late times of infection (27).Open in a separate windowFIG. 1.(A) HCMV UL37 isoforms. UL37 proteins share N-terminal UL37x1 MTS, including a moderately hydrophobic MTSα leader (aa 1 to 22, cylinder), MTSβ proximal basic residues (aa 23 to 29, ++++), downstream acidic (aa 81 to 108, —) and basic (aa 134 to 151, +++) domains. The unique C-terminal sequences encoded by UL37 exon 3 contain an N-glycosylation domain (aa 206 to 391, branches) as well as two additional TM domains (aa 178 to 196 and aa 433 to 459, cylinders). The fusion proteins carrying the full length (pUL37x1 wt_1-163) or MTS (wt_1-36-YFP) with C-terminal fluorophores are represented below. The two UL37x1 antiapoptotic domains are also shown (17). (B) Kinetics of pUL37x1 mitochondrial importation. HFFs were cotransfected with plasmids encoding pUL37x1 wt_1-163-YFP and DsRed1-mito (Clontech). After 2 h, anisomycin (70 μM) was added to the medium. After 12 h, the cells were either fixed with 100% methanol (0 min) or washed with 1× PBS and overlaid with fresh, anisomycin-free medium. The cells were incubated for the indicated times before methanol fixation and confocal imaging. The images were obtained by using comparable settings of aperture and laser power. (C) Colocalization of newly synthesized pUL37x1 with a mitochondrial marker. HFFs transiently transfected with pUL37x1 wt_1-163-YFP (green) were treated with anisomycin-containing medium as in panel B for 12 h. Inhibitor-containing medium was removed, and the cells were washed and overlaid with fresh, anisomycin-free medium for 45 min. At that time, 50 nM MitoTracker Red CMXRos (red, Invitrogen) was added to the medium, followed by incubation for 15 min at 37°C, prior to methanol fixation. The cells were then imaged by confocal microscopy. The panels on the left and center are grayscale. The panel on the right is the color merge of both channels. The small insets are enlargements of the indicated region of interest in the cell. (D) UL37x1 MTS is sufficient for mitochondrial import. HFFs were transiently transfected with expression vectors for wt_1-36-YFP and treated with MitoTracker Red (top row) as described above or for wt_1-163-YFP and DsRed1-mito (bottom). Cells were harvested 24 h later and imaged by confocal microscopy. The left and center panels are grayscale. The panels on the right show merged images of both channels.To accomplish their multiple functions in the cell, HCMV UL37 proteins sequentially traffic from the ER to mitochondria (4, 9, 17, 24-26, 45). The amino-terminal UL37x1 antiapoptotic domain serves as a mitochondrial targeting sequence (MTS) (16, 17, 24, 26). UL37 proteins first translocate into the ER, traffic through the mitochondria-associated membrane (MAM) subcompartment of the ER, and then to the OMM (9, 11, 24-26, 45). The MAM is a lipid-rich subdomain of the ER, which directly contacts mitochondria, allowing for the transfer of lipids from the ER to the OMM and the inner mitochondrial membrane (41), and functionally provides microdomains for efficient coupling of ER to mitochondria calcium transfer (37, 42).The HCMV UL37x1 bipartite MTS includes a weakly hydrophobic leader (MTSα, amino acids [aa] 1 to 22) that is required for ER translocation and mitochondrial import, as well as downstream sequences (MTSβ, aa 23 to 34) that are additionally required for its OMM importation (24) (Fig. ​(Fig.2A).2A). The HCMV UL37 MTS is conserved in the homologous primate CMV UL37x1 genes (28).Open in a separate windowFIG. 2.(A) Conservation of UL37x1 MTS among the primate cytomegaloviruses. The sequences of HCMV, chimpanzee CMV (CCMV), rhesus monkey CMV (RhCMV), and African green monkey (AgmCMV) are shown (top). The boxed areas enclose MTSα, the predicted alpha-helical domain, based upon HMMTOP analysis, within each leader. The MTSβ spans downstream residues 23 to 36. The boldfacing and filled circles indicate identity among primate CMV UL37x1 genes. The HCMV UL37x1 hydrophobic leader was mutated to lowered hydrophobicity by replacement of nonconserved residues V4G, L8G, and L14G while maintaining the same length of the TM in the LH mutant (bottom). The predicted hydrophobicity scores (grand average of hydropathicity, GRAVY, Kyte-Doolittle scale) were calculated for the boxed residues of the wt and LH mutant using ProtParam application on the ExPASy Proteomics Server. (B) Colocalization of UL37x1 LH_1-36-YFP with MitoTracker. HFFs transiently transfected with a vector expressing pUL37x1 LH_1-36-YFP for 24 h were treated with 50 nM MitoTracker as described above and imaged by confocal microscopy. Shown on the left and middle panels are the grayscale images, while the panel on the right is the overlay both channels. The small insets are enlargements of the indicated regions of interest. (C) ER translocation and mitochondrial import of pUL37x1 LH_1-36-YFP and LH_1-163-YFP. HeLa cells were transfected with expression vectors of wt_1-36-YFP, LH_1-36-YFP, or YFP vector alone (top) or wt_1-163-YFP, LH_1-163-YFP, or YFP vector alone (bottom). ER and mitochondrial fractions were isolated as described previously (8, 9). (Top) 10 μg (wt_1-36-YFP and YFP vector alone) or 40 μg (LH_1-36-YFP) of each fraction was analyzed by Westerns with anti-GFP (1:200) antibody. (Bottom) 20 μg of each fraction was analyzed by Western analysis with anti-UL37x1 (DC35, 1:2,500) or Grp75 (1:1,000) antibodies.In contrast, most signal-anchored proteins of the OMM are synthesized in the cytosol as precursors with NH₂-terminal sequences that directly target them to mitochondria (31). Signal-anchored OMM proteins, such as the translocase of the OMM subunits, Tom20 and Tom70 (43, 46), are similar in topology to pUL37x1 and the NH₂-terminal cleavage product, pUL37_NH2, of the UL37 glycoprotein (gpUL37) (26). Tom20 and Tom70 are anchored to the OMM by short NH₂-terminal transmembrane (TM) domains with the bulk of the polypeptides exposed to the cytosol in a type I orientation (21). The important structural elements of their signal anchor sequences are (i) moderate hydrophobicity of the TM domain and (ii) positively charged amino acids in its flanking domain (21, 43). Tom20 is targeted from the cytosol to the OMM by a moderately hydrophobic NH₂-terminal leader (score = 1.826) with a minimal requirement for a net basic charge within one to five residues downstream of the leader (21). The juxtaposed basic residues release the Tom20 hydrophobic leader from the ER-targeting signal recognition particle (SRP) and allow for its direct targeting to the OMM. This arrangement of the Tom20 intracellular sorting signals (20, 41) is similar to that of the MTS of pUL37x1 (22), whose leader, while lower in hydropathy (score = 1.289), is nonetheless ER translocated rather than imported from the cytosol directly into the OMM (24, 26).Our studies were undertaken to define the sequence requirements for pUL37x1 sequential targeting to the ER and to the OMM and to determine whether these signals are distinct from those of other OMM proteins. We examined the potential role of conventional OMM targeting signals (leader hydrophobicity and proximal basic residues) as well as sequences conserved in the homologues of primate CMVs. Unpredictably, UL37x1 MTSβ (aa 23 to 36) did not act analogously to the Tom20 mitochondrial targeting leader. Rather, HCMV UL37x1 sequences retargeted the Tom20 hydrophobic leader to sequential ER to OMM import. Moreover, mutation of conventional mitochondrial targeting basic residues did not markedly alter pUL37x1 mitochondrial import. Similarly, UL37x1 lowered hydrophobicity MTSα mutants dually trafficked to the ER and mitochondria. Conversely, pUL37x1 trafficking was altered by increased hydropathy, which effectively blocked mitochondrial import. From these studies, we conclude that weak hydrophobicity of the pUL37x1 MTSα and downstream residues play a role in directing translocation but involve more complex interplay than previously appreciated. Importantly, two previously unrecognized MTS signals, the consensus MTSα posttranslational modification (PTM) site (²¹SY) and a downstream MTSβ proline-rich domain (PRD, aa 33 to 36), regulated pUL37x1 mitochondrial import.(These studies were performed by C.D.W. in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)     

Analysis of a Charge Cluster Mutation of Herpes Simplex Virus Type 1 UL34 and Its Extragenic Suppressor Suggests a Novel Interaction between pUL34 and pUL31 That Is Necessary for Membrane Curvature around Capsids

Interaction between pUL34 and pUL31 is essential for targeting both proteins to the inner nuclear membrane (INM). Sequences mediating the targeting interaction have been mapped by others with both proteins. We have previously reported identification of charge cluster mutants of herpes simplex virus type 1 UL34 that localize properly to the inner nuclear membrane, indicating interaction with UL31, but fail to complement a UL34 deletion. We have characterized one mutation (CL04) that alters a charge cluster near the N terminus of pUL34 and observed the following. (i) The CL04 mutant has a dominant-negative effect on pUL34 function, indicating disruption of some critical interaction. (ii) In infections with CL04 pUL34, capsids accumulate in close association with the INM, but no perinuclear enveloped viruses, cytoplasmic capsids, or virions or cell surface virions were observed, suggesting that CL04 UL34 does not support INM curvature around the capsid. (iii) Passage of UL34-null virus on a stable cell line that expresses CL04 resulted in selection of extragenic suppressor mutants that grew efficiently using the mutant pUL34. (iv) All extragenic suppressors contained an R229→L mutation in pUL31 that was sufficient to suppress the CL04 phenotype. (v) Immunolocalization and coimmunoprecipitation experiments with truncated forms of pUL34 and pUL31 confirm that N-terminal sequences of pUL34 and a C-terminal domain of pUL31 mediate interaction but not nuclear membrane targeting. pUL34 and pUL31 may make two essential interactions—one for the targeting of the complex to the nuclear envelope and another for nuclear membrane curvature around capsids.Egress of herpesvirus capsids from the nucleus occurs by envelopment of capsids at the inner nuclear membrane (INM) and is followed by de-envelopment at the outer nuclear membrane (ONM). This process can be broken down into a pathway of discrete steps that begin with recruitment of the viral envelopment apparatus to the INM. Herpes simplex virus type 1 (HSV-1) UL34 and UL31 and their homologs in other herpesviruses are required for efficient envelopment at the INM (7, 13, 22, 23, 29). HSV-1 pUL31 and pUL34 are targeted specifically to the INM by a mechanism that requires their interaction with each other (27, 28), and this mutual dependence is a conserved feature of herpesvirus envelopment (9, 14, 27, 28, 32, 33, 39). Localization of these two proteins at the INM results in the recruitment of other proteins, including protein kinase C delta and pUS3, to the nuclear membrane (22, 24, 30). The sequences in HSV-1 pUL34 that mediate interaction with UL31 and that lead to nuclear envelope targeting were mapped to amino acids (aa) 137 to 181 (16). The sequences in the murine cytomegalovirus (MCMV) homolog of UL31, M53, that mediate the nuclear envelope targeting interaction with the UL34 homolog, M50, were mapped to the N-terminal third of the protein in the first of four conserved regions (17), and Schnee et al. subsequently showed that this same region of pUL31 homologs from other families of herpesviruses mediates interaction with the corresponding pUL34 homologs (33).After the targeting of the pUL34/pUL31 complex to the INM, subsequent steps in nuclear egress include, it is thought, (i) local disruption of the nuclear lamina to allow capsid access to the INM, (ii) recognition and docking of capsids by the envelopment apparatus at the INM, (iii) curvature of the inner and outer nuclear membranes around the capsid, (iv) scission of the INM to create an enveloped virion in the space between the INM and ONM, (v) fusion of the virion envelope with the outer nuclear membrane, and (vi) capsid release into the cytoplasm.At least some of the viral and cellular factors critical for nuclear lamina disruption and for de-envelopment fusion have been identified. pUL34, pUL31, and pUS3 of HSV-1 have all been implicated in changes in localization, interaction, and phosphorylation of nuclear lamina components, including lamins A/C and B and the lamina-associated protein, emerin (3, 15, 19, 20, 24, 26, 34, 35). pUS3, pUL31, and glycoproteins B and H have been implicated in de-envelopment of primary virions at the ONM (8, 21, 28, 30, 38).pUL34 and pUL31 are thought to be involved in steps between lamina disruption and de-envelopment, but genetic evidence in infected cells has so far been lacking. Klupp et al. have shown that overexpression of alphaherpesvirus pUL31 and pUL34 in the absence of other viral proteins can induce formation of small vesicles derived from the INM, suggesting a role for these two proteins in membrane curvature around the capsid (12). Tight membrane curvature is an energetically unfavorable event and is thought to be accomplished by coupling curvature to energetically favorable interactions between membrane-bound proteins or protein complexes (reviewed in reference 40). The data of Klupp et al. suggest the possibility that upon recognition of a capsid, pUL31 and pUL34 may interact in a way that induces tight curvature of the INM. Here we present data in support of this hypothesis, showing that a specific point mutation in UL34 induces accumulation of docked capsids at the INM, extragenic suppression of the mutant phenotype is associated with a mutation in UL31, and pUL31 and pUL34 can interact via sequences that are not involved in their INM targeting interaction.We previously published a characterization of a library of 19 charge cluster mutants of pUL34. In each of these mutants, one charge cluster (defined as a group of five consecutive amino acids in which two or more of the residues have charged side chains) was mutated such that the charged residues were replaced by alanine. Six of the 19 charge cluster mutants tested failed to complement replication of UL34-null virus, indicating that they disrupt essential functions of pUL34. Interestingly, five of the six noncomplementing mutants were synthesized at levels comparable to that of wild-type UL34 and localized normally to the nuclear envelope, suggesting that they were unimpaired in their ability to make a nuclear envelope targeting interaction with UL31. In order to identify essential functions of pUL34 downstream of nuclear envelope targeting, we have undertaken a detailed study of the behavior and interactions of these mutants.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus

The herpes simplex virus type 1 (HSV-1) gene UL12 encodes a conserved alkaline DNase with orthologues in all herpesviruses. The HSV-1 UL12 gene gives rise to two separately promoted 3′ coterminal mRNAs which encode distinct but related proteins: full-length UL12 and UL12.5, an amino-terminally truncated form that initiates at UL12 codon 127. Full-length UL12 localizes to the nucleus where it promotes the generation of mature viral genomes from larger precursors. In contrast, UL12.5 is predominantly mitochondrial and acts to trigger degradation of the mitochondrial genome early during infection. We examined the basis for these very different subcellular localization patterns. We confirmed an earlier report that the amino-terminal region of full-length UL12 is required for nuclear localization and provide evidence that multiple nuclear localization determinants are present in this region. In addition, we demonstrate that mitochondrial localization of UL12.5 relies largely on sequences located between UL12 residues 185 and 245 (UL12.5 residues 59 to 119). This region contains a sequence that resembles a typical mitochondrial matrix localization signal, and mutations that reduce the positive charge of this element severely impaired mitochondrial localization. Consistent with matrix localization, UL12.5 displayed a detergent extraction profile indistinguishable from that of the matrix protein cyclophilin D. Mitochondrial DNA depletion required the exonuclease activity of UL12.5, consistent with the idea that UL12.5 located within the matrix acts directly to destroy the mitochondrial genome. These results clarify how two highly related viral proteins are targeted to different subcellular locations with distinct functional consequences.All members of the Herpesviridae encode a conserved alkaline DNase that displays limited homology to bacteriophage λ red α (2, 24), an exonuclease that acts in conjunction with the synaptase red β to catalyze homologous recombination between DNA molecules (23). The most thoroughly characterized member of the herpesvirus alkaline nuclease family is encoded by the herpes simplex virus type 1 (HSV-1) gene UL12 (7, 9, 22). HSV-1 UL12 has both endo- and exonuclease activity (15-17, 36) and binds the viral single-stranded DNA binding protein ICP8 (37, 39) to form a recombinase that displays in vitro strand exchange activity similar to that for red α/β (27). UL12 localizes to the nucleus (26) where it plays an important, but as-of-yet ill-defined, role in promoting the production of mature packaged unit-length linear progeny viral DNA molecules (12, 20), perhaps via a recombination mechanism (27, 28). The importance of UL12 is documented by the observation that UL12 null mutants display a ca. 1,000-fold reduction in the production of infectious progeny virions (41).HSV-1 also produces an amino-terminally truncated UL12-related protein termed UL12.5, which is specified by a separately promoted mRNA that initiates within UL12 coding sequences (7, 9, 21). UL12.5 is translated in the same reading frame as UL12 but initiates at UL12 codon 127 and therefore lacks the first 126 amino acid residues of the full-length protein. UL12.5 retains the nuclease and ICP8 binding activities of UL12 (4, 14, 26) but does not accumulate to high levels in the nucleus (26) and is unable to efficiently substitute for UL12 in promoting viral genome maturation (14, 21). We recently showed that UL12.5 localizes predominantly to mitochondria, where it triggers massive degradation of the host mitochondrial genome early during HSV infection (31). Mammalian mitochondrial DNA (mt DNA) is a 16.5-kb double-stranded circle located within the mitochondrial matrix that encodes 13 proteins involved in oxidative phosphorylation and the RNA components of the mitochondrial translational apparatus (reviewed in reference 10). Inherited mutations that inactivate or deplete mt DNA impair oxidative phosphorylation, leading to a wide range of pathological conditions, including neuropathy and myopathy (reviewed in references 8 and 40). Thus, although the contribution of mt DNA depletion to the biology of HSV infection has yet to be determined, it likely has a major negative impact on host cell functions.UL12 and UL12.5 provide a striking example of a pair of highly related proteins that share a common biochemical activity yet differ markedly in subcellular location and biological function. The basis for their distinct subcellular localization patterns is of considerable interest, as the only difference in the primary sequences is that UL12.5 lacks the first 126 residues of UL12. Reuven et al. (26) demonstrated that this UL12-specific region contains one or more signals able to target enhanced green fluorescent protein (eGFP) to the nucleus. However, the determinants of the mitochondrial localization of UL12.5 have not been previously examined. Most proteins that are imported into the mitochondrial matrix bear a matrix targeting sequence that is located at or close to the amino terminus (reviewed in references 25 and 38). We speculated that UL12.5 bears such an amino-terminal matrix targeting sequence and that the function of this element is masked in the full-length UL12 protein by the UL12-specific amino-terminal extension, which contains the nuclear localization signal(s) (NLS). Our results broadly support this hypothesis and indicate that the mitochondrial localization sequence of UL12.5 is located ca. 60 residues from its N terminus.     

The Flexible Loop of the Human Cytomegalovirus DNA Polymerase Processivity Factor ppUL44 Is Required for Efficient DNA Binding and Replication in Cells

Phosphoprotein ppUL44 of the human cytomegalovirus (HCMV) DNA polymerase plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit pUL54 by tethering it to the DNA. Here, for the first time, we examine in living cells the function of the highly flexible loop of ppUL44 (UL44-FL; residues 162 to 174 [PHTRVKRNVKKAP¹⁷⁴]), which has been proposed to be directly involved in ppUL44''s interaction with DNA. In particular, we use a variety of approaches in transfected cells to characterize in detail the behavior of ppUL44Δloop, a mutant derivative in which three of the five basic residues within UL44-FL are replaced by nonbasic amino acids. Our results indicate that ppUL44Δloop is functional in dimerization and binding to pUL54 but strongly impaired in binding nuclear structures within the nucleus, as shown by its inability to form nuclear speckles, reduced nuclear accumulation, and increased intranuclear mobility compared to wild-type ppUL44. Moreover, analysis of cellular fractions after detergent and DNase treatment indicates that ppUL44Δloop is strongly reduced in DNA-binding ability, in similar fashion to ppUL44-L86A/L87A, a point mutant derivative impaired in dimerization. Finally, ppUL44Δloop fails to transcomplement HCMV oriLyt-dependent DNA replication in cells and also inhibits replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for double-stranded DNA binding. UL44-FL thus emerges for the first time as an important determinant for HCMV replication in cells, with potential implications for the development of novel antiviral approaches by targeting HCMV replication.The Betaherpesviridae subfamily member human cytomegalovirus (HCMV) is a major human pathogen, causing serious disease in newborns following congenital infection and in immunocompromised individuals (28, 42). Replication of its double-stranded DNA (dsDNA) genome occurs in the nuclei of infected cells via a rolling-circle process mediated by 11 virally encoded proteins (32, 33), including a viral DNA polymerase holoenzyme, comprising a catalytic subunit, pUL54, and a proposed processivity factor, ppUL44 (14). ppUL44 is readily detectable in virus-infected cells as a 52-kDa phosphoprotein of 433 amino acids with strong dsDNA-binding ability (30, 45). Defined as a “polymerase accessory protein” (PAP) whose function is highly conserved among herpesviruses, ppUL44 is an essential factor for viral replication in cultured cells and hence represents a potential therapeutic target to combat HCMV infection (39). It is a multifunctional protein capable of self-associating (5, 10), as well as interacting with a plethora of viral and host cell proteins, including the viral kinase pUL97 (29), the viral transactivating protein pUL84 (15), the viral uracil DNA glycosylase ppUL114 (37), and the host cell importin α/β (IMPα/β) heterodimer, which is responsible for its transport into the nucleus (4). The activities of ppUL44 as a processivity factor, including the ability to dimerize, as well as bind to, pUL54 and DNA, reside in the N-terminal portion (26, 45), whereas the C terminus is essential for phosphorylation-regulated, IMPα/β-dependent nuclear targeting of ppUL44 monomers and dimers (4-6). Once within the nucleus, ppUL44 is thought to tether the DNA polymerase holoenzyme to the DNA, thus increasing its processivity (14).Recent studies have identified specific residues responsible for ppUL44 interaction with pUL54, as well as for the interaction with IMPα/β and homodimerization (4, 10, 27, 41). The crystal structure of ppUL44''s N-terminal domain (Fig. ​(Fig.1A)1A) reveals striking similarity to that of other processivity factors, such as proliferating cell nuclear antigen (PCNA) and its herpes simplex virus type 1 (HSV-1) homologue UL42 (10, 46). Unlike the PCNA trimeric ring, however, both ppUL44 and UL42, which bind to dsDNA as dimers and monomers, respectively, have an open structure, which is believed to be the basis for their ability to bind to dsDNA in the absence of clamp loaders and ATP (9, 10, 46). Both ppUL44 and UL42 share a very basic “back” face, which appears to be directly involved in DNA binding via electrostatic interactions (19, 22, 23, 38, 46). One striking difference between ppUL44 and UL42 is the presence on the former of an extremely basic flexible loop (UL44-FL, PHTRVKRNVKKAP¹⁷⁴) protruding from the basic back face of the protein (Fig. ​(Fig.1A).1A). Comparison of ppUL44 homologues from different betaherpesviruses, including human herpesvirus 6 (HHV-6) and 7 (HHV-7), showed that all possess similar sequences in the same position (44) (Fig. ​(Fig.1B),1B), implying functional significance.Open in a separate windowFIG. 1.The highly conserved flexible loop (residues 162 to 174) within ppUL44 protrudes from ppUL44 basic face and is important for efficient nuclear accumulation and localization in nuclear speckles. (A) Schematic representation of ppUL44 N-terminal domain (residues 9 to 270, protein data bank accession no. 1T6L) generated using the Chimera software based on the published crystal structure (10, 35). Color: yellow, β-sheets; red, α-helices. Residues involved in ppUL44 dimerization (P85, L86, L87, L93, F121, and M123), as well as basic residues potentially involved in DNA binding (K21, R28, K32, K35, K128, K158, K224, and K237), are represented as spacefill in orange and green, respectively. Residues P162 and C175, in black, are indicated by arrowheads, while residues 163 to 174 are not visible in the electron density maps and could potentially extend in the cavity formed by ppUL44''s basic face to directly contact DNA. Residues forming ppUL44 connector loop (128-142) are in blue. (B) Sequence alignment between HCMVUL44-FL and the corresponding region of several betaherpesvirus ppUL44 homologues. The single-letter amino acid code is used, with basic residues in boldface. (C) COS-7 cells were transfected to express the indicated GFP fusion proteins and imaged live 16 h after transfection using CLSM and a 40× water immersion objective lens. (D) Quantitative results for the Fn/c and speckle formation for GFP-UL44 fusion proteins. The data for the Fn/c ratios represent the mean Fn/c relative to each protein indicated as a percentage of the mean Fn/c relative to GFP-UL44wt ± the standard error of the mean, with the number of analyzed cells in parentheses. (E) HEK 293 cells expressing the indicated GFP-UL44 fusion proteins were lysed, separated by PAGE, and analyzed by Western blotting as described in Materials and Methods, using either the anti-GFP or the anti-α-tubulin MAbs.A recent study revealed that substitution of UL44-FL basic residues with alanine residues strongly impairs the ability of a bacterially expressed N-terminal fragment of UL44 to bind 30-bp dsDNA oligonucleotides in vitro, suggesting that UL44-FL could be involved in dsDNA-binding during viral replication (22). However, the role of UL44-FL in mediating the binding of full-length UL44 to dsDNA in cells and its role in DNA replication have not been investigated. We use here a variety of approaches to delineate the role of UL44-FL in living cells, our data revealing that UL44-FL is not required for ppUL44 dimerization or binding to the catalytic subunit pUL54 but is crucial for HCMV oriLyt-dependent DNA replication, being required for the formation of nuclear aggregates, nuclear accumulation/retention, and DNA binding of ppUL44. Importantly, ppUL44Δloop exhibits a transdominant-negative phenotype, inhibiting HCMV oriLyt-dependent DNA replication in the presence of wild-type ppUL44, possibly via formation of heterodimers defective for dsDNA binding. This underlines ppUL44-FL as an important determinant for HCMV replication in a cellular context for the first time, with potential implications for the development of novel antiviral approaches.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Onset of Human Cytomegalovirus Replication in Fibroblasts Requires the Presence of an Intact Vimentin Cytoskeleton

Like all viruses, herpesviruses extensively interact with the host cytoskeleton during entry. While microtubules and microfilaments appear to facilitate viral capsid transport toward the nucleus, evidence for a role of intermediate filaments in herpesvirus entry is lacking. Here, we examined the function of vimentin intermediate filaments in fibroblasts during the initial phase of infection of two genotypically distinct strains of human cytomegalovirus (CMV), one with narrow (AD169) and one with broad (TB40/E) cell tropism. Chemical disruption of the vimentin network with acrylamide, intermediate filament bundling in cells from a patient with giant axonal neuropathy, and absence of vimentin in fibroblasts from vimentin^−/− mice severely reduced entry of either strain. In vimentin null cells, viral particles remained in the cytoplasm longer than in vimentin^+/+ cells. TB40/E infection was consistently slower than that of AD169 and was more negatively affected by the disruption or absence of vimentin. These findings demonstrate that an intact vimentin network is required for CMV infection onset, that intermediate filaments may function during viral entry to facilitate capsid trafficking and/or docking to the nuclear envelope, and that maintenance of a broader cell tropism is associated with a higher degree of dependence on the vimentin cytoskeleton.Human cytomegalovirus (CMV) is a ubiquitous herpesvirus that can cause serious disease in immunocompromised individuals (8, 58). Virtually all cell types, with the exception of lymphocytes and polymorphonuclear leukocytes, can support CMV replication in vivo (80), and this remarkably broad tropism is at the basis of the numerous clinical manifestations of CMV infection (8, 58). The range of permissive cells in vitro is more limited, with human fibroblasts (HF) and endothelial cells being the most widely used for propagation of clinical isolates. Two extensively studied strains, AD169 and Towne, were generated by serial passage of tissue isolates in HF for the purpose of vaccine development (22, 68). During this process, both strains accumulated numerous genomic changes (11) and lost the ability to grow in cell types other than HF. By contrast, propagation in endothelial cells produced strains with more intact genomes and tropism, such as TB40/E, VR1814, TR, and PH (59, 80).The viral determinants of endothelial and epithelial cell tropism have recently been mapped to the UL128-UL131A (UL128-131A) genomic locus (32, 92, 93). Each of the products of the UL128, UL130, and UL131A genes is independently required for tropism and participates in the formation of a complex at the surface of the virion with the viral glycoproteins gH and gL (74, 93), which can also independently associate with gO (45). The gH/gL/UL128-131A complex appears to be required for entry into endothelial cells by endocytosis, followed by low-pH-dependent fusion of the virus envelope with endosomal membranes (73, 74) although some virus strains expressing the UL128-UL131A genes do not require endosome acidification for capsid release (66, 79).HF-adapted strains consistently contain mutations in the UL128-131A genes (32). Loss of endothelial cell tropism in AD169 has been associated with a frameshift mutation in the UL131A gene, leading to the production of a truncated protein and to the loss of the gH/gL/UL128-131A complex, but not the gH/gL/gO complex, from the surface of AD169 virions (1, 3, 92). Reestablishment of wild-type UL131A expression in AD169 by repair of the UL131A gene mutation or by cis-complementation yielded viruses with restored tropism for endothelial cells but with reduced replication capacities in HF (1, 92). Interestingly, the efficiencies of entry of wild-type and repaired or complemented AD169 viruses were comparable, suggesting that the presence of UL131A did not interfere with the initial steps of infection in HF but negatively affected virion release (1, 92).The cellular determinants of CMV tropism are numerous and have not been fully identified. Virus entry begins with virion attachment to the ubiquitously expressed heparan sulfate proteoglycans at the cell surface (17), followed by engagement of one or more receptor(s) including the integrin heterodimers α2β1, α6β1, and α_Vβ3 (23, 39, 94); the platelet-derived growth factor-α receptor (84); and the epidermal growth factor receptor, whose role in CMV entry is still debated (38, 95).Subsequent delivery of capsids into the cytoplasm requires fusion of the virus envelope with cellular membranes. Release of AD169 capsids in HF occurs mainly by fusion at the plasma membrane at neutral pH although incoming virions have also been found within phagolysosome-like vacuoles (16, 83). Fusion with the plasmalemma appears to be mediated by the gH/gL/gO complex as AD169 virions do not contain the gH/gL/UL128-131A complex, and infectivity of a gO mutant was severely reduced (37). The mechanism used by strain TB40/E to penetrate into HF has not been described but was assumed to be similar to that of AD169 (80) even though TB40/E virions contain both gH/gL/gO and gH/gL/UL128-131A complexes.Transport of released, de-enveloped capsids toward the nucleus is mediated by cellular microtubules, and treatment of Towne-infected HF with microtubule-depolymerizing agents substantially reduced expression levels of the viral nuclear immediate-early protein 1 (IE1) (64). Depolymerization of actin microfilaments was also observed in HF as early as 10 to 20 min postinfection with the Towne strain while stress fiber disappearance was evident at 3 to 5 h postinfection (hpi) with AD169 (4, 42, 54), suggesting that microfilament rearrangement may be required to facilitate capsid transition through the actin-rich cell cortex.The role of intermediate filaments (IF) in CMV infection not been studied. In vivo, expression of the IF protein vimentin is specific to cells of mesenchymal origin like HF and endothelial cells (12). Although the phenotype of vimentin^−/− (vim⁻) mice appears to be mild (15), vimentin-null cells display numerous defects including fragmentation of the Golgi apparatus (26), development of nuclear invaginations in some instances (76), and reduced formation of lipid droplets, glycolipids, and autophagosomes (29, 52, 87). Vimentin IF interact with integrins α2β1, α6β4, and α_Vβ3 at the cell surface and participate in recycling of integrin-containing endocytic vesicles (40, 41). They also accompany endocytic vesicles during their perinuclear accumulation (34), regulate endosome acidification by binding to the adaptor complex AP-3 (86), control lysosome distribution into the cytoplasm (87), and promote directional mobility of cellular vesicles (69). The vimentin cytoskeleton is tightly associated with the nuclear lamina (10) and was shown to anchor the nucleus within the cell, to mediate force transfer from the cell periphery to the nucleus, and to bind to repetitive DNA sequences as well as to supercoiled DNA and histones in the nuclear matrix (56, 89, 90). Microtubules and vimentin IF form close connections in HF (30). Drug-induced disassembly of the microtubule network alters IF synthesis and organization, leading to the collapse of vimentin IF into perinuclear aggregates (2, 25, 30, 70). By contrast, coiling of IF after injection of antivimentin antibodies has no effect on the structure of microtubules (28, 46, 53), indicating that the interaction between vimentin IF and microtubules is functionally unidirectional.In this work, we sought to assess the role of the vimentin cytoskeleton in CMV entry. We hypothesized that vimentin association with integrins at the cell surface, with endosomes and microtubules in the cytoplasm, and with the lamina and matrix in the nucleus might facilitate viral binding and penetration, capsid transport toward the nucleus, and nuclear deposition of the viral genome.We found that, akin to microtubules, vimentin IF do not depolymerize during entry of either AD169 or TB40/E. In comparison to AD169, onset of TB40/E infection in HF was delayed, and the proportion of infected cells was reduced. Virus entry was negatively affected by the disruption of vimentin networks after exposure to acrylamide (ACR), by IF bundling in cells from patients with giant axonal neuropathy (GAN), and by the absence of vimentin IF in vim⁻ mouse embryo fibroblasts (MEF). In vim⁻ cells, the efficiency of particles trafficking toward the nucleus appeared significantly lower than in vimentin^+/+ (vim⁺) cells, and in each instance the negative effects were more pronounced in TB40/E-infected cells than in AD169-infected cells. These data show that vimentin is required for efficient entry of CMV into HF and that the endotheliotropic strain TB40/E is more reliant on the presence and integrity of vimentin IF than the HF-adapted strain AD169.     

Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.The herpesvirus virion is composed of an icosahedral nucleocapsid containing the viral genome, an envelope of cellular origin with inserted viral (glyco)proteins, and a tegument which links nucleocapsid and envelope comparable to the matrix of RNA viruses. The herpesvirus tegument contains a multitude of viral and cellular proteins (reviewed in references 45 and 46). Tegument proteins execute various regulatory and structural functions, including activation of viral gene expression (2), modulation of the host cell for virus replication (26, 51, 55), and mediation of posttranslational modification of proteins (10, 27, 50). Numerous interactions have been identified among tegument proteins, between tegument and capsid proteins, and between tegument and envelope proteins (7, 14, 16, 18, 33, 36, 42, 53, 58-61).The largest tegument proteins found in the herpesviruses are homologs of pUL36 of herpes simplex virus type 1 (HSV-1). Pseudorabies virus (PrV) pUL36 consists of 3,084 amino acids (aa) with a molecular mass of 324 kDa (33). PrV and HSV-1 pUL36 are essential for viral replication (13, 15). In their absence, nonenveloped nucleocapsids accumulate in the cytoplasm. Whereas in several studies nuclear stages like cleavage and packaging of the viral DNA as well as nuclear egress were not found affected (13, 15), another study indicated an effect of pUL36 deletion on PrV nuclear egress (41).pUL36 homologs complex with another tegument protein, pUL37, as has been shown for HSV-1 (59), PrV (15, 33), and human cytomegalovirus (3, 23), and the interacting region on pUL36 has been delineated for PrV (33) and identified at the amino acid level for HSV-1 (47). Deletion of the pUL37 interaction domain from PrV pUL36 impedes virion formation in the cytosol but does not block it completely, yielding a phenotype similar to that of a pUL37 deletion mutant (31). This indicates an important but nonessential role for pUL37 and the pUL37 interaction domain in pUL36 in virion formation (15). In contrast, absence of pUL37 completely blocks virion formation in HSV-1 (11, 38).pUL36 is stably attached to the nucleocapsid (39, 43, 56), remains associated with incoming particles during transport along microtubules to the nuclear pore (21, 40, 52), and is required for intracellular nucleocapsid transport during egress (41). In contrast, absence of pUL37 delays nuclear translocation of incoming PrV nucleocapsids but does not abolish it (35). HSV-1 pUL36 is involved not only in transport but also in docking of nucleocapsids to the nuclear pore (9), and proteolytic cleavage of pUL36 appears to be necessary for release of HSV-1 DNA into the nucleus (24).Immunoelectron microscopical studies of PrV-infected cells showed that pUL36 is added to nucleocapsids prior to the addition of pUL37 (33). Since neither pUL36 nor pUL37 was detected on primary enveloped PrV virions, it was concluded that acquisition of tegument takes place in the cytoplasm (20). However, conflicting data exist whether pUL36 is present in the nucleus, and whether it is already added onto the capsids in this cellular compartment. Indirect immunofluorescence, immunoelectron microscopy and mass spectrometry of intranuclear capsids yielded discrepant results. By immunofluorescence HSV-1 pUL36 was detected both in the cytoplasm and in the nucleus (1, 42, 48). However, whereas one study detected the protein on nuclear C-capsids by Western blotting (6), it was not found by cryo-electron microscopy and mass spectrometry (57). In contrast, the C terminus of PrV pUL36 was suggested to direct pUL36 to capsid assemblons in the nucleus (37) by binding to capsid-associated pUL25 (8), although pUL36 could not be detected in the nucleus during PrV infection (33). These differing results in HSV-1 and between HSV-1 and PrV might be due to the fact that pUL36 could be processed during the replication cycle and that the resulting subdomains may exhibit selective localization patterns (24, 28).Amino acid sequence analyses of HSV-1 and PrV pUL36 revealed several putative nuclear localization signals (NLS) (1, 4, 5, 49). HSV-1 pUL36 contains four of these NLS motifs (49). Functionality in nuclear localization of a reporter protein was shown for the NLS motif at aa 425 (1). This motif is highly conserved in herpesvirus pUL36 homologs pointing to an important function (1). Besides this conserved NLS (designated in this report as NLS1), two other NLS motifs are predicted in PrV pUL36. One is located adjacent to NLS1 (aa 288 to 296) at aa 315 to 321 (NLS2), and a third putative NLS motif is present in the C-terminal half of the protein (aa 1679 to 1682; NLS3) (4). Whereas this may be indicative for a role for pUL36 inside the nucleus, NLS motifs might also be involved in transport to the nucleus along microtubules (54) and docking at the nuclear pore complex (49).The discrepancy in pUL36 localization and the putative presence of pUL36 cleavage products with specialized functions and localization prompted us to generate monospecific antisera covering the major part of PrV pUL36 and to study localization of PrV pUL36 by immunofluorescence during viral replication and after transient transfection and by immunoelectron microscopy of infected cells.