Secondary Envelopment Research Articles

Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.

ABSTRACTVaricella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome.IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral exocytosis and autophagy pathways. Specifically, both LC3-II and Rab11 proteins copurified with some infectious VZV particles. The results suggested that a subpopulation of VZV particles were carried to the cell surface in single-walled vesicles with attributes of an amphisome, an organelle formed from the fusion of an endosome and an autophagosome. Our results also addressed the interpretation of autophagy/xenophagy results with mutated herpes simplex virus lacking its ICP34.5 neurovirulence gene (HSVΔ34.5). The VZV genome lacks an ICP34.5 ortholog, yet we found no evidence of VZV particles housed in a double-membraned autophagosome. In other words, xenophagy, a degradative process documented after infection with HSVΔ34.5, was not observed in VZV-infected cells.

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions.

High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production after secondary envelopment of viral capsids, the encapsidation of HCMV capsids by a lipid bilayer that occurs before virions exit the cell.

Protein-Protein Interactions Suggest Novel Activities of Human Cytomegalovirus Tegument Protein pUL103.

Human cytomegalovirus (HCMV) is an enveloped double-stranded DNA virus that causes severe disease in newborns and immunocompromised patients. During infection, the host cell endosecretory system is remodeled to form the cytoplasmic virion assembly complex (cVAC). We and others previously identified the conserved, multifunctional HCMV virion tegument protein pUL103 as important for cVAC biogenesis and efficient secondary envelopment. To help define its mechanisms of action and predict additional functions, we used two complementary methods, coimmunoprecipitation (co-IP) and proximity biotinylation (BioID), to identify viral and cellular proteins that interact with pUL103. By using the two methods in parallel and applying stringent selection criteria, we identified potentially high-value interactions of pUL103 with 13 HCMV and 18 cellular proteins. Detection of the previously identified pUL103-pUL71 interaction, as well as verification of several interactions by reverse co-IP, supports the specificity of our screening process. As might be expected for a tegument protein, interactions were identified that suggest distinct roles for pUL103 across the arc of lytic infection, including interactions with proteins involved in cellular antiviral responses, nuclear activities, and biogenesis and transport of cytoplasmic vesicles. Further analysis of some of these interactions expands our understanding of the multifunctional repertoire of pUL103: we detected HCMV pUL103 in nuclei of infected cells and identified an ALIX-binding domain within the pUL103 sequence. Human cytomegalovirus (HCMV) is able to reconfigure the host cell machinery to establish a virion production factory, the cytoplasmic virion assembly complex (cVAC). cVAC biogenesis and operation represent targets for development of novel HCMV antivirals. We previously showed that the HCMV tegument protein pUL103 is required for cVAC biogenesis. Using pUL103 as bait, we investigated viral and cellular protein-protein interactions to identify and understand the range of pUL103 functions. We found that pUL103 interacts with cellular antiviral defense systems and proteins involved in organelle biogenesis and transport of cytoplasmic vesicles and is present in infected cell nuclei. These results expand our understanding of the functional repertoire of pUL103 to include activities that extend from the earliest stages of infection through virion assembly and egress.

Morphological development of the structures related to annualism in the ovarian follicle of the killifish Millerichthys robustus (Costa,1995) (Teleostei: Cyprinodontiformes).

Ovaries of five females of the annual fish teleost species Millerichthys robustus were processed, and the development of the cortical alveoli, zona pellucida and secondary envelope during oogenesis were described. We also documented the origin of the cortical alveoli in time and space similar to the Balbiani body; the synthesis of three generations of cortical alveoli and an active zona pellucida prior to vitellogenesis, which is implicated in the entry of oils to the interior of the oocyte. We found that in this species, the diameter of the alveoli is greater than in the other teleost fish species reported in the literature, except for Fundulus heteroclitus, in which the diameter is similar. The thickness of the zona pellucida recorded in M. robustus is the greatest reported to date. Likewise, two periods of secondary envelope deposition were documented: filaments during pre-vitellogenesis and, subsequently, trapeze-shaped projections during the maturation of the oocytes. We report about development of structures that are considered key for the survival of embryos in annual fish during the long periods of diapause in their extreme habitats. The development of peripheral structures described here probably reflects the changes in the physiology of the oocytes in M. robustus. J. Morphol. 277:1219-1230, 2016. © 2016 Wiley Periodicals, Inc.

Conserved Tryptophan Motifs in the Large Tegument Protein pUL36 Are Required for Efficient Secondary Envelopment of Herpes Simplex Virus Capsids.

Herpes simplex virus (HSV) replicates in the skin and mucous membranes, and initiates lytic or latent infections in sensory neurons. Assembly of progeny virions depends on the essential large tegument protein pUL36 of 3,164 amino acid residues that links the capsids to the tegument proteins pUL37 and VP16. Of the 32 tryptophans of HSV-1-pUL36, the tryptophan-acidic motifs (1766)WD(1767) and (1862)WE(1863) are conserved in all HSV-1 and HSV-2 isolates. Here, we characterized the role of these motifs in the HSV life cycle since the rare tryptophans often have unique roles in protein function due to their large hydrophobic surface. The infectivity of the mutants HSV-1(17(+))Lox-pUL36-WD/AA-WE/AA and HSV-1(17(+))Lox-CheVP26-pUL36-WD/AA-WE/AA, in which the capsid has been tagged with the fluorescent protein Cherry, was significantly reduced. Quantitative electron microscopy shows that there were a larger number of cytosolic capsids and fewer enveloped virions compared to their respective parental strains, indicating a severe impairment in secondary capsid envelopment. The capsids of the mutant viruses accumulated in the perinuclear region around the microtubule-organizing center and were not dispersed to the cell periphery but still acquired the inner tegument proteins pUL36 and pUL37. Furthermore, cytoplasmic capsids colocalized with tegument protein VP16 and, to some extent, with tegument protein VP22 but not with the envelope glycoprotein gD. These results indicate that the unique conserved tryptophan-acidic motifs in the central region of pUL36 are required for efficient targeting of progeny capsids to the membranes of secondary capsid envelopment and for efficient virion assembly. Herpesvirus infections give rise to severe animal and human diseases, especially in young, immunocompromised, and elderly individuals. The structural hallmark of herpesvirus virions is the tegument, which contains evolutionarily conserved proteins that are essential for several stages of the herpesvirus life cycle. Here we characterized two conserved tryptophan-acidic motifs in the central region of the large tegument protein pUL36 of herpes simplex virus. When we mutated these motifs, secondary envelopment of cytosolic capsids and the production of infectious particles were severely impaired. Our data suggest that pUL36 and its homologs in other herpesviruses, and in particular such tryptophan-acidic motifs, could provide attractive targets for the development of novel drugs to prevent herpesvirus assembly and spread.

The Mach stem phenomenon for shaped obstacles buried in soil

The paper investigates the explosion characteristics of a below ground event of an explosive charge in proximity to a rigid cylindrical obstacle. The two-dimensional study simulates a line explosive and a parallel long cylindrical structure. The investigation shows that the unloading branch has a negligible effect on the peak pressure envelope whereas shear behavior and explosives burning have a considerable effect and should not be disregarded. The effect of the soil's equation of state and especially the full locking parameter on the pressure distribution on an obstacle has been studied. At a short standoff distance where a steep pressure growth beyond the full compaction point is developed, the pressure distributions envelope shows three maxima values that are located at some distance away from the axis of symmetry. It is different than the common single peak along the axis, in the case of a distant explosion. This effect is more pronounced for a medium having sharper pressure growth in the EOS beyond the full compaction point and for smaller charge standoff distances. The pressure distributions analysis indicates that the appearance of second (absolute-primary) and third (secondary) peaks are caused by the Mach stem effect appearing in a soil medium with full locking. The secondary peak pressure envelope maximum corresponds to the secondary Mach stem phenomenon that does not appear in the case of a planar wall, where the incident angle depends on the wave front curvature only.

Marek's disease virus undergoes complete morphogenesis after reactivation in a T-lymphoblastoid cell line transformed by recombinant fluorescent marker virus.

T-lymphocytes are central targets of Marek's disease, a major chicken disease induced by the oncogenic alphaherpesvirus Marek's disease virus (MDV). T-lymphocyte infection is also associated with immunosuppression and virus latency. To decipher viral morphogenesis in T-lymphocytes, we used the recombinant vRB-1B 47EGFP marker virus to generate a new lymphoblastoid cell line, 3867K, that exhibited typical properties of other MDV-transformed chicken cell lines in term of cell markers, reactivation rate and infectivity. Examination of reactivating EGFP-positive 3867K cells by transmission electron microscopy revealed the presence of most types of herpesvirus particles inside the cells but no extracellular ones. Quantification of virion types indicated only 5% cytoplasmic particles, with 0.5% being mature. This study demonstrated that MDV morphogenesis is complete upon reactivation in T-lymphocytes, albeit with poor efficiency, with a defect in the exit of virions from the nucleus and secondary envelopment, as occurs in infected fibroblasts.

Tegument Assembly and Secondary Envelopment of Alphaherpesviruses.

Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei.

Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication.

It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this protein is co-opted by HSV.

Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids. Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for understanding the biology of HCMV and may help to identify targets for antiviral intervention. Here, we identified tegument protein pUL47 to function in tegumentation and proper trafficking of capsids during late phases of infection. Although pUL47 is not essential for the generation and release of infectious virions, its absence led to massive accumulations of partially tegumented capsids at the cell periphery. Detection of pUL48 at these accumulations indicated a pUL47-independent attachment of pUL48 to the capsid. On the other hand, localization of pUL47 to the vAC during infection appeared to be dependent on tegument protein pUL48, which suggests an intricate interplay of these proteins for normal generation of infectious virus progeny.

Insights into Herpesvirus Tegument Organization from Structural Analyses of the 970 Central Residues of HSV-1 UL36 Protein

The tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions.

An innovative intelligent system based on automatic diagnostic feature extraction for diagnosing heart diseases

An innovative intelligent diagnostic system is proposed in this study, which is primarily reflected in first heart sound (S1) and second heart sound (S2) automatic extraction, frequency feature matrix (FFM) automatic extraction, diagnostic feature y1 and y2 generation based on principal components analysis (PCA) and diagnostic method definition based on the classification boundary curves. Four stages corresponding to the diagnostic system implementation are summarized as follows. Stage 1 describes an envelope ET extraction from heart sound signals. In stage 2, heart sound segmentation points and peaks are first automatically located based on a novel method STMHT, and then S1 and S2 are automatically extracted according to the relationship between the systolic time interval and the diastolic time interval. In stage 3, in the frequency domain, a novel method is first proposed to generate the secondary envelopes SES1 and SES2 for S1 and S2, respectively, and then an STMHT-based FFM is automatically extracted from SES1 and SES2. Finally, the PCA-based diagnostic features y1 and y2 are generated from the FFM. In stage 4, support vector machine (SVM)-based classification curves for the dataset consisting of y1 and y2 are first generated, and then, based on the classification curves, the scatter diagram diagnostic result (SDDR) and numerical diagnostic result (NDR) are defined for diagnosis of heart diseases. The proposed intelligent diagnosis system is validated by sounds from online heart sound databases and by sounds from clinical heart diseases. As a result, the classification accuracies (CA) achieved are 91.7%, 98.8%, 98.4%, 99.8%, 98.7%, 97.8%, 98.1% and 96.5% for the detection of atrial fibrillation (AF), aortic regurgitation(AR), mitral regurgitation (MR), normal sound (NM), pulmonary stenosis (PS), small ventricular septal defect (SVSD), medium ventricular septal defect (MVSD) and large ventricular septal defect (LVSD), respectively.

Deletion of the ORF9p acidic cluster impairs the nuclear egress of varicella-zoster virus capsids.

The protein encoded by ORF9 is essential for varicella-zoster virus (VZV) replication. Previous studies documented its presence in the trans-Golgi network and its involvement in secondary envelopment. In this work, we deleted the ORF9p acidic cluster, destroying its interaction with ORF47p, and this resulted in a nuclear accumulation of both proteins. This phenotype results in an accumulation of primary enveloped capsids in the perinuclear space, reflecting a capsid de-envelopment defect.

Maturation and vesicle-mediated egress of primate gammaherpesvirus rhesus monkey rhadinovirus require inner tegument protein ORF52.

The tegument layer of herpesviruses comprises a collection of proteins that is unique to each viral species. In rhesus monkey rhadinovirus (RRV), a close relative of the human oncogenic pathogen Kaposi's sarcoma-associated herpesvirus, ORF52 is a highly abundant tegument protein tightly associated with the capsid. We now report that ORF52 knockdown during RRV infection of rhesus fibroblasts led to a greater than 300-fold reduction in the viral titer by 48 h but had little effect on the number of released particles and caused only modest reductions in the levels of intracellular viral genomic DNA and no appreciable change in viral DNA packaging into capsids. These data suggested that the lack of ORF52 resulted in the production and release of defective particles. In support of this interpretation, transmission electron microscopy (TEM) revealed that without ORF52, capsid-like particles accumulated in the cytoplasm and were unable to enter egress vesicles, where final tegumentation and envelopment normally occur. TEM also demonstrated defective particles in the medium that closely resembled the accumulating intracellular particles, having neither a full tegument nor an envelope. The disruption in tegument formation from ORF52 suppression, therefore, prevented the incorporation of ORF45, restricting its subcellular localization to the nucleus and appearing, by confocal microscopy, to inhibit particle transport toward the periphery. Ectopic expression of small interfering RNA (siRNA)-resistant ORF52 was able to partially rescue all of these phenotypic changes. In sum, our results indicate that efficient egress of maturing virions and, in agreement with studies on murine gammaherpesvirus 68 (MHV-68), complete tegumentation and secondary envelopment are dependent on intact ORF52. The tegument, or middle layer, of herpesviruses comprises both viral and cellular proteins that play key roles in the viral life cycle. A subset of these proteins is present only within members of one of the three subfamilies (alphaherpesviruses, betaherpesviruses, or gammaherpesviruses) of Herpesviridae. In this report, we show that the gammaherpesvirus-specific tegument protein ORF52 is critical for maturation of RRV, the closest relative of Kaposi's sarcoma-associated herpesvirus (KSHV) (a human cancer-causing pathogen) that has undergone this type of analysis. Without ORF52, the nascent subviral particles are essentially stuck in maturation limbo, unable to acquire the tegument or outer (envelope) layers. This greatly attenuates infectivity. Our data, together with earlier work on a murine homolog, as well as a more distantly related human homolog, provide a more complete understanding of how early protein interactions involving virus-encoded tegument proteins are critical for virus assembly and are also, therefore, potentially attractive therapeutic targets.

Nelfinavir Inhibits Maturation and Export of Herpes Simplex Virus 1

Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment. We confirmed the inhibitory effects of NFV on HSV-1 replication by plaque assay and found that treatment with NFV did not affect capsid assembly, activity of the HSV-1 maturational protease, or formation of DNA-containing capsids in the nucleus. Confocal and electron microscopy studies showed that these capsids were transported to the cytoplasm but failed to complete secondary envelopment and subsequent exit from the cell. Consistent with the microscopy results, a light-scattering band corresponding to enveloped virions was not evident following sucrose gradient rate-velocity separation of lysates from drug-treated cells. Evidence of a possibly related effect of NFV on viral glycoprotein maturation was also discovered. NFV also inhibited the replication of an HSV-1 thymidine kinase mutant resistant to nucleoside analogues such as acyclovir. Given that NFV is neither a nucleoside mimic nor a known inhibitor of nucleic acid synthesis, this was expected and suggests its potential as a coinhibitor or alternate antiviral therapeutic agent in cases of resistance. Nelfinavir (NFV) is a clinically important antiviral drug that inhibits production of infectious HIV. It was reported to inhibit herpesviruses in cell culture. Herpes simplex virus 1 (HSV-1) infections are common and often associated with several diseases. The studies we describe here confirm and extend earlier findings by investigating how NFV interferes with HSV-1 replication. We show that early steps in virus formation (e.g., assembly of DNA-containing capsids in the nucleus and their movement into the cytoplasm) appear to be unaffected by NFV, whereas later steps (e.g., final envelopment in the cytoplasm and release of infectious virus from the cell) are severely restricted by the drug. Our findings provide the first insight into how NFV inhibits HSV-1 replication and suggest that this drug may have applications for studying the herpesvirus envelopment process. Additionally, NFV may have therapeutic value alone or in combination with other antivirals in treating herpesvirus infections.

The interaction of the HSV-1 tegument proteins pUL36 and pUL37 is essential for secondary envelopment during viral egress

The herpes simplex virus type 1 (HSV-1) tegument proteins pUL36 (VP1/2) and pUL37 are essential for viral egress. We previously defined a minimal domain in HSV-1 pUL36, residues 548–572, as important for binding pUL37. Here, we investigated the role of this region in binding to pUL37 and facilitating viral replication. We deleted residues 548–572 in frame in a virus containing a mRFP tag at the N-terminus of the capsid protein VP26 and an eGFP tag at the C-terminus of pUL37 (HSV-1pUL36∆548-572). This mutant virus was unable to generate plaques in Vero cells, indicating that deletion of this region of pUL36 blocks viral replication. Imaging of HSV-1pUL36∆548-572-infected Vero cells, in comparison to parental and resucant, revealed a block in secondary envelopment of cytoplasmic capsids. In addition, immunoblot analysis suggested that failure to bind pUL37 affected the stability of pUL36. This study provides further insight into the role of this essential interaction.

Murine gammaherpesvirus-68 ORF38 encodes a tegument protein and is packaged into virions during secondary envelopment

Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that interactions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gammaherpesviruses including Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvirus-68 (MHV-68) is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion morphogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network (TGN), a site where secondary envelopment takes place.

An Endothelial Cell-Specific Requirement for the UL133-UL138 Locus of Human Cytomegalovirus for Efficient Virus Maturation

Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb' region of the HCMV genome as important for the latent infection in CD34(+) hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138(NULL) virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138(NULL) virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138(NULL) virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138(NULL) virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138(NULL) virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.

Cytosolic herpes simplex virus capsids not only require binding inner tegument protein pUL36 but also pUL37 for active transport prior to secondary envelopment

As the inner tegument proteins pUL36 and pUL37 of alphaherpesviruses may contribute to efficient intracellular transport of viral particles, we investigated their role in cytosolic capsid motility during assembly of herpes simplex virus type 1 (HSV1). As reported previously for pUL36, untagged pUL37 and UL37GFP bound to cytosolic capsids before these acquired outer tegument and envelope proteins. Capsids tagged with CheVP26 analysed by live cell imaging were capable of directed long-distance cytoplasmic transport during the assembly of wild-type virions, while capsids of the HSV1-ΔUL37 or HSV1-ΔUL36 deletion mutants showed only random, undirected motion. The HSV1-ΔUL37 phenotype was restored when UL37GFP had been overexpressed prior to infection. Quantitative immunoelectron microscopy revealed that capsids of HSV1-ΔUL37 still recruited pUL36, whereas pUL37 did not colocalize with capsids of HSV1-ΔUL36. Nevertheless, the cytosolic capsids of neither mutant could undergo secondary envelopment. Our data suggest that pUL36 and pUL37 are important prior to their functions in linking the inner to the outer tegument. Efficient capsid transport to the organelle of secondary envelopment requires recruitment ofpUL37 onto capsids, most likely via its interaction with pUL36, while capsid-associated pUL36 alone is insufficient.

Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy

Electron microscopy (EM) allows visualization of viruses in fixed cells with high resolution. High-pressure freezing for sample fixation in combination with freeze substitution and embedding in resin improves significantly the preservation of cellular structures and specifically of membranes. This advancement allows better visualization of human cytomegalovirus (HCMV) morphogenesis occurring at membranes. To obtain comprehensive information on viral phenotypes from ultrastructural images it is important to also quantify morphological phenotypes. This again can be much refined by three-dimensional visualization after serial sectioning. For elucidation of dynamic processes three-dimensional tomography is extremely helpful. We analysed interaction of HCMV particles with host cell membranes during final envelopment. Both wild-type virus and a viral mutant with impaired envelopment were analysed in fibroblasts, but also using in vivo relevant human endothelial cells and macrophages. The quantification of the EM data showed similar ultrastructural phenotypes regarding the envelopment efficiency in the different cell types indicating similar mechanisms in late stages of virus morphogenesis. Furthermore, thorough analysis of the viral assembly complex (AC) - a virus-induced cytosolic structure - by using three-dimensional visualization techniques combined with a quantitative analysis revealed that the events of final envelopment are equally distributed within the AC irrespective of different local membrane composition.