The UL48 tegument protein of pseudorabies virus is critical for intracytoplasmic assembly of infectious virions

The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-DeltaUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-DeltaUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.     

Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration.

We examined the entry process of herpes simplex virus type 1 (HSV-1) by using infectious virus and previously characterized noninfectious viruses that can bind to cells but cannot penetrate as a result of inactivation of essential viral glycoprotein D (gD) or H (gH). After contact of infectious virus with the cell plasma membrane, discernible changes of the envelope and tegument could be seen by electron microscopy. Noninfectious virions were arrested at distinct steps in interactions with cells. Viruses inactivated by anti-gD neutralizing antibodies attached to cells but were arrested prior to initiation of a visible fusion bridge between the virus and cell. As judged from its increased sensitivity to elution, virus lacking gD was less stably bound to cells than was virus containing gD. Moreover, soluble gD could substantially reduce virus attachment when added to cells prior to or with the addition of virus. Virus inactivated by anti-gH neutralizing antibodies attached and could form a fusion bridge but did not show expansion of the fusion bridge or extensive rearrangement of the envelope and tegument. We propose a model for infectious entry of HSV-1 by a series of interactions between the virion envelope and the cell plasma membrane that trigger virion disassembly, membrane fusion, and capsid penetration. In this entry process, gD mediates a stable attachment that is likely required for penetration, and gH seems to participate in fusion initiation or expansion.     

Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Actin is a component of the compensation mechanism in pseudorabies virus virions lacking the major tegument protein VP22

Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.     

Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49

Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells. In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A. R. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1. However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49. Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles. In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression. In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.     

Murine gammaherpesvirus 68 ORF52 encodes a tegument protein required for virion morphogenesis in the cytoplasm

The tegument, a semiordered matrix of proteins overlying the nucleocapsid and underlying the virion envelope, in viruses in the gamma subfamily of Herpesviridae is poorly understood. Murine gammaherpesvirus 68 (MHV-68) is a robust model for studying gammaherpesvirus virion structure, assembly, and composition, as MHV-68 efficiently completes the lytic phase and productively infects cultured cells. We have found that MHV-68 ORF52 encodes an abundant tegument protein conserved among gammaherpesviruses. Detergent sensitivity experiments revealed that the MHV-68 ORF52 protein is more tightly bound to the virion nucleocapsid than the ORF45 tegument protein but could be dissociated from particles that retained the ORF65 small capsomer protein. ORF52, tagged with enhanced green fluorescent protein or FLAG epitope, localized to the cytoplasm. A recombinant MHV-68 bacterial artificial chromosome mutant with a nonsense mutation incorporated into ORF52 exhibited viral DNA replication, expression of late lytic genes, and capsid assembly and packaging at levels near those of the wild type. However, the MHV-68 ORF52-null virus was deficient in the assembly and release of infectious virion particles. Instead, partially tegumented capsids produced by the ORF52-null mutant accumulated in the cytoplasm, containing conserved capsid proteins, the ORF64 and ORF67 tegument proteins, but virtually no ORF45 tegument protein. Thus, ORF52 is essential for the tegumentation and egress of infectious MHV-68 particles in the cytoplasm, suggesting an important conserved function in gammaherpesvirus virion morphogenesis.     

Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions

The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.     

Role of tegument proteins in herpesvirus assembly and egress

Morphogenesis and maturation of viral particles is an essential step of viral replication. An infectious herpesviral particle has a multilayered architecture, and contains a large DNA genome, a capsid shell, a tegument and an envelope spiked with glycoproteins. Unique to herpesviruses, tegument is a structure that occupies the space between the nucleocapsid and the envelope and contains many virus encoded proteins called tegument proteins. Historically the tegument has been described as an amorphous structure, but increasing evidence supports the notion that there is an ordered addition of tegument during virion assembly, which is consistent with the important roles of tegument proteins in the assembly and egress of herpesviral particles. In this review we first give an overview of the herpesvirus assembly and egress process. We then discuss the roles of selected tegument proteins in each step of the process, i.e., primary envelopment, deenvelopment, secondary envelopment and transport of viral particles. We also suggest key issues that should be addressed in the near future.     

Proteomic characterization of pseudorabies virus extracellular virions

Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virions execute important functions during viral spread and pathogenesis, but a detailed understanding of the composition of PRV virions has been lacking. In this report, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry using two complementary approaches. To exclude proteins present in the extracellular medium that may nonspecifically associate with virions, we also analyzed virions treated with proteinase K and samples prepared from mock-infected cells. Overall, we identified 47 viral proteins associated with PRV virions, 40 of which were previously localized to the capsid, tegument, and envelope layers using traditional biochemical approaches. Additionally, we identified seven viral proteins that were previously undetected in virions, including pUL8, pUL20, pUL32, pUL40 (RR2), pUL42, pUL50 (dUTPase), and Rsp40/ICP22. Furthermore, although we did not enrich for posttranslational modifications, we detected phosphorylation of four virion proteins: pUL26, pUL36, pUL46, and pUL48. Finally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways such as intracellular signaling, mRNA translation and processing, cytoskeletal dynamics, and membrane organization. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.     

Pseudorabies virus envelope glycoproteins gp50 and gII are essential for virus penetration, but only gII is involved in membrane fusion.

To investigate the function of the envelope glycoproteins gp50 and gII of pseudorabies virus in the entry of the virus into cells, we used linker insertion mutagenesis to construct mutant viruses that are unable to express these proteins. In contrast to gD mutants of herpes simplex virus, gp50 mutants, isolated from complementing cells, were able to form plaques on noncomplementing cells. However, progeny virus released from these cells was noninfectious, although the virus was able to adsorb to cells. Thus, the virus requires gp50 to penetrate cells but does not require it in order to spread by cell fusion. This finding indicates that fusion of the virus envelope with the cell membrane is not identical to fusion of the cell membranes of infected and uninfected cells. In contrast to the gp50 mutants, the gII mutant was unable to produce plaques on noncomplementing cells. Examination by electron microscopy of cells infected by the gII mutant revealed that enveloped virus particles accumulated between the inner and outer nuclear membranes. Few noninfectious virus particles were released from the cell, and infected cells did not fuse with uninfected cells. These observations indicate that gII is involved in several membrane fusion events, such as (i) fusion of the viral envelope with the cell membrane during penetration, (ii) fusion of enveloped virus particles with the outer nuclear membrane during the release of nucleocapsids into the cytoplasm, and (iii) fusion of the cell membranes of infected and uninfected cells.     

Cytoplasmic envelopment of human cytomegalovirus requires the postlocalization function of tegument protein pp28 within the assembly compartment

The assembly of herpesvirus remains incompletely defined due to the structural complexity of these viruses. Although the assembly of the capsid of these large DNA viruses is well studied and reasonably well conserved for all members of this diverse family of viruses, the cytoplasmic processes of tegumentation and envelopment are not well understood. The virion of the largest human herpesvirus, human cytomegalovirus (HCMV), contains over 70 virus-encoded proteins that are incorporated during a nuclear and cytoplasmic phase of assembly. Envelopment of this virus requires the function of at least one tegument protein, pp28, the product of the UL99 open reading frame. However, the role of pp28 in the envelopment of HCMV remains undefined. We have generated a pp28 mutant virus that encodes only the first 50 amino acids (aa) of this 190-aa virion protein. This virus is replication impaired and is defective in virus assembly. Characterization of both intracellular and extracellular virions from cells infected with this viral mutant indicated that the decrease in production of infectious virus was secondary to a defect in envelopment and the accumulation of tegumented, noninfectious intracellular particles. Image analysis using fluorescence recovery after photobleaching indicated that the pp28 mutant protein encoded by this virus failed to efficiently accumulate in the virus assembly compartment (AC). Our results suggest that pp28 must accumulate in the AC for efficient envelopment of the particle and provide evidence for a direct role of this tegument protein in the late stages of assembly, such as envelopment.     

Assembly of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22

VP22, the 301-amino-acid phosphoprotein product of the herpes simplex virus type 1 (HSV-1) U(L)49 gene, is incorporated into the tegument during virus assembly. We previously showed that highly modified forms of VP22 are restricted to infected cell nuclei (L. E. Pomeranz and J. A. Blaho, J. Virol. 73:6769-6781, 1999). VP22 packaged into infectious virions appears undermodified, and nuclear- and virion-associated forms are easily differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994). As VP22 packaging-associated undermodification is unique among HSV-1 tegument proteins, we sought to determine the role of VP22 during viral replication. We now show the following. (i) VP22 modification occurs in the absence of other viral factors in cell lines which stably express its gene. (ii) RF177, a recombinant HSV-1 strain generated for this study, synthesizes only the amino-terminal 212 amino acids of VP22 (Delta212). (iii) Delta212 localizes to the nucleus and incorporates into virions during RF177 infection of Vero cells. Thus, the carboxy-terminal region is not required for nuclear localization of VP22. (iv) RF177 synthesizes the tegument proteins VP13/14, VP16, and VHS (virus host shutoff) and incorporates them into infectious virions as efficiently as wild-type virus. However, (v) the loss of VP22 in RF177 virus particles is compensated for by a redistribution of minor virion components. (vi) Mature RF177 virions are identical to wild-type particles based on electron microscopic analyses. (vii) Single-step growth kinetics of RF177 in Vero cells are essentially identical to those of wild-type virus. (viii) RF177 plaque size is reduced by nearly 40% compared to wild-type virus. Based on these results, we conclude that VP22 is not required for tegument formation, virion assembly/maturation, or productive HSV-1 replication, while the presence of full-length VP22 in the tegument is needed for efficient virus spread in Vero cell monolayers.     

Virion proteins of Kaposi's sarcoma-associated herpesvirus

The proteins that compose a herpesvirus virion are thought to contain the functional information required for de novo infection, as well as virion assembly and egress. To investigate functional roles of Kaposi's sarcoma-associated herpesvirus (KSHV) virion proteins in viral productive replication and de novo infection, we attempted to identify virion proteins from purified KSHV by a proteomic approach. Extracellular KSHV virions were purified from phorbol-12-tetradecanoate-13-acetate-induced BCBL-1 cells through double-gradient ultracentrifugation, and their component proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty prominent protein bands were excised and subjected to high-performance liquid chromatography ion trap mass spectrometric analysis. This study led to the identification of 24 virion-associated proteins. These include five capsid proteins, eight envelope glycoproteins, six tegument proteins, and five proteins whose locations in the virions have not yet been defined. Putative tegument proteins encoded by open reading frame 21 (ORF21), ORF33, and ORF45 were characterized and found to be resistant to protease digestion when purified virions were treated with trypsin, confirming that they are located within the virion particles. The ORF64-encoded large tegument protein was found to be associated with capsid but sensitive to protease treatment, suggesting its unique structure and array in KSHV virions. In addition, cellular beta-actin and class II myosin heavy chain type A were found inside KSHV virions and associated with tegument-capsid structure. Identification of KSHV virion proteins makes it possible to study the functional roles of these virion proteins in KSHV replication and pathogenicity.     

Deletion of the Human Cytomegalovirus US17 Gene Increases the Ratio of Genomes per Infectious Unit and Alters Regulation of Immune and Endoplasmic Reticulum Stress Response Genes at Early and Late Times after Infection

Human cytomegalovirus (HCMV) employs numerous strategies to combat, subvert, or co-opt host immunity. One evolutionary strategy for this involves capture of a host gene and then its successive duplication and divergence, forming a family of genes, many of which have immunomodulatory activities. The HCMV US12 family consists of 10 tandemly arranged sequence-related genes in the unique short (US) region of the HCMV genome (US12 to US21). Each gene encodes a protein possessing seven predicted transmembrane domains, patches of sequence similarity with cellular G-protein-coupled receptors, and the Bax inhibitor 1 family of antiapoptotic proteins. We show that one member, US17, plays an important role during virion maturation. Microarray analysis of cells infected with a recombinant HCMV isolate with a US17 deletion (the ΔUS17 mutant virus) revealed blunted host innate and interferon responses at early times after infection (12 h postinfection [hpi]), a pattern opposite that previously seen in the absence of the immunomodulatory tegument protein pp65 (pUL83). Although the ΔUS17 mutant virus produced numbers of infectious particles in fibroblasts equal to the numbers produced by the parental virus, it produced >3-fold more genome-containing noninfectious viral particles and delivered increased amounts of pp65 to newly infected cells. These results suggest that US17 has evolved to control virion composition, to elicit an appropriately balanced host immune response. At later time points (96 hpi), ΔUS17 mutant-infected cells displayed aberrant expression of several host endoplasmic reticulum stress response genes and chaperones, some of which are important for the final stages of virion assembly and egress. Our results suggest that US17 modulates host pathways to enable production of virions that elicit an appropriately balanced host immune response.     

Differing roles of inner tegument proteins pUL36 and pUL37 during entry of herpes simplex virus type 1

Studies with herpes simplex virus type 1 (HSV-1) have shown that secondary envelopment and virus release are blocked in mutants deleted for the tegument protein gene UL36 or UL37, leading to the accumulation of DNA-containing capsids in the cytoplasm of infected cells. The failure to assemble infectious virions has meant that the roles of these genes in the initial stages of infection could not be investigated. To circumvent this, cells infected at a low multiplicity were fused to form syncytia, thereby allowing capsids released from infected nuclei access to uninfected nuclei without having to cross a plasma membrane. Visualization of virus DNA replication showed that a UL37-minus mutant was capable of transmitting infection to all the nuclei within a syncytium as efficiently as the wild-type HSV-1 strain 17⁺ did, whereas infection by UL36-minus mutants failed to spread. Thus, these inner tegument proteins have differing functions, with pUL36 being essential during both the assembly and uptake stages of infection, while pUL37 is needed for the formation of virions but is not required during the initial stages of infection. Analysis of noninfectious enveloped particles (L-particles) further showed that pUL36 and pUL37 are dependent on each other for incorporation into tegument.     

鸭病毒性肠炎病毒强毒株的形态发生学与超微病理学研究

应用透射电镜和超薄切片技术,研究鸭病毒性肠炎病毒(duck enteritis virus,DEV)CH强毒株人工感染成年鸭后,病毒在宿主细胞内的形态发生及各组织器官的超微结构变化.结果表明,感染后不同时间剖杀及发病后死亡鸭的肝、肠、脾、胸腺、法氏囊等组织器官中,均观察到典型的疱疹病毒粒子.病毒主要的靶细胞为淋巴细胞、网状内皮细胞、成纤维细胞、巨噬细胞、血管内皮细胞、肠道上皮细胞、肠道平滑肌细胞和肝细胞等.DEV的核衣壳有空心型、致密核心型、双环型和内壁附有颗粒型四种形态,存在胞核和胞浆两种装配方式.病毒核衣壳可在核内获得皮层,通过核内膜获得囊膜成为成熟病毒;也可通过内外核膜进入胞浆,在其中获得皮层,然后在各种质膜上获得囊膜,最后成熟病毒释放到细胞外.伴随着病毒的复制、装配和成熟,细胞中出现多种核内和胞浆包涵体、核内致密病毒核酸颗粒、微管和中空短管以及胞浆内膜包裹的电子致密小体、双层管等病毒相关结构.超微研究表明,组织细胞有坏死和凋亡两种变化.坏死细胞肿胀甚至破裂,线粒体肿胀空泡化,粗面内质网扩张,核糖体脱落,有的细胞器甚至完全崩解,染色质或固缩或溶解.凋亡细胞则染色质聚集,胞浆凝聚深染,细胞膜上有大量空泡,并有凋亡小体形成.细胞坏死与凋亡往往同时存在,疾病发生过程中,脾、胸腺、法氏囊以及小肠固有层中的淋巴细胞凋亡数量明显增多.     

The herpes simplex virus type 1 DNA packaging protein UL17 is a virion protein that is present in both the capsid and the tegument compartments

The UL17 protein of herpes simplex virus type 1 is essential for packaging the viral genome into the procapsid, a spherical assembly intermediate, and is present in the mature virus particle. We have examined the distribution of UL17 in various assembly products and virions to determine which component of the virus particle UL17 is associated with and at what stage in capsid assembly UL17 is required. UL17 was present in the procapsid, in the DNA-containing angularized C capsid, and in two other angularized capsid forms, A and B, that lack DNA and are thought to be dead-end products. The results suggest that UL17 is a minor capsid protein which is incorporated into the procapsid during assembly of the particle. UL17 was also found in virions and in noninfectious structures known as light (L) particles, which possess a tegument and envelope but lack a capsid. The level of UL17 in these particles was much greater than the amount that could be attributed to capsid contamination of the purified L-particle preparation, suggesting that UL17 is also a tegument protein. The finding that virions contain approximately twofold more UL17 than do C capsids provided further support for the idea that UL17 is present in two different structural components within the mature virion. The UL25 packaging protein, which is also present in virions, was not found in significant amounts in L particles, indicating that it is associated only with the capsid. UL6, the third virion-associated packaging protein, was present in slightly increased levels in L particles.     

Glycoprotein D-negative pseudorabies virus can spread transneuronally via direct neuron-to-neuron transmission in its natural host, the pig, but not after additional inactivation of gE or gI.

Envelope glycoprotein D (gD) is essential for entry of pseudorabies virus (PRV) into cells but is not required for the subsequent steps in virus replication. Phenotypically complemented gD mutants can infect cells and can spread, both in vitro and in mice, by direct cell-to-cell transmission. Progeny virions released by infected cells are noninfectious because they lack gD. The aim of this study was to determine the role of gD in the neuropathogenicity of PRV in its natural host, the pig. We investigated whether gD-negative PRV can spread transneuronally via synaptically linked neurons of the olfactory and trigeminal routes. High doses of a phenotypically complemented gD mutant and gD mutants that are unable to express either gI or gI plus gE were inoculated intranasally in 3- to 5-week-old pigs. Compared with the wild-type virus, the virulence of the gD mutant was reduced. However, pigs inoculated with the gD mutant still developed fever and respiratory signs. Additional inactivation of either gI or gI plus gE further decreased virulence for pigs. Immunohistochemical examination of infected pigs showed that a PRV gD mutant could replicate and spread transneuronally into the central nervous system (CNS). Compared with the wild-type virus, the gD mutant had infected fewer neurons of the CNS on day 2. Nevertheless, on day 3, the gD-negative PRV had infected more neurons and viral antigens were present in second- and third-order neurons in the olfactory bulb, brain stem, and medulla oblongata. In contrast, gD mutants which are unable to express either gI or gI plus gE infected a limited number of first-order neurons in the olfactory epithelium and in the trigeminal ganglion and did not spread transneuronally or infect the CNS. Thus, transsynaptic spread of PRV in pigs can occur independently of gD. Possible mechanisms of transsynaptic transport of PRV are discussed.     

A null mutation in the UL36 gene of herpes simplex virus type 1 results in accumulation of unenveloped DNA-filled capsids in the cytoplasm of infected cells

The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KDeltaUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KDeltaUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KDeltaUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KDeltaUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KDeltaUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.