Nucleolin is required for an efficient herpes simplex virus type 1 infection

Productive infection by herpes simplex virus type 1 (HSV-1), which occurs in the host cell nucleus, is accompanied by dramatic modifications of the nuclear architecture, including profound alterations of nucleolar morphology. Here, we show that the three most abundant nucleolar proteins--nucleolin, B23, and fibrillarin--are redistributed out of the nucleoli as a consequence of HSV-1 infection. We show that the amount of nucleolin increases progressively during the course of infection. We demonstrate for the first time that a nucleolar protein, i.e., nucleolin, colocalizes with ICP8 in the viral replication compartments, at the time when viral replication is effective, suggesting an involvement of nucleolin in the HSV-1 DNA replication process. At later times of infection, a granular form of nucleolin localizes to the cytoplasm, in structures that display the characteristic features of aggresomes, indicating that this form of nucleolin is very probably destined for degradation. The delocalization of nucleolin from the nucleoli requires the viral ICP4 protein or a factor(s) whose expression involves ICP4. Using small interfering RNA technology, we show that viral replication requires a high level of nucleolin expression, demonstrating for the first time a direct role for a nucleolar protein in herpes simplex virus biology.     

The fibroblast growth factor receptor is not required for herpes simplex virus type 1 infection.

The early events mediating herpes simplex virus type 1 (HSV-1) infection include virion attachment to cell surface heparan sulfates and subsequent penetration. Recent evidence has suggested that the high-affinity fibroblast growth factor (FGF) receptor mediates HSV-1 entry. This report presents three lines of experimental evidence showing that the high-affinity FGF receptor is not required for HSV-1 infection. First, rat L6 myoblasts lacking FGF receptors were as susceptible to HSV-1 infection as L6 cells genetically engineered to express the FGF receptor. Second, a soluble FGF receptor fragment that inhibited FGF binding and receptor activation did not inhibit HSV-1 infection. Finally, basic FGF (but not acidic FGF) inhibited HSV-1 infection in L6 cells lacking FGF receptors, presumably by blocking cell surface heparan sulfates also required for HSV-1 infection. These results show that the high-affinity FGF receptor is not required for HSV-1 infection but instead that specific low-affinity basic FGF binding sites are used for HSV-1 infection.     

The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.     

Sialic acid on herpes simplex virus type 1 envelope glycoproteins is required for efficient infection of cells

Herpes simplex virus type 1 (HSV-1) envelope proteins are posttranslationally modified by the addition of sialic acids to the termini of the glycan side chains. Although gC, gD, and gH are sialylated, it is not known whether sialic acids on these envelope proteins are functionally important. Digestion of sucrose gradient purified virions for 4 h with neuraminidases that remove both alpha2,3 and alpha2,6 linked sialic acids reduced titers by 1,000-fold. Digestion with a alpha2,3-specific neuraminidase had no effect, suggesting that alpha2,6-linked sialic acids are required for infection. Lectins specific for either alpha2,3 or alpha2,6 linkages blocked attachment and infection to the same extent. In addition, the mobility of gH, gB, and gD in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was altered by digestion with either alpha2,3 specific neuraminidase or nonspecific neuraminidases, indicating the presence of both linkages on these proteins. The infectivity of a gC-1-null virus, DeltagC2-3, was reduced to the same extent as wild-type virus after neuraminidase digestion, and attachment was not altered. Neuraminidase digestion of virions resulted in reduced VP16 translocation to the nucleus, suggesting that the block occurred between attachment and entry. These results show for the first time that sialic acids on HSV-1 virions play an important role in infection and suggest that targeting virion sialic acids may be a valid antiviral drug development strategy.     

Recombination during early herpes simplex virus type 1 infection is mediated by cellular proteins

Homologous recombination was examined in cells infected with herpes simplex virus type I. Circular and linear DNA with directly repeated sequences was introduced as recombination substrates into cells. Recombination was measured either by origin-dependent amplification of recombination products or by recombination-dependent expression of luciferase from a disrupted gene. Homologous recombination in baby hamster kidney cells converted linear DNA to circular templates for DNA replication and luciferase expression in the complete absence of virus. The products of homologous recombination were efficiently amplified by the viral replication apparatus. The efficiency of recombination was dependent on the structure of the substrate as well as the cell type. Linear DNA with the direct repeats at internal positions failed to recombine in Balb/c 3T3 cells and induced p53-dependent apoptosis. In contrast, linear DNA with directly repeated sequences precisely at the ends recombined and replicated in 3T3 cells. Homologous recombination in baby hamster kidney cells did not depend on the position of the repeated sequences. We conclude that homologous recombination is independent of viral gene functions and that it is likely to be carried out by cellular proteins. We suggest that homologous recombination between directly repeated sequences in the linear herpes simplex virus type 1 chromosome may help to avoid p53-dependent apoptosis and to promote viral DNA replication.     

Linker histones are mobilized during infection with herpes simplex virus type 1

Histones interact with herpes simplex virus type 1 (HSV-1) genomes and localize to replication compartments early during infections. However, HSV-1 genomes do not interact with histones in virions and are deposited in nuclear domains devoid of histones. Moreover, late viral replication compartments are also devoid of histones. The processes whereby histones come to interact with HSV-1 genomes, to be later displaced, remain unknown. However, they would involve the early movement of histones to the domains containing HSV-1 genomes and the later movement away from them. Histones unbind from chromatin, diffuse through the nucleoplasm, and rebind at different sites. Such mobility is upregulated by, for example, phosphorylation or acetylation. We evaluated whether HSV-1 infection modulates histone mobility, using fluorescence recovery after photobleaching. All somatic H1 variants were mobilized to different degrees. H1.2, the most mobilized, was mobilized at 4 h and further so at 7 h after infection, resulting in increases in its "free" pools. H1.2 was mobilized to a "basal" degree under conditions of little to no HSV-1 protein expression. This basal mobilization required nuclear native HSV-1 genomes but was independent of HSV-1 proteins and most likely due to cellular responses. Mobilization above this basal degree, and increases in H1.2 free pools, however, depended on immediate-early or early HSV-1 proteins, but not on HSV-1 genome replication or late proteins. Linker histone mobilization is a novel consequence of cell-virus interactions, which is consistent with the dynamic interactions between histones and HSV-1 genomes during lytic infection; it may also participate in the regulation of viral gene expression.     

ICP0 is required for efficient reactivation of herpes simplex virus type 1 from neuronal latency

Relative to wild-type herpes simplex virus type 1 (HSV-1), ICP0-null mutant viruses reactivate inefficiently from explanted, latently infected mouse trigeminal ganglia (TG), indicating that ICP0 is not essential for reactivation but plays a central role in enhancing the efficiency of reactivation. The validity of these findings has been questioned, however, because the replication of ICP0-null mutants is impaired in animal models during the establishment of latency, such that fewer mutant genomes than wild-type genomes are present in latently infected mouse TG. Therefore, the reduced number of mutant viral genomes available to reactivate, rather than mutations in the ICP0 gene per se, may be responsible for the reduced reactivation efficiency of ICP0-null mutants. We have recently demonstrated that optimization of the size of the ICP0 mutant virus inoculum and transient immunosuppression of mutant-infected mice with cyclophosphamide can be used to establish wild-type levels of ICP0-null mutant genomes in latently infected TG (W. P. Halford and P. A. Schaffer, J. Virol. 74:5957-5967, 2000). Using this procedure to equalize mutant and wild-type genome numbers, the goal of the present study was to determine if, relative to wild-type virus, the absence of ICP0 function in two ICP0-null mutants, n212 and 7134, affects reactivation efficiency from (i) explants of latently infected TG and (ii) primary cultures of latently infected TG cells. Although equivalent numbers of viral genomes were present in TG of mice latently infected with either wild-type or mutant viruses, reactivation of n212 and 7134 from heat-stressed TG explants was inefficient (31 and 37% reactivation, respectively) relative to reactivation of wild-type virus (KOS) (95%). Similarly, n212 and 7134 reactivated inefficiently from primary cultures of dissociated TG cells plated directly after removal from the mouse (7 and 4% reactivation, respectively), relative to KOS (60% reactivation). The efficiency and kinetics of reactivation of KOS, n212, and 7134 from cultured TG cells (treated with acyclovir to facilitate the establishment of latency) in response to heat stress or superinfection with a nonreplicating HSV-1 ICP4(-) mutant, n12, were compared. Whereas heat stress induced reactivation of KOS from 69% of latently infected TG cell cultures, reactivation of n212 and 7134 was detected in only 1 and 7% of cultures, respectively. In contrast, superinfection with the ICP4(-) virus, which expresses high levels of ICP0, resulted in the production of infectious virus in nearly 100% of cultures latently infected with KOS, n212, or 7134 within 72 h. Thus, although latent mutant viral genome loads were equivalent to that of wild-type virus, in the absence of ICP0, n212 and 7134 reactivated inefficiently from latently infected TG cells during culture establishment and following heat stress. Collectively, these findings demonstrate that ICP0 is required to induce efficient reactivation of HSV-1 from neuronal latency.     

Modulation of apoptosis during herpes simplex virus infection in human cells

Human herpes simplex virus (HSV) is cytolytic and has profound impacts on its host cells. Consequences of HSV infection include the induction of apoptosis and the concomitant synthesis of proteins which act to block this process. We review recent evidence showing how this important human pathogen modulates the fundamental cell death process.     

The cytoplasmic domain of herpes simplex virus type 1 glycoprotein C is required for membrane anchoring.

The herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) gene was altered so that it encoded a truncated glycoprotein lacking a cytoplasmic domain but retaining 20 of 23 amino acids of the transmembrane domain. No additional amino acid residues were introduced into the glycoprotein encoded by the altered gene. The gene was recombined into the HSV-1 genome by marker transfer. Two recombinant viruses, dl1 and dl2, that expressed the mutant gene were isolated. Characterization of these viruses showed that a substantial fraction of the mutant glycoprotein was secreted from infected cells. Pulse-chase experiments showed that the kinetics of posttranslational modification of the mutant glycoprotein were similar to those of the wild type. However, comparison of the kinetics of secretion of gC by dl2 and gC-3, a gC mutant lacking both the transmembrane and cytoplasmic domains, showed that dl2 gC was secreted much more slowly than gC-3 gC. Iodination of plasma membrane glycoproteins showed that dl2 gC was initially expressed on the cell surface as a membrane protein and subsequently was slowly released from the membrane into the medium. These data indicate that a major function of the cytoplasmic domain of gC is to ensure the stable anchoring of the glycoprotein in plasma membranes. In contrast to these major changes in the membrane-anchoring properties of gC, characterization of the virions produced by dl1 and dl2 showed that they contain significant amounts of gC. Thus the cytoplasmic domain does not appear to be essential for incorporation of this glycoprotein into virions.     

UL36p is required for efficient transport of membrane-associated herpes simplex virus type 1 along microtubules

Microtubule-mediated anterograde transport is essential for the transport of herpes simplex virus type 1 (HSV-1) along axons, yet little is known regarding the mechanism and the machinery required for this process. Previously, we were able to reconstitute anterograde transport of HSV-1 on microtubules in an in vitro microchamber assay. Here we report that the large tegument protein UL36p is essential for this trafficking. Using a fluorescently labeled UL36 null HSV-1 strain, KΔUL36GFP, we found that it is possible to isolate a membrane-associated population of this virus. Although these viral particles contained normal amounts of tegument proteins VP16, vhs, and VP22, they displayed a 3-log decrease in infectivity and showed a different morphology compared to UL36p-containing virions. Membrane-associated KΔUL36GFP also displayed a slightly decreased binding to microtubules in our microchamber assay and a two-thirds decrease in the frequency of motility. This decrease in binding and motility was restored when UL36p was supplied in trans by a complementing cell line. These findings suggest that UL36p is necessary for HSV-1 anterograde transport.     

The UL45 gene product is required for herpes simplex virus type 1 glycoprotein B-induced fusion.

Herpes simplex virus type 1 (HSV-1) syncytial (syn) mutants cause formation of giant polykaryocytes and have been utilized to identify genes promoting or suppressing cell fusion. We previously described an HSV-1 recombinant, F1 (J.L. Goodman, M. L. Cook, F. Sederati, K. Izumi, and J. G. Stevens, J. Virol. 63:1153-1161, 1989), which has unique virulence properties and a syn mutation in the carboxy terminus of glycoprotein B (gB). We attempted to replace this single-base-pair syn mutation through cotransfection with a 379-bp PCR-generated fragment of wild-type gB. The nonsyncytial viruses isolated were shown by DNA sequencing not to have acquired the expected wild-type gB sequence. Instead, they had lost their cell-cell fusion properties because of alterations mapping to the UL45 gene. The mutant UL45 gene is one nonsyncytial derivative of F1, A4B, was found to have a deletion of a C at UL45 nucleotide 230, resulting in a predicted frame shift and termination at 92 rather than 172 amino acids. Northern (RNA) analysis showed that the mutant UL45 gene was normally transcribed. However, Western immunoblotting showed no detectable UL45 gene product from A4B or from another similarly isolated nonsyncytial F1 derivative, A61B, while another such virus, 1ACSS, expressed reduced amounts of UL45. When A4B was cotransfected with the wild-type UL45 gene, restoration of UL45 expression correlated with restoration of syncytium formation. Conversely, cloned DNA fragments containing the mutant A4B UL45 gene transferred the loss of cell-cell fusion to other gB syn mutants, rendering them UL45 negative and nonsyncytial. We conclude that normal UL45 expression is required to allow cell fusion induced by gB syn mutants and that the nonessential UL45 protein may play an important role as a mediator of fusion events during HSV-1 infection.     

Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.     

Study of early events during herpes simplex virus type 1 infection by confocal microscopy

Laser scanning confocal microscopy is a powerful technique that can be applied to study the localisation and behaviour of proteins and nucleic acids in many experimental situations. It is a particularly useful technique for the study of virus infections because of the changes that occur in the distribution and amounts of both viral and cellular proteins as infection develops. These changes reflect key stages and important regulatory events that govern the efficiency of infection. Using herpes simplex virus type 1 infected cells as an experimental model, this article provides guidance for users new to confocal microscopy on basic principles and techniques. The emphasis is on recognising, diagnosing and avoiding potential artifacts, and the workflow of the production of high quality, technically correct images.     

Non-invasive bioluminescence imaging for monitoring herpes simplex virus type 1 hematogenous infection

Traditional studies on viral neuroinvasiveness and pathogenesis have generally relied on murine models that require the sacrifice of infected animals to determine viral distributions and titers. The present paper reports the use of in vivo bioluminescence imaging to monitor the replication and tropism of KOS strain HSV-1 viruses expressing the firefly luciferase reporter protein in hematogenously infected mice. Following intraperitoneal injection, a comparison was made between real-time PCR determinations of HSV-1 DNA concentrations (requiring the sacrifice of the experimental animals) and in vivo bioluminescence emissions in living animals. For further comparison, in vitro light emission was also measured in the ovaries and adrenal glands of sacrificed mice. After infection, HSV-1 spread preferentially to the ovaries and adrenal glands (these organs showed the highest virus levels). Both the PCR and bioluminescence methods detected low viral loads in the nervous system, where the virus was restricted to the spinal cord. The concentrations of viral DNA measured correlated with the magnitude of bioluminescence in vivo, and with the photon flux determined by the in vitro luciferase enzyme assay. The results show that bioluminescence imaging can be used for non-invasive, real-time monitoring of HSV-1 hematogenous infection in living mice, but that coupling this methodology with conventional techniques aids in the characterization of the infection.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.