Characterization of the UL16 gene product of herpes simplex virus type 2

Summary.  We have raised rabbit polyclonal antisera against a His-tagged herpes simplex virus type 1 (HSV-1) UL16 fusion protein, one of which very specifically reacted with 40 kDa and 41 kDa proteins in the lysates of HSV-1 and HSV-2-infected cells, respectively. Since its reactivity to the 41 kDa protein was clearly eliminated by pre-adsorption with E. coli lysates expressing the UL16 fusion protein, the antiserum was used to characterize the UL16 products of HSV-2. The HSV-2 UL16 protein was produced at the late phase of infection in a manner highly dependent on viral DNA synthesis and was distributed in both the nuclei and the cytoplasma of infected cells. In immunofluorescence studies, the UL16-specific fluorescence in the nuclei was shown to be detected as small discrete granules. On the other hand, the cytoplasmic fluorescence was diffusely distributed around the nucleus at 8 h postinfection but, at later times of infection, it was mainly detected as a mass at a perinuclear region. The analysis on its association with capsids has revealed that the UL16 protein copurified with C capsids but not B and A capsids, and that the association with C capsids was not tight. Moreover, our experiments have shown that a detectable level of the UL16 protein was not associated with extracellular virions, and that the partially purified UL16 proteins had a DNA-binding activity. These observations are consistent with the hypothesis that the UL16 protein plays a role in capsid maturation including DNA packaging/cleavage. We have also determined the complete nucleotide sequence of the HSV-2 UL16 gene and found that a nonstandard initiation codon may be used for its translation. Accepted January 12, 1998 Received October 31, 1997     

Characterization of the UL4 gene product of herpes simplex virus type 2

Summary.  We have identified the herpes simplex virus type 2 (HSV-2) UL4 gene product using a rabbit polyclonal antiserum raised against a recombinant 6xHis-UL4 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 27-kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor, suggesting that the UL4 gene was expressed as a γ₂ gene. Indirect immunofluorescence studies localized the UL4 protein within the nucleus as discrete punctate forms at late times postinfection. However, when expressed in the absence of other viral proteins, the UL4 protein was limited to the cytoplasm, indicating that an interaction with one or more other virus-induced proteins was responsible for the nuclear localization during infection. Subnuclear fractionation studies showed that the protein was released from the nuclear structure of infected cells by high salt treatment. Moreover, the UL4 protein was detected in purified virions and light particles. Received December 24, 1997 Accepted February 4, 1998     

Identification and Characterization of the UL24 Gene Product of Herpes Simplex Virus Type 2

The UL24 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 281 amino acid protein with a molecular mass of 30.5kDa. In this study, the HSV-2 UL24 gene product has been identified by using a rabbit polyclonal antiserum produced against a recombinant protein containing the full-length UL24 gene product of HSV-2 fused to glutathione-S-transferase. The antiserum reacted specifically with a 32kDa protein in HSV-2 186-infected Vero cells and with 31 and 32kDa proteins in UL24-expressing Cos-7 cells. Accumulation of UL24 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. UL24 protein was found to be associated with purified HSV-2 virions and C capsids. Indirect immunofluorescence analysis demonstrated that the UL24-specific fluorescence was detected in perinuclear regions of the cytoplasm and/or in the nucleus as small discrete granules from 9h post infection (hpi). Furthermore, the UL24 protein expressed singly was detected predominantly in the nucleus and slightly in the cytoplasm at 24h after transfection, with branch-like cytoplasmic protruding structures. Strong nucleolus staining was visible in partial cells.     

Herpes simplex virus UL17 protein is associated with B capsids and colocalizes with ICP35 and VP5 in infected cells

Summary.  A previous study using a mutant lacking the UL17 gene has suggested that the UL17 protein of herpes simplex virus type 1 (HSV-1) is required for the cleavage/packaging of viral DNA. In this study, we have raised a rabbit polyclonal antiserum which specifically reacted with the UL17 protein which has an apparent molecular mass of 78-kDa in the lysates of HSV types 1- and 2-infected Vero cells. Western blot analysis of intracellular capsids demonstrates that the UL17 protein was associated with B and C capsids. Indirect immunofluorescence studies reveal that it colocalized with the major capsid protein VP5 and the scaffoling protein ICP35 within the nucleus. These results suggest that the association of the UL17 protein with immature B-type capsids is important for its role in cleavage/packaging. Accepted June 11, 1999/Received April 26, 1999     

Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.     

Herpes simplex virus type 1 gene UL13 encodes a phosphoprotein that is a component of the virion.

The UL13 open reading frame of herpes simplex virus type 1 (HSV-1) has been expressed in insect cells by a recombinant baculovirus and in Escherichia coli. In the latter case, the UL13 gene was fused to the gene for glutathione S-transferase (GST) to allow high-level expression of an 80-kDa GST-UL13 fusion protein. Antibody raised against the fusion protein reacted specifically with the 55-kDa UL13 gene product expressed by the recombinant baculovirus. This antibody also recognized a late phosphoprotein in HSV-1-infected cell lysates and a component of purified HSV-1 virions, both with the same electrophoretic mobility as the baculovirus-expressed protein. The virion component was efficiently phosphorylated in vitro by a virion-associated protein kinase. Using the same antibody, the probable homolog of the UL13 gene product was identified in HSV-2-infected cells and purified virions.     

Identification of the UL56 Protein of Herpes Simplex Virus Type 1 within the Virion by Immuno Electron Microscopy

Recently the UL56 protein of herpes simplex virus type 1 (HSV-1) was shown to be associated with the virion of HSV-1 as determined by Western blot analysis. The detection of the UL56 protein in infected cells and its association with virions of HSV-1 is of particular importance, pointing to a possible involvement of UL56 protein in virus-host interactions. In order to investigate the properties of the UL56 protein further immuno-localization was performed using rabbit hyperimmune serum against fusion recombinant UL56 protein and purified virions of HSV-1 strain F. The UL56 protein was detected in the HSV-1 virions by immuno gold negative staining. This revised version was published online in July 2006 with corrections to the Cover Date.     

Synthesis, subcellular localization and VP16 interaction of the herpes simplex virus type 2 UL46 gene product

Summary.  We developed a rabbit polyclonal antiserum reactive against a recombinant 6x His-UL46 fusion protein expressed in*Escherichia coli, and using this antiserum identified the UL46 gene product of herpes simplex virus type 2 (HSV-2) to be phosphoproteins with apparent molecular masses of 82-, 84-, and 86-kDa in infected Vero cells. The UL46 protein was produced in the late phase of infection in a manner highly dependent on viral DNA synthesis, and was mainly distributed at the edge of the nucleus in the cytoplasm. Although its kinetics of production and its progress of distribution were different from those of the major tegument protein VP16 (the UL48 gene product or α-trans-inducing factor (αTIF)), most of the UL46 protein colocalized with VP16 in the late phase of infection, and copurified with it in column chromatography. Moreover, our data showed that the HSV-2 UL46 protein, when coexpressed with VP16, enhanced α4 promotor-regulated gene expression in a transient luciferase reporter assay, while the expression of the UL46 protein alone suppressed it. Received December 16, 1999 Accepted February 23, 2000     

Early shutoff of host protein synthesis in cells infected with herpes simplex viruses

Herpes simplex viruses 1 (HSV-1) and 2 (HSV-2) are capable of suppressing the host cell protein synthesis even without viral gene expression. This phenomenon is known as the early shutoff or as the virion-associated host shutoff (vhs) to emphasize that it is mediated by a component of infecting virions which is a product of the UL41 (vhs) gene. The UL41 encoded protein is a functional tegument protein also present in light (L) particles and is not essential for virus replication. The major product of UL41 gene is a 58 K phosphoprotein. At least two forms of UL41 protein differing in the extent of phosphorylation are present in HSV-1-infected cells. HSV-2 compared to HSV-1 strains display a stronger vhs phenotype. However, in superinfection experiments the less strong vhs phenotype is dominant. UL41 protein triggers disruption of polysomes and rapid degradation of all host and viral mRNAs and blocks a reporter gene expression without other HSVs proteins. The available evidence suggests that UL41 protein is either itself a ribonuclease (RNase) or a subunit of RNase that contains also one or more cellular subunits. UL41 protein is capable of interacting with a transactivator of an alpha-gene, the alpha-transinducing factor (alpha-TIF). Interaction of UL41 protein with alpha-TIF down regulates the UL41 (vhs) gene activity during lytic infection. The possible role of other viral proteins in the shutoff is discussed.     

Complex mechanisms for the packaging of the UL16 tegument protein into herpes simplex virus

The conserved UL16 tegument protein of herpes simplex virus exhibits dynamic capsid-binding properties with a release mechanism that is triggered during initial virus attachment events. In an effort to understand the capsid association and subsequent release of UL16, we sought to define the mechanism by which this protein is packaged into virions. The data presented here support a model for the addition of some UL16 to capsids prior to their arrival at the TGN. UL16 was found on capsids isolated from cells infected with viruses lacking UL36, UL37 or gE/gD, which are defective for budding and accumulate non-enveloped capsids in the cytoplasm. Additionally, membrane-flotation experiments showed that UL16 co-purified with cytoplasmic capsids that are not associated with membranes. Moreover, the amount of UL16 packaged into extracellular particles was severely reduced in the absence of two conserved binding partners, UL21 or UL11.     

Identification and characterization of the duck enteritis virus UL51 gene

Compared to the UL51 gene of other alphaherpesviruses, the duck enteritis virus (DEV) UL51 gene contains ten conserved motifs and has a close evolutionary relationship with members of the genus Mardivirus. The DEV UL51 gene product was identified using a rabbit polyclonal antiserum raised against a 6-His-UL51 fusion protein expressed in Escherichia coli as a 34-kDa protein. Western blotting and RT-(real time) PCR analysis of DEV-infected cells showed that the protein was produced at the late stage of infection and that its production was highly dependent on viral DNA synthesis, suggesting that the gene should be classified as γ2 class. Analysis of extracellular virions revealed that the protein was a component of extracellular mature DEV virions. Indirect immunofluorescence studies localized most of the protein to the juxtanuclear region. These results will provide a basis for further functional analysis of the gene. Chan-Juan Shen, An-Chun Cheng, Ming-Shu Wang and Yu-Fei Guo were contributed equally to this work.     

Enhancement of herpes simplex virus type 2 (HSV-2) DNA synthesis in infected cells that constitutively express the BglII-N region of the HSV-2 genome

The BglII-N fragment of the herpes simplex virus type-2 (HSV-2) genome encodes one of two known transforming regions of this DNA virus. In this study, we report the derivation of HeLa S3 cells (2DC₄) that stably express the HSV-2 BglII-N region, including the small subunit of HSV-2 ribonucleotide reductase (RR). Superinfection of the 2DC₄ cells with wild-type HSV-2 resulted in the efficient induction of HSV-2-encoded ICP10, DNA polymerase, and thymidine kinase. The amount of HSV-2 DNA synthesis in 8-hr HSV-2-infected 2DC₄ cells was enhanced 2.6±0.6-fold relative to infected control cells. Furthermore, the replication kinetics of HSV-2 DNA in 2DC₄ cells were accelerated relative to HeLa S3 cells; HSV-2 DNA synthesis was detectable as early as 3 hr postinfection in 2DC₄ cells as compared to 6 hr postinfection in HeLa S3 cells. These results suggest that the BglII-N region of HSV-2 encodes function(s) that activate the viral DNA synthesis apparatus and that this activation could relate to the transforming ability of this DNA region.     

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

Role of the UL28 homologue of bovine herpesvirus 1 in viral DNA cleavage and packaging

Summary.  In the aim to study the function of the bovine herpesvirus 1 (BoHV-1) UL28 protein during the replicative cycle, we characterized a UL28 deletion mutant of BoHV-1, BoHV-1 Δ UL28. Productive growth of BoHV-1 Δ UL28 was only observed in a specifically engineered complementing cell line expressing the native UL28 protein, demonstrating that UL28 is essential for virus replication. UL28 deficiency did not compromised viral protein synthesis of the late class as shown by the detection of the viral alpha gene trans-inducing factor protein encoded by UL48, a gene of the γ2 class. Southern blotting analyses of total DNA extracted from BoHV-1 Δ UL28-infected normal cells revealed that viral DNA replication was not compromised but the process of cleavage of the newly synthesized DNA was defective. Transmission electron microscopy of non-complementing BoHV-1 Δ UL28-infected cells revealed an accumulation of capsids devoid of DNA, suggesting that the DNA packaging was impaired. We conclude that the BoHV-1 UL28 protein is essential for viral replication and is necessary for the formation of mature capsid. Received October 24, 2002; accepted November 29, 2002     

The herpes simplex virus UL33 gene product is required for the assembly of full capsids.

Phenotypic analysis of the herpes simplex virus type 1 temperature-sensitive DNA-positive mutant, ts1233, revealed that the mutant had a structural defect at the nonpermissive temperature (NPT). Cells infected with ts1233 at the NPT contained large numbers of intermediate capsids, lacking dense cores but possessing some internal structure. No full capsids or enveloped virus particles were detected. In contrast to the defect in another packaging-deficient mutant ts1201, the block in the formation of dense-cored, DNA-containing capsids in ts1233-infected cells at the NPT could not be reversed by transferring the cells to the permissive temperature in the presence of a protein synthesis inhibitor. Furthermore, the capsids produced by ts1233 at the NPT had more compact internal structures than those of the gene UL26 mutant ts1201. Southern blot analysis of viral DNA in ts1233-infected cells confirmed that the mutant DNA was not encapsidated at the NPT and showed that the unpackaged DNA was not cleaved into genome-length molecules. The ts1233 mutation was mapped by marker rescue to the vicinity of genes UL32 and UL33. Sequence analysis of the DNA in this region from the mutant and two independently isolated revertants for growth revealed that ts1233 had a single base-pair change at the amino-terminal end of UL33, resulting in the substitution of an isoleucine with an asparagine. The nucleotide sequence of the revertants in this part of the genome was identical to that of wild-type virus.     

Characterization of the U(L)33 gene product of herpes simplex virus 1

The U(L)33 protein is one of six genes (including U(L)6, U(L)15, U(L)17, U(L)28, and U(L)32) required for cleavage of viral concatemeric DNA into unit-length genomes and packaging of the virus genomes into preformed capsids. The U(L)25 gene product is dispensable for cleavage of viral DNA but essential for packaging of DNA into capsids. A polyclonal antiserum was produced against an affinity-purified protein containing the full-length U(L)33 gene product of herpes simplex virus 1 fused to glutathione-S-transferase. A protein of approximate M(r) 19,000 that reacted with the antiserum was detected in immunoblots of herpes simplex virus 1-infected cellular lysates. This protein was not detected in lysates of mock-infected cells or cells infected with a mutant virus containing a stop codon in U(L)33, indicating that the 19,000 M(r) protein is the product of the U(L)33 open reading frame. The U(L)33 gene product was not detected in purified virions or capsids. Accumulation of the U(L)33 protein to detectable levels required viral DNA synthesis, indicating that the protein was regulated as a late gene. Indirect immunofluorescence analysis demonstrated that U(L)33 protein accumulated predominantly within replication compartments in the central domains of infected cell nuclei and within the cytoplasm. Localization of the U(L)33 gene product in replication compartments was maintained in cells infected with a variety of cleavage/packaging mutants.     

The HSV-1 tegument protein pUL46 associates with cellular membranes and viral capsids

The molecular mechanisms responsible for the addition of tegument proteins into nascent herpesvirus particles are poorly understood. To better understand the tegumentation process of herpes simplex virus type 1 (HSV-1) virions, we initiated studies that showed the tegument protein pUL46 (VP11/12) has a similar cellular localization to the membrane-associated tegument protein VP22. Using membrane flotation analysis we found that pUL46 associates with membranes in both the presence and absence of other HSV-1 proteins. However, when purified virions were stripped of their envelope, the majority of pUL46 was found to associate with the capsid fraction. This strong affinity of pUL46 for capsids was confirmed by an in vitro capsid pull-down assay in which purified pUL46-GST was able to interact specifically with capsids purified from the nuclear fraction of HSV-1 infected cells. These results suggest that pUL46 displays a dynamic interaction between cellular membranes and capsids.     

UL5, a protein required for HSV DNA synthesis: genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies

The mutations in two DNA-negative ts mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg, 1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that the 99-kDa protein contains a consensus sequence for an ATP binding site. Possible roles of this protein in viral DNA synthesis are discussed.     

Herpes simplex virus type 1 genes in human mononuclear cells and affecting cell-mediated immunity

To evaluate whether herpes simplex virus type 1 (HSV-1) replicates in freshly isolated human mononuclear cells (HMNC) and results in cellmediated immunity, HMNC purified from the peripheral blood of a healthy HSV-1-seronegative individual were infected with HSV-1 (KOS strain). The kinetic data indicated that HSV-1 particles were detected in HMNC at 9 hr postinfection, and the highest HSV-1 titer was obtained at 24 hr postinfection. It was found that the newly synthesized HSV-1 DNA increased in HMNC corresponding to the duration of time postinfection. The HSV-1 tk mRNA was detected in HSV-1-infected HMNC by Northern blot analysis. The Western blot analysis indicated that the HSV-1 infected HMNC could express the viral proteins. These results suggest that HSV-1 replicates in freshly isolated HMNC and expresses its DNA, RNA, and proteins. Interleukin-2 (IL-2) production and cell-mediated cytotoxicity (CMC) were also determined in HSV-1-infected HMNC but were lower than those found in uninfected HMNC. On the other hand, HSV-1 DNA was detectable in HMNC of patients suffering from recurrent HSV-1 infection. The results show that, in the recrudescent phase, the patients'; HMNC contain more HSV-1 DNA than in the convalescent phase. We conclude that the impairments of immune responses are related to the infection of HMNC by HSV-1.