Absence Of Viral DNA Synthesis Research Articles

Identification of human cytomegalovirus genes important for biogenesis of the cytoplasmic virion assembly complex.

Human cytomegalovirus (HCMV) has many effects on cells, including remodeling the cytoplasm to form the cytoplasmic virion assembly complex (cVAC), the site of final virion assembly. Viral tegument, envelope, and some nonstructural proteins localize to the cVAC, and cytoskeletal filaments radiate from a microtubule organizing center in the cVAC. The endoplasmic reticulum (ER)-to-Golgi intermediate compartment, Golgi apparatus, and trans-Golgi network form a ring that outlines the cVAC. The center of the cVAC ring is occupied by numerous vesicles that share properties with recycling endosomes. In prior studies, we described the three-dimensional structure and the extensive remodeling of the cytoplasm and shifts in organelle identity that occur during development of the cVAC. The objective of this work was to identify HCMV proteins that regulate cVAC biogenesis. Because the cVAC does not form in the absence of viral DNA synthesis, we employed HCMV-infected cells transfected with synthetic small interfering RNAs (siRNAs) that targeted 26 candidate early-late and late protein-coding genes required for efficient virus replication. We identified three HCMV genes (UL48, UL94, and UL103) whose silencing had major effects on cVAC development, including failure to form the Golgi ring and dispersal of markers of early and recycling endosomes. To confirm and extend the siRNA results, we constructed recombinant viruses in which pUL48 and pUL103 are fused with a regulatable protein destabilization domain (dd-FKBP). In the presence of a stabilizing ligand (Shield-1), the cVAC appeared to develop normally. In its absence, cVAC development was abrogated, verifying roles for pUL48 and pUL103 in cVAC biogenesis. Human cytomegalovirus (HCMV) is an important human pathogen that causes disease and disability in immunocompromised individuals and in children infected before birth. Few drugs are available for treatment of HCMV infections. HCMV remodels the interior of infected cells to build a factory for assembling new infectious particles (virions), the cytoplasmic virion assembly complex (cVAC). Here, we identified three HCMV genes (UL48, UL94, and UL103) as important contributors to cVAC development. In addition, we found that mutant viruses that express an unstable form of the UL103 protein have defects in cVAC development and production of infectious virions and produce small plaques and intracellular virions with aberrant appearances. Of these, only the reduced production of infectious virions is not eliminated by chemically stabilizing the protein. In addition to identifying new functions for these HCMV genes, this work is a necessary prelude to developing novel antivirals that would block cVAC development.

Murine Cytomegalovirus Protein pM79 Is a Key Regulator for Viral Late Transcription

Herpesvirus genes are temporally expressed during permissive infections, but how their expression is regulated at late times is poorly understood. Previous studies indicate that the human cytomegalovirus (CMV) gene, UL79, is required for late gene expression. However, the mechanism remains to be fully elucidated, and UL79 homologues in other CMVs have not been studied. Here, we characterized the role of the conserved murine CMV (MCMV) gene M79. We showed that M79 encoded a protein (pM79) which was expressed with early-late kinetics and localized to nuclear viral replication compartments. M79 transcription was significantly decreased in the absence of viral DNA synthesis but markedly stimulated by pM79. To investigate its role, we created the recombinant virus SMin79, in which pM79 expression was disrupted. While marker-rescued virus grew efficiently in fibroblasts, SMin79 failed to produce infectious progeny but was rescued by pM79 expression in trans. During SMin79 infection, representative viral immediate-early and early gene products as well as viral DNA accumulated sufficiently. Formation of viral replication compartments also appeared normal. Pulsed-field gel electrophoresis analysis indicated that the overall structure of replicating viral DNA was indistinguishable between wild-type and SMin79 infection. Viral tiled array and quantitative PCR analysis revealed that many late transcripts sensitive to a viral DNA synthesis inhibitor (phosphonoacetic acid) were markedly reduced by pM79 mutation. This study indicates that cytomegaloviruses use a conserved mechanism to promote transcription at late stages of infection and that pM79 is a critical regulator for at least a subset of viral DNA synthesis-dependent transcripts.

Identification of an ICP27-Responsive Element in the Coding Region of a Herpes Simplex Virus Type 1 Late Gene

During productive herpes simplex virus type 1 (HSV-1) infection, a subset of viral delayed-early (DE) and late (L) genes require the immediate-early (IE) protein ICP27 for their expression. However, the cis-acting regulatory sequences in DE and L genes that mediate their specific induction by ICP27 are unknown. One viral L gene that is highly dependent on ICP27 is that encoding glycoprotein C (gC). We previously demonstrated that this gene is posttranscriptionally transactivated by ICP27 in a plasmid cotransfection assay. Based on our past results, we hypothesized that the gC gene possesses a cis-acting inhibitory sequence and that ICP27 overcomes the effects of this sequence to enable efficient gC expression. To test this model, we systematically deleted sequences from the body of the gC gene and tested the resulting constructs for expression. In so doing, we identified a 258-bp "silencing element" (SE) in the 5' portion of the gC coding region. When present, the SE inhibits gC mRNA accumulation from a transiently transfected gC gene, unless ICP27 is present. Moreover, the SE can be transferred to another HSV-1 gene, where it inhibits mRNA accumulation in the absence of ICP27 and confers high-level expression in the presence of ICP27. Thus, for the first time, an ICP27-responsive sequence has been identified in a physiologically relevant ICP27 target gene. To see if the SE functions during viral infection, we engineered HSV-1 recombinants that lack the SE, either in a wild-type (WT) or ICP27-null genetic background. In an ICP27-null background, deletion of the SE led to ICP27-independent expression of the gC gene, demonstrating that the SE functions during viral infection. Surprisingly, the ICP27-independent gC expression seen with the mutant occurred even in the absence of viral DNA synthesis, indicating that the SE helps to regulate the tight DNA replication-dependent expression of gC.

Characterization of Human Herpesvirus 7 U27 Gene Product and Identification of Its Nuclear Localization Signal

A monoclonal antibody, 5H4, that recognizes human herpesvirus 7 (HHV-7) was used in Western analysis to probe HHV-7-infected SupT1 cells. This antibody recognizes a 40-kDa virus-specific polypeptide that is expressed in the absence of viral DNA synthesis. By screening a λgt11 HHV-7 cDNA library, the gene encoding the protein was identified as the U27 open reading frame previously reported [J. Virol. (1996) 70, 5975–5989]. Immunofluorescent studies showed a punctate nuclear localization of the protein in both HHV-7-infected cells and transfected cells. A computer program predicted two classic nuclear localization signals (NLSs) in the middle and C-terminal regions of the protein. A C-terminal deletion mutant of the protein could not enter the nucleus, whereas green fluorescent protein or maltose binding protein fused to the C-terminal region of the protein was transported into the nucleus. These findings demonstrate that the predicted C-terminal, but not middle, NLS of the protein actually function as NLS. In addition, nuclear transport of a maltose binding protein-fusion protein containing the C-terminal NLS of the U27 protein was inhibited by both wheat germ agglutinin and a Q69L Ran-GTP mutant, indicating that the U27 protein is transported into the nucleus from the cytoplasm by means of classic nuclear transport machinery. Interestingly, this NLS motif is highly conserved at the C-termini of all herpesvirus DNA polymerase processivity factors that have been examined.

Early expression of herpes simplex virus (HSV) proteins and reactivation of latent infection.

During the last decade, new data accumulated describing the early events during herpes simplex virus 1 (HSV-1) replication occurring before capsid formation and virion envelopment. The HSV virion carries its own specific transcription initiation factor (alpha-TIF), which functions together with other components of the cellular transcriptase complex to mediate virus-specific immediate early (IE) transcription. The virus-coded IE proteins are the transactivator and regulatory elements modulating early transcription and subsequent translation of nonstructural virus-coded proteins needed mainly for viral DNA synthesis and for the supply of corresponding nucleoside components. They also cooperate at the late transcription and translation of the virion (capsid, tegument and envelope) proteins. In addition, the transactivator IE proteins down-regulate their own transcription, while others facilitate viral mRNA processing or interfere with the presentation of newly synthesized virus antigens. Establishment of latency is closely related to the transcription of a separate category of transcripts, termed latency-associated (LAT). Formation of LATs occurs mainly in nondividing neurons which are metabolically less active and express lower levels of cellular transcription factors (nonpermissive cells). Expression of the stable non-spliced (2 kb), and especially of stable spliced (1.5 and 1.45 kb) LATs is a prerequisite for HSV reactivation. Different HSV genomes (from various HSV strains) do not undergo IE transcription at the same rate. Restricted IE transcription and the absence of viral DNA synthesis favors LAT formation and persistence of the silenced genome. Uneven levels of LAT expression and differences in the metabolic state of carrier neurons influence the reactivation competence. Under artificial or natural activation conditions, sufficient amounts of IE transactivator proteins and proteins promoting nucleoside metabolism are synthesized even in the absence of the viral alpha-TIF facilitating reactivation.

Modulation of RANTES production by human cytomegalovirus infection of fibroblasts.

Chemokines play a major role in inflammatory responses and affect hematopoiesis both negatively and positively. We show that fresh isolates and laboratory strains (Towne and Ad-169) of human cytomegalovirus (HCMV) induce production of the CC chemokine RANTES in fibroblasts. Induction of extracellular RANTES production occurred as early as 8 h after infection, peaked around 24 h after infection, and was almost undetectable by 48 and 72 h. Upregulation occurred in the absence of viral DNA synthesis, suggesting that it was due to immediate-early-early HCMV gene expression. CMV infection stimulated RANTES transcription, since reverse transcription-PCR detected a sharp increase in RANTES RNA which persisted even when extracellular RANTES was no longer detected. Induction of RANTES in fibroblasts was not due to prior induction of tumor necrosis factor alpha or interleukin 1 beta. Down-regulation required an active viral genome. Decrease of RANTES in culture supernatants may be associated with the appearance of the HCMV CC chemokine receptor US28, since we show that this gene is transcribed as early as 8 h after infection. Modulation of CC chemokine production early during CMV infection might have a regulatory effect on viral replication, as well as affect immune surveillance.

The human cytomegalovirus glycoprotein B gene (ORF UL55) is expressed early in the infectious cycle.

Northern hybridizations were carried out using mRNA preparations of human cytomegalovirus (HCMV)-infected cultures and gene-specific antisense RNA probes for transcriptional analysis of the gene cluster composed of genes for DNA polymerase, glycoprotein B (gB), herpes simplex virus-infected cell protein 18.5 homologue p130 and a major DNA-binding protein corresponding to open reading frames (ORFs) UL54-UL57, respectively. Monocistronic transcripts of 5 kb and 3.7 kb were found for ORFs UL54 and UL55, respectively, and five additional high molecular mass overlapping transcripts of 14 kb, 10 kb, 10 kb, 8 kb and 6 kb were found. Mapping of 5' ends showed that transcription was initiated at the expected distance downstream of predicted TATA elements; in the case of a UL56-specific transcript two potential initiation sites were identified. Transcription was found to terminate at the expected distance downstream of either of two prominent polyadenylation consensus motifs in the region of UL54. All transcripts were identified early in the infectious cycle, except for the UL55 (gB)-specific transcript of 3.7 kb which was not synthesized until late post-infection. However, specific immunoreactions demonstrated the presence of a gB-specific polypeptide early after infection in the absence of viral DNA synthesis. It is suggested that a bicistronic transcript of 8 kb encoded by ORFs UL55 and UL54 is involved in biosynthesis of early HCMV gB.

Assembly of Herpes Simplex Virus Replication Proteins at Two Distinct Intranuclear Sites

Herpes simplex virus DNA replication proteins amplify the viral genome in large globular replication compartments within infected cell nuclei. In the absence of viral DNA synthesis, the replication proteins accumulate at punctate foci throughout the nucleus referred to as prereplicative sites. To more thoroughly understand the nature of this nuclear assembly process, we have examined the viral and cellular factors involved. First, we demonstrate that six viral replication proteins are sufficient for formation of functional replication compartments in transfected cells in the absence of viral origin-containing DNA. Second, we show that the viral replication proteins form two distinct types of prereplicative sites within infected cells. One type of punctate structure assembles in S-phase cells, colocalizes with cellular DNA synthesis, and contains components of the host-cell replication apparatus as indicated by the presence of Replication Protein A. However, the other class of prereplicative sites is independent of host-cell DNA synthesis as evidenced by their formation in cells arrested in G1 byn-butyrate. These complexes are significantly less abundant and closely correspond with cellular Nuclear Domain 10 structures to which viral DNA has recently been demonstrated to be targeted early in infection (G. G. Maul, A. M. Ishov, and R. D. Everett, 1996,Virology217, 67–75). Hence, this second type appears to represent the subset of prereplicative sites destined to become replication compartments.

Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNA-binding protein (65KDBP), the major DNA-binding protein (infected cell polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA- ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication protein to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.

Kinetics of expression of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1, encoded by gene UL42, is required for herpes simplex virus origin-dependent DNA replication (C.A. Wu, N.J. Nelson, D.J. McGeoch, and M.D. Challberg, J. Virol. 62:435-443, 1988). We found by indirect immunofluorescence with monoclonal antibody to 65KDBP that the protein was first detectable at 3 h postinfection. It localized first to the inner periphery of the nucleus, but accumulated in large globular compartments within the nucleus by 6 h postinfection in a pattern similar to that displayed by the major DNA-binding protein ICP8. Immune electron microscopy revealed that 65KDBP was associated with the marginated heterochromatin at the early times, but migrated further into the nucleus at late times when the only discernible areas devoid of 65KDBP were the nucleoli and heterochromatin. The 65KDBP gene is a member of the beta kinetic class as determined by the ability of the mRNA to be expressed at significant levels even in the absence of viral DNA synthesis. Furthermore, in the presence or absence of the DNA polymerase inhibitor phosphonoacetic acid, the patterns of accumulation of protein as well as mRNA were virtually indistinguishable from those displayed by the model beta genes encoding ICP8 and thymidine kinase. Nuclear run-on experiments demonstrated that maximum rates of 65KDBP gene transcription occurred prior to the maximum rate of progeny viral DNA synthesis and confirmed that the expression of the 65KDBP gene is regulated at the level of transcriptional initiation.

Activation of herpes simplex virus 1 γ 2 genes by viral DNA replication

The expression of γ 2 or true γ genes resident in the herpes simplex virus 1 genome requires functional products of genes expressed earlier in infection and viral DNA synthesis. To determine whether the requirement for viral DNA synthesis for the expression of γ 2 genes reflects a trans-acting function of a product of one or a few genes made or activated during viral DNA synthesis or whether it reflects a cis effect of DNA synthesis at specific sites, 143 thymidine kinase (TK) minus cells were sequentially infected with a virus carrying a 700-bp deletion of the TK gene and 6 hr later with wild-type or recombinant viruses carrying an α-TK or a γ 2-TK gene in either the presence or absence of viral DNA synthesis. These experiments indicated that the γ 2-TK gene contained in an unreplicated viral genome was not expressed by trans-acting factors specified before and during DNA synthesis by the first infecting virus, and, therefore, the induction of γ 2 genes appears to be mediated by a cis-acting function associated with viral DNA synthesis.

Synthesis of the gB and gC glycoproteins of herpes simplex virus type 1 in the absence of viral DNA synthesis

Abstract Using an inhibitor of DNA synthesis as well as a temperature-sensitive (ts), DNA negative mutant of herpes simplex virus type 1 (HSV-1), we have examined the role of viral DNA synthesis on the expression of the virus-specific glycoproteins. Analysis by immunoblotting employing monospecific antisera revealed that only gC glycoprotein synthesis was completely inhibited in the absence of viral DNA synthesis. In contrast, the gB glycoprotein was detectable in significant, albeit somewhat reduced, amounts. These data suggest that gC is a ‘true-late’ protein of HSV-1 while gB is not. In addition, temperature shift-up experiments with the ts mutant (tsA1) suggest that some viral gene product(s) is needed continuously to achieve wild-type levels of gC synthesis.

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus)

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.

Human cytomegalovirus DNA: Restriction enzyme cleavage maps and map locations for immediate-early, early, and late RNAs

Complete physical maps for human cytomegalovirus (Davis strain) DNA were constructed from fragments produced by restriction endonucleases HindIII, XbaI, and EcoRI. The results showed that the human cytomegalovirus genome has a structural organization similar to that of the herpes simplex virus genome: a long (L) segment, comprising 82% of the genome, joined to a short (S) segment, comprising the remaining 18% of the genome. Permutations of the S and L segments produce four different molecular arrangements which are represented in equimolar proportions in virion DNA preparations. Heterogeneity in the size of the DNA was detected within fragments which map at one terminus of the S segment and also in fragments which map at one terminus of the L segment. The regions of the genome from which RNA synthesized at various times after infection originates was determined. Immediate-early RNA (RNA synthesized in the presence of cycloheximide) was restricted to a few regions on the genome but hybridized in abundance to a region which mapped between 0.686 and 0.733 units (within the L segment). Early RNA (synthesized in the absence of viral DNA synthesis) hybridized to most areas of the genome, but in particular abundance to a small region which mapped between 0 and 0.046 units (also within the L segment). RNA which accumulated during the late phase of infection was not characterized by particular abundances to any region of the genome.

Viral DNA synthesis is required for the efficient expression of specific herpes simplex virus type 1 mRNA species

Inhibition of HSV-1 DNA synthesis with either arabinosyladenine plus the adenosine deaminase inhibitor pentostatin, or with arabinosylthymine, showed a viral mRNA population identical to that seen prior to viral DNA replication (early) by the criteria of quantitative hybridization, specific mRNA species identifiable by Southern blot hybridization of size-fractionated RNA, and migration of polypeptides resolved by in vitro translation of purified viral mRNA. The amount of viral mRNA associated with infected cell polyribosomes was determined by quantitative DNA excess solution hybridization. At 2 hr postinfection (p.i.) (before viral DNA synthesis) and in drug-treated cells at 6 hr p.i., the majority of the polyadenylated RNA was cell specific with some virus-specific RNA detectable. In contrast, at 6 hr p.i., in the absence of drugs (during maximum viral DNA synthesis), nearly all the polyadenylated polyribosomal RNA was viral. Blot hybridization of size-fractionated viral RNA confirmed several specific differences between the viral mRNA species occurring before and after HSV-1 DNA synthesis, which have been reported previously from this laboratory. These differences also were reflected in the in vitro translation products encoded by the viral mRNAs. The mRNA species and the encoded polypeptides that were present in the absence of viral DNA synthesis are a subset of those viral mRNA species and polypeptides expressed in the presence of viral DNA synthesis. The viral mRNA species fall into several groups based on their relative abundance at various times of infection. These data suggest that, in the normal virus infection cycle, the onset of viral DNA synthesis is necessary for normal expression of later viral genes.

Partial Characterization of a Soluble Antigen Preparation from Cells Infected with Human Cytomegalovirus: Properties of Antisera Prepared to the Antigen

Soluble antigen (SA) preparations were obtained from cell cultures infected with either the Davis or AD169 strains of cytomegalovirus (CMV). Fractionation of SA preparations through Sephadex G-200 resulted in a molecular weight value ranging from 67,000 to 85,000. Rate-zonal centrifugation produced an approximate value of 5.5S for the CMV antigenic material. Antisera to SA prepared from either AD169- or Davis-infected cells lacked neutralizing activity but produced specific fluorescence confined to CMV intranuclear inclusion material when used in the indirect fluorescent antibody test (IFA). The specific fluorescing inclusion reaction was seen when either AD169 or Davis antisera were used with cells infected with the Davis, AD169, Kerr, or C-87 strains of CMV. Fluorescence was not observed in cells infected with a strain of Herpes simplex type 1, varicella-zoster virus, an EBV transformed lymphocyte line, the Cx-90-3B human CMV transformed hamster embryo cell line or CMV-infected cell cultures treated with cytosine arabinoside (Ara-C) and showing only antigens expressed in the absence of viral DNA synthesis. Antisera prepared to SA preparations obtained from CMV-infected cells apparently react with specific CMV antigens that are dependent on viral DNA synthesis and are common to several strains.

Radiobiological studies of vaccinia virus

Radiation studies of vaccinia virus were carried out using two types of radiation, Co 60 gamma rays and ultraviolet light (UVL). When virus particles were irradiated with gamma rays a two-component survival curve was obtained. The capacity of Earle's L cells to support the growth of vaccinia virus was relatively sensitive to gamma rays. Infectious centres formed by infecting L cells with vaccinia virus were irradiated at various times during the eclipse period of virus growth (the Luria-Latarjet experiment). When irradiated with gamma rays, infectious centres exhibited an initial sensitization to inactivation which persisted for at least 3 hours. At times later than 3 hours after infection the infectious centres became progressively more resistant to inactivation by gamma rays (“stabilization”); the survival curves remained almost single-hit throughout the eclipse period. Studies with FUdR, an inhibitor of DNA synthesis, showed little stabilization in the absence of viral DNA synthesis. Free particles of vaccinia virus were inactivated according to single-hit kinetics by UVL. The capacity of L cells to form virus was relatively resistant to UVL. In the Luria-Latarjet experiment using UVL, the survival curves of the infectious centres remained single-hit for the first 3 hours of the eclipse period, with slopes about half that of the survival curve for the free virus. Starting between 3 and 4 hours after infection, the survival curves became multihit, but their slopes remained parallel to the slopes of the survival curves of the early infectious centres. Possible interpretations of these results are discussed.