UL26-deficient human cytomegalovirus produces virions with hypophosphorylated pp28 tegument protein that is unstable within newly infected cells

The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.     

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.     

Functional analysis of the pseudorabies virus UL51 protein

Homologs of the UL51 protein of herpes simplex virus have been identified in all herpesvirus subfamilies, but until now, no function has been assigned to any of them. To investigate function of the UL51 gene product of the alphaherpesvirus pseudorabies virus (PrV), we isolated and analyzed a mutant lacking the major part of the open reading frame, PrV-DeltaUL51F, and a rescuant. One-step growth analysis of PrV-DeltaUL51F revealed only slightly reduced titers, but plaque size was notably diminished and reached only approximately 30% the plaque size of wild-type PrV. Ultrastructurally, intracytoplasmic capsids were found in large numbers either without envelope or in different stages of envelopment, indicating that secondary envelopment in the cytoplasm was less efficient. However, neuroinvasion in the mouse trigeminal pathway after intranasal infection was only slightly delayed. A PrV UL11 mutant also showed a defect in secondary envelopment (M. Kopp, H. Granzow, W. Fuchs, B. G. Klupp, E. Mundt, A. Karger, and T. C. Mettenleiter, J. Virol. 77:5339-5351, 2003). Since both proteins are part of the viral tegument and are predicted to be membrane associated, they may serve similar, possibly redundant functions during viral morphogenesis. Therefore, we also isolated a mutant simultaneously lacking UL51 and UL11. This mutant exhibited further reduced plaque size compared to the single-deletion mutants, but viral titers were comparable to those for the UL11 mutant. In electron microscopic analyses, the observed defect in secondary envelopment was similar to that found in the UL11 single-deletion mutant. In conclusion, both conserved tegument proteins, either singly or in combination, are involved in virion morphogenesis in the cytoplasm but are not essential for viral replication in vitro and in vivo.     

Human cytomegalovirus pUS24 is a virion protein that functions very early in the replication cycle

We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.     

A specific subform of the human cytomegalovirus transactivator protein pUL69 is contained within the tegument of virus particles.

The polypeptide encoded by the open reading frame UL69 of human cytomegalovirus (HCMV), which is homologous to the immediate-early regulator ICP27 of herpes simplex virus, has recently been identified as a transactivator protein that exerts a broad stimulatory effect on gene expression (M. Winkler, S. A. Rice, and T. Stamminger, J. Virol. 68:3943-3954, 1994). Here, we provide evidence that pUL69 is a phosphorylated tegument protein of HCMV. This finding could be demonstrated by Western blot (immunoblot) analyses with purified virions and a specific antiserum against pUL69. These experiments revealed that one phosphorylated subform of the three pUL69 polypeptides that are synthesized in infected fibroblast cells is contained within the HCMV virion. After the treatment of purified virions with detergents, pUL69 could not be detected within the membrane fraction, suggesting that it is either a capsid or a tegument protein. Its presence within dense bodies, however, shows that pUL69 is a constituent of the viral tegument.     

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.     

Mutant human cytomegalovirus lacking the immediate-early TRS1 coding region exhibits a late defect

The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. ADsubIRS1 failed to produce the related IRS1 and IRS1(263) proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in trans of the IRS1(263) protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. ADsubTRS1 failed to produce the TRS1 protein, and it generated an approximately 200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in ADsubTRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within ADsubTRS1-infected cells. Further, infectious ADsubTRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the ADsubTRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.     

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function

The human cytomegalovirus (HCMV) virion is comprised of a linear double-stranded DNA genome, proteinaceous capsid and tegument, and a lipid envelope containing virus-encoded glycoproteins. Of these components, the tegument is the least well defined in terms of both protein content and function. Several of the major tegument proteins are phosphoproteins (pp), including pp150, pp71, pp65, and pp28. pp28, encoded by the UL99 open reading frame (ORF), traffics to vacuole-like cytoplasmic structures and was shown recently to be essential for envelopment. To elucidate the UL99 amino acid sequences necessary for its trafficking and function in the HCMV replication cycle, two types of viral mutants were analyzed. Using a series of recombinant viruses expressing various UL99-green fluorescent protein fusions, we demonstrate that myristoylation at glycine 2 and an acidic cluster (AC; amino acids 44 to 57) are required for the punctate perinuclear and cytoplasmic (vacuole-like) localization observed for wild-type pp28. A second approach involving the generation of several UL99 deletion mutants indicated that at least the C-terminal two-thirds of this ORF is nonessential for viral growth. Furthermore, the data suggest that an N-terminal region of UL99 containing the AC is required for viral growth. Regarding virion incorporation or UL99-encoded proteins, we provide evidence that suggests that a hypophosphorylated form of pp28 is incorporated, myristoylation is required, and sequences within the first 57 amino acids are sufficient.     

The UL14 tegument protein of herpes simplex virus type 1 is required for efficient nuclear transport of the alpha transinducing factor VP16 and viral capsids

The protein encoded by the UL14 gene of herpes simplex virus type 1 (HSV-1) and HSV-2 is expressed late in infection and is a minor component of the virion tegument. An UL14-deficient HSV-1 mutant (UL14D) forms small plaques and exhibits an extended growth cycle at low multiplicities of infection (MOI) compared to wild-type virus. Although UL14 is likely to be involved in the process of viral maturation and egress, its precise role in viral replication is still enigmatic. In this study, we found that immediate-early viral mRNA expression was decreased in UL14D-infected cells. Transient coexpression of UL14 and VP16 in the absence of infection stimulated the nuclear accumulation of both proteins. We intended to visualize the fate of VP16 released from the infected virion and constructed UL14-null (14D-VP16G) and rescued (14R-VP16G) viruses that expressed a VP16-green fluorescent protein (GFP) fusion protein. Synchronous high-multiplicity infection of the viruses was performed at 4°C in the absence of de novo protein synthesis. We found that the presence of UL14 in the virion had an enhancing effect on the nuclear accumulation of VP16-GFP. The lack of UL14 did not significantly alter virus internalization but affected incoming capsid transport to the nuclear pore. These observations suggested that UL14 (i) enhanced VP16 nuclear localization at the immediately early phase, thus indirectly regulating the expression of immediate-early genes, and (ii) was associated with efficient nuclear targeting of capsids. The tegument protein UL14 could be part of the machinery that regulates HSV-1 replication.     

The cytoplasmic tail of glycoprotein M (gpUL100) expresses trafficking signals required for human cytomegalovirus assembly and replication

The virion envelope of human cytomegalovirus (HCMV) is complex and consists of an incompletely defined number of glycoproteins. The gM/gN protein complex is the most abundant protein component of the envelope. Studies have indicated that deletion of the viral gene encoding either gM or gN is a lethal mutation. Analysis of the amino acid sequence of gM disclosed a C-terminal acidic cluster of amino acids and a tyrosine-containing trafficking motif, both of which are well-described trafficking/sorting signals in the cellular secretory pathway. To investigate the roles of these signals in the trafficking of the gM/gN complex during virus assembly, we made a series of gM (UL100 open reading frame) mutants in the AD169 strain of HCMV. Mutant viruses that lacked the entire C-terminal cytoplasmic tail of gM were not viable, suggesting that the cytoplasmic tail of gM is essential for virus replication. In addition, the gM mutant protein lacking the cytoplasmic domain exhibited decreased protein stability. Mutant viruses with a deletion of the acidic cluster or alanine substitutions in tyrosine-based motifs were viable but exhibited a replication-impaired phenotype suggestive of a defect in virion assembly. Analysis of these mutant gMs using static immunofluorescence and fluorescence recovery after photobleaching demonstrated delayed kinetics of intracellular localization of the gM/gN protein to the virus assembly compartment compared to the wild-type protein. These data suggest an important role of the glycoprotein gM during virus assembly, particularly in the dynamics of gM trafficking during viral-particle assembly.     

Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E

Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.     

The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression

We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.     

The human cytomegalovirus (HCMV) tegument protein UL94 is essential for secondary envelopment of HCMV virions

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood. However, several tegument proteins are known to be essential for proper particle assembly and maturation. Despite intense investigation, the function of many tegument proteins remains unknown. The HCMV UL94 gene is conserved among all herpesviruses and encodes a virion protein of unknown function. We demonstrate here that UL94 is a tegument protein that is expressed with true-late kinetics and localizes to the viral assembly complex during infection. To elucidate the function of UL94, we constructed a UL94-null mutant, designated UL94stop. This mutant is completely defective for replication, demonstrating that UL94 is essential. Phenotypic analysis of the UL94stop mutant shows that in the absence of UL94, viral gene expression and genome synthesis occur at wild-type levels. However, analysis of the localization of viral proteins to the cytoplasmic assembly complex shows that the essential tegument protein UL99 (pp28) exhibits aberrant localization in cells infected with the UL94stop mutant. Finally, we show that there is a complete block in secondary envelopment in the absence of UL94. Taken together, our data suggest that UL94 functions late in infection to direct UL99 to the assembly complex, thereby facilitating secondary envelopment of virions.     

Interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication

The human cytomegalovirus UL82-encoded pp71 protein is required for efficient virus replication and immediate-early gene expression when cells are infected at a low multiplicity. Functions attributed to pp71 include the ability to enhance the infectivity of viral DNA, bind to and target hypophosphorylated Rb family member proteins for degradation, drive quiescent cells into the cell cycle, and bind to the cellular protein hDaxx. Using UL82 mutant viruses, we demonstrate that the LXCXD motif within pp71 is not necessary for efficient virus replication in fibroblasts, suggesting that pp71's ability to degrade hypophosphorylated Rb family members and induce quiescent cells into the cell cycle is not responsible for the growth defect associated with a UL82 deletion mutant. However, UL82 mutants that cannot bind to hDaxx are unable to induce immediate-early gene expression and are severely attenuated for viral replication. These results indicate that the interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.     

Control of cytoplasmic maturation events by cytomegalovirus tegument protein pp150

Cytomegalovirus replication depends upon a betaherpesvirus-conserved 150-kDa tegument phosphoprotein (pp150; encoded by UL32) that supports the final steps in virion maturation at cytoplasmic assembly compartments. Amino acid substitutions were introduced into conserved region 1 (CR1) and CR2 of pp150, affecting a region that may interact with nucleocapsids. Two independent CR2 point mutants (N201A and G207A) failed to support viral replication in evaluations by a transient complementation assay or after reconstruction into recombinant viruses. An assembly compartment-like cytoplasmic inclusion developed in UL32 mutant virus-infected cells that was similar to that of wild-type virus-infected cells. The cellular localization of the trans-Golgi marker Golgin-97 suggested differences in the organization of the assembly compartment compared to that of wild-type virus-infected cells. Replication-defective CR2 point mutants exhibited the same phenotype as that of a virus carrying a complete deletion of the UL32 open reading frame in these assays. Electron micrographs of fibroblasts at 3 or 5 days postinfection with a deletion mutant (ΔUL32) grown on UL32-complementing cells showed a similar number and morphology of capsids in the nucleus, but the cytoplasmic region associated with virion assembly appeared highly vesiculated and contained few recognizable nucleocapsids or complete virus particles. These data demonstrate that the principle role of pp150 is to retain nucleocapsid organization through secondary envelopment at the assembly compartment.