Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication: characterization of a potential site of virus assembly

The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with the trans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.     

Sequence requirements for localization of human cytomegalovirus tegument protein pp28 to the virus assembly compartment and for assembly of infectious virus

The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.     

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.     

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.     

Multimerization of tegument protein pp28 within the assembly compartment is required for cytoplasmic envelopment of human cytomegalovirus

Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.     

Betaherpesvirus-conserved cytomegalovirus tegument protein ppUL32 (pp150) controls cytoplasmic events during virion maturation

The UL32 gene of human cytomegalovirus (CMV) encodes a prominent betaherpesvirus-conserved virion tegument protein, called pp150 (basic phosphoprotein/ppUL32), that accumulates within a cytoplasmic inclusion adjacent to the nucleus at late times during infection. Using a UL32 deletion mutant (DeltaUL32-BAC) (where BAC is bacterial artificial chromosome), we demonstrate that pp150 is critical for virion maturation in the cytoplasmic compartment. Cotransfection of a pp150 expression plasmid with DeltaUL32-BAC DNA led to complementation of the replication defect with focus formation due to secondary spread. Deletion of the amino terminus of pp150 or disruption of the betaherpesvirus conserved regions, CR1 and CR2, revealed these regions to be critical for replication. In contrast, deletion of the carboxyl terminus only partially compromised maturation while disruption of glycosylation sites had no effect. An African green monkey CMV UL32 homolog complemented DeltaUL32-BAC replication but murine CMV M32 failed to complement, consistent with evolutionary divergence of rodent and primate cytomegaloviruses. Infection with DeltaUL32-BAC showed normal expression of all kinetic classes of viral genes and replication of viral DNA, with accumulation of viral DNA-containing particles in the cytoplasm; however, mutant virus did not spread to adjacent cells. In contrast to this block in virion infectivity, cell-to-cell transfer of pp65-containing particles was observed, suggesting that release of dense bodies continued in the absence of pp150. These observations demonstrate that pp150 is critical for virion egress, possibly at the stage of final envelopment.     

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.     

Cytomegalovirus pUL96 is critical for the stability of pp150-associated nucleocapsids

Maturation of human cytomegalovirus (HCMV) initiates with nucleocapsids that egress from the nucleus and associate with a juxtanuclear cytoplasmic assembly compartment, where virion envelopment and release are orchestrated. Betaherpesvirus conserved proteins pp150 (encoded by UL32) and pUL96 are critical for HCMV growth in cell culture. pp150 is a capsid-proximal tegument protein that preserves the integrity of nucleocapsids during maturation. pUL96, although expressed as an early protein, acts late during virus maturation, similar to pp150, based on the comparable antigen distribution in UL96, UL32, or UL96/UL32 dual mutant virus-infected cells. pp150 associates with nuclear capsids prior to DNA encapsidation, whereas both pp150 and pUL96 associate with extracellular virus, suggesting that pUL96 is added after pp150. In the absence of pUL96, capsid egress from the nucleus continues; however, unlike wild-type virus infection, pp150 accumulates in the nuclear, as well as in the cytoplasmic, compartment. Ultrastructural evaluation of a UL96 conditional mutant revealed intact nuclear stages but aberrant nucleocapsids accumulating in the cytoplasm comparable to the known phenotype of UL32 mutant virus. In summary, pUL96 preserves the integrity of pp150-associated nucleocapsids during translocation from the nucleus to the cytoplasm.     

The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture.

The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses. The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies. Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed. However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses. In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.     

Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

In response to virus infection, cells can alter protein expression to modify cellular functions and limit viral replication. To examine host protein expression during infection with human cytomegalovirus (HCMV), an enveloped DNA virus, we performed a semiquantitative, temporal analysis of the cell surface proteome in infected fibroblasts. We determined that resident low density lipoprotein related receptor 1 (LRP1), a plasma membrane receptor that regulates lipid metabolism, is elevated early after HCMV infection, resulting in decreased intracellular cholesterol. siRNA knockdown or antibody-mediated inhibition of LRP1 increased intracellular cholesterol and concomitantly increased the infectious virus yield. Virions produced under these conditions contained elevated cholesterol, resulting in increased infectivity. Depleting cholesterol from virions reduced their infectivity by blocking fusion of the virion envelope with the cell membrane. Thus, LRP1 restricts HCMV infectivity by controlling the availability of cholesterol for the virion envelope, and increased LRP1 expression is likely a defense response to infection.     

Large-Molecular-Weight Precursors of Sindbis Virus Proteins

Infection of chicken embryo fibroblasts with a temperature-sensitive mutant of Sindbis virus at the nonpermissive temperature leads to the accumulation of a large-molecular-weight protein. We have shown that this protein contains (14)C-arginine tryptic peptides present in the three virion proteins. We have also found that a slightly smaller protein which is detected in Sindbis-infected BHK cells contains the (14)C-arginine tryptic peptides of the two envelope proteins but not those of the capsid protein. Pulse-chase experiments indicate that the Sindbis virus protein in BHK cells is cleaved to the envelope proteins.     

Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment

Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for alpha- and beta-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [(3)H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.     

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

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

The human cytomegalovirus-specific UL1 gene encodes a late-phase glycoprotein incorporated in the virion envelope

We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.     

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.     

Nuclear envelope breakdown can substitute for primary envelopment-mediated nuclear egress of herpesviruses

Herpesvirus nucleocapsids assemble in the nucleus but mature to infectious virions in the cytoplasm. To gain access to this cellular compartment, nucleocapsids are translocated to the cytoplasm by primary envelopment at the inner nuclear membrane and subsequent fusion of the primary envelope with the outer nuclear membrane. The conserved viral pUL34 and pUL31 proteins play a crucial role in this process. In their absence, viral replication is strongly impaired but not totally abolished. We used the residual infectivity of a pUL34-deleted mutant of the alphaherpesvirus pseudorabies virus (PrV) for reversion analysis. To this end, PrV-ΔUL34 was serially passaged in rabbit kidney cells until final titers of the mutant virus PrV-ΔUL34Pass were comparable to those of wild-type PrV. PrV-ΔUL34Pass produced infectious progeny independently of the pUL34/pUL31 nuclear egress complex and the pUS3 protein kinase. Ultrastructural analyses demonstrated that this effect was due to virus-induced disintegration of the nuclear envelope, thereby releasing immature and mature capsids into the cytosol for secondary envelopment. Our data indicate that nuclear egress primarily serves to transfer capsids through the intact nuclear envelope. Immature and mature intranuclear capsids are competent for further virion maturation once they reach the cytoplasm. However, nuclear egress exhibits a strong bias for nucleocapsids, thereby also functioning as a quality control checkpoint which is abolished by herpesvirus-induced nuclear envelope breakdown.     

The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.     

Phosphorylation of tegument protein pp28 contributes to trafficking to the assembly compartment in human cytomegalovirus infection

Human cytomegalovirus (HCMV) UL99 encodes a late tegument protein pp28 that is essential for envelopment and production of infectious virus. This protein is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells but it localizes to the cytoplasmic assembly compartment (AC) in HCMV-infected cells. Trafficking of pp28 to the AC is required for the assembly of infectious virus. The N-terminal domain (aa 1–61) of pp28 is sufficient for trafficking and function of the wild type protein during viral infection. However, residues required for authentic pp28 trafficking with the exception of the acidic cluster in the N-terminal domain of pp28 remain undefined. Monitoring protein migration on SDS-PAGE, we found that pp28 is phosphorylated in the virus-infected cells and dephosphorylated in the viral particles. By generating substitution mutants of pp28, we showed that three serine residues (aa 41–43) and a tyrosine residue (aa 34) account for its phosphorylation. The mutant forms of pp28 were localized to the plasma membrane as well as the ERGIC in transfected cells. Likewise, these mutant proteins were localized to the plasma membrane as well as the AC in virus-infected cells. These results suggested that phosphorylation of pp28 contributes to its intracellular trafficking and efficient viral assembly and incorporation.