Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, causes a lifelong subclinical infection in healthy adults but leads to significant morbidity and mortality in neonates and immunocompromised individuals. Its ability to grow in different cell types is responsible for HCMV-associated diseases, including mental retardation and retinitis, and vascular disorders. To globally assess viral gene function for replication in cells, we determined the genomic sequence of a bacterial artificial chromosome (BAC)-based clone of HCMV Towne strain and used this information to delete each of its 162 unique ORFs and generate a collection of viral mutants. The growth of these mutants in different cultured cells was examined to systematically investigate the necessity of each ORF for replication. Our results showed that 45 ORFs are essential for viral replication in fibroblasts and 117 are nonessential. Some genes were found to be required for viral replication in retinal pigment epithelial cells and microvascular endothelial cells, but not in fibroblasts, indicating their role as tropism factors. Interestingly, several viral mutants grew 10- to 500-fold better than the parental strain in different cell types, suggesting that the deleted ORFs encode replication temperance or repressing functions. Thus, HCMV encodes supportive and suppressive growth regulators for optimizing its replication in human fibroblasts, epithelial, and endothelial cells. Suppression of viral replication by virus-encoded temperance factors represents a novel mechanism for regulating the growth of an animal virus, and may contribute to HCMV's optimal infection of different tissues and successful proliferation among the human population.

https://www.pnas.org/content/pnas/100/24/14223.full.pdf

Functional profiling of a human cytomegalovirus genome

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.

Accumulation of Virion Tegument and Envelope Proteins in a Stable Cytoplasmic Compartment during Human Cytomegalovirus Replication: Characterization of a Potential Site of Virus Assembly

Human cytomegalovirus (HCMV) is a common, medically relevant human herpesvirus. The tegument layer of herpesvirus virions lies between the genome-containing capsids and the viral envelope. Proteins within the tegument layer of herpesviruses are released into the cell upon entry when the viral envelope fuses with the cell membrane. These proteins are fully formed and active and control viral entry, gene expression, and immune evasion. Most tegument proteins accumulate to high levels during later stages of infection, when they direct the assembly and egress of progeny virions. Thus, viral tegument proteins play critical roles at the very earliest and very last steps of the HCMV lytic replication cycle. This review summarizes HCMV tegument composition and structure as well as the known and speculated functions of viral tegument proteins. Important directions for future investigation and the challenges that lie ahead are identified and discussed.

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Tegument Proteins of Human Cytomegalovirus

The human cytomegalovirus UL99-encoded pp28 is a myristylated phosphoprotein that is a constituent of the virion. The pp28 protein is positioned within the tegument of the virus particle, a protein structure that resides between the capsid and envelope. In the infected cell, pp28 is found in a cytoplasmic compartment derived from the Golgi apparatus, where the virus buds into vesicles to acquire its final membrane. We have constructed two mutants of human cytomegalovirus that fail to produce the pp28 protein, a substitution mutant (BADsubUL99) and a point mutant (BADpmUL99), and we have propagated them by complementation in pp28-expressing fibroblasts. Both mutant viruses are profoundly defective for growth in normal fibroblasts; no infectious virus could be detected after infection. Whereas normal levels of viral DNA and late proteins were observed in mutant virus-infected cells, large numbers of tegument-associated capsids accumulated in the cytoplasm that failed to acquire an envelope. We conclude that pp28 is required for the final envelopment of the human cytomegalovirus virion in the cytoplasm.

Human Cytomegalovirus UL99-Encoded pp28 Is Required for the Cytoplasmic Envelopment of Tegument-Associated Capsids

The human cytomegalovirus UL82 gene encodes a protein (pp71) that is localized in the tegument domain of the virus particle. The UL82 gene product is delivered to the nucleus at the time of infection, and it is believed to function in gene activation. We have constructed a human cytomegalovirus mutant, ADsubUL82, that lacks a substantial portion of the UL82 coding region. It was propagated on human diploid fibroblasts expressing the UL82 gene product, and it was possible to produce a mutant virus lacking the UL82 protein by passaging virus stocks for one cycle of growth on normal, noncomplementing fibroblasts. The UL82-deficient mutant displays a multiplicity-dependent growth defect in normal human fibroblasts. The growth of ADsubUL82 is severely restricted at low input multiplicities (0.01-0.1 plaque-forming units per cell), producing a yield that is reduced by a factor of about 10(5) in comparison to wild-type virus. At higher input multiplicities (10 plaque-forming units per cell), ADsubUL82 grew nearly as well as the wild-type virus. By using a human cytomegalovirus gene array, we demonstrated that UL82 functions to facilitate virus mRNA accumulation very early during the human cytomegalovirus replication cycle. The growth phenotype associated with the UL82 mutant seems to result from its inability to efficiently activate human cytomegalovirus immediate early genes.

https://www.pnas.org/content/pnas/97/26/14506.full.pdf

UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells.

A polyclonal antiserum, raised against a pp71 fusion protein, was prepared in order to investigate the biosynthesis and localization of the matrix protein pp71 of human cytomegalovirus (HCMV), the UL82 gene product, during the HCMV infectious cycle in human fibroblasts. Transcription of the pp71-specific bicistronic 4·0 kb mRNA and pp71 biosynthesis exhibited a biphasic pattern during one round of the HCMV infectious cycle, with a first peak at 12 h and a second at 72 h post-infection (p.i.). Cycloheximide treatment of infected human fibroblasts revealed that the presence of pp71 in total cell extracts prior to 3 h p.i. was due to the input virus inoculum. Transcription of the two specific pp71 mRNAs commenced 5–7 h p.i. as shown by Northern blot analysis of total cellular RNA. Western blot analysis of isolated nuclei and indirect immuno-fluorescence experiments indicated that pp71, like the major tegument protein pp65, is present in the nucleus shortly after infection as well as during the late phase of viral morphogenesis. Also, after transient transfection of UL82 into U373MG cells, pp71 was found to be present in the nucleus of the transfected cells. By immunogold labelling, pp71 was detected in the nucleoplasm in association with nucleocapsids in electron-dense nuclear skein structures at late stages of the infection cycle. These findings suggest functions of pp71 in viral maturation in addition to that as an early transactivator of viral gene transcription described recently.

Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82): evidence for its translocation into the nucleus.

The human cytomegalovirus (HCMV) basic phosphoprotein pp150, encoded by the UL32 gene, together with the two other major phosphoproteins, pp65 (ppUL83) and pp71 (ppUL82) and several minor structural proteins, form the tegument around the viral nucleocapsid. Experiments were undertaken to locate the area of assembly of tegument proteins pp150 and pp65 and nucleocapsids in fibroblasts, in order to assess the functional role of these two structural proteins in HCMV morphogenesis. Whereas pp150 expression starts during the cytoplasmic maturation of HCMV, pp65 is expressed in the early and late phases of HCMV gene transcription. Western blot analysis of isolated cell fractions showed that pp150 is initially (48 h post-infection) localized in the nucleus, associated either with the nuclear membrane or with viral assembly regions, and later (72 h post-infection) in the cytoplasm. By indirect immunofluorescence, pp150 and pp65 could be detected in nuclear subcompartments and were strongly associated with the nuclear membrane. Using immunogold analysis by electron microscopy, pp65 was exclusively detected within the matrix of cytoplasmic and extracellular dense bodies and of dense body-like structures in the nucleoplasm. These were localized in close contact with hypertrophic nucleoli, in the proximity of developing nucleocapsids and in special patches at the inner nuclear membrane. Positive immunostaining of pp150 was observed at the surface of developing nucleocapsids concentrated within viral assembly regions in the nucleoplasm. Additionally, the tegument of cytoplasmic and extracellular virions was stained, whereas dense bodies or nuclear dense body-like structures did not react. Thus, the acquisition of the tegument protein pp150 seems to start in special nuclear subcompartments of the HCMV-infected fibroblasts.

Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppul32)

Human cytomegalovirus (HCMV) open reading frame UL32 codes for the basic phosphoprotein pp150 (ppUL32), an abundant constituent of the virion tegument. In order to study its potential role in the assembly and/or transport of progeny particles, astrocytoma cell lines (U373MG) were generated, stably expressing a 2.1 kb 5' fragment of UL32 in antisense orientation under the control of the HCMV major immediate early promoter. The steady-state level of the UL32 sense mRNA and pp150 synthesis were strongly reduced in infected antisense cell lines. Neither immediate early and early gene expression, nor viral DNA replication, was inhibited; the expression of the late gene product gB (gpUL55) was also reduced, but mainly at the level of translation. Control experiments indicated that this differential effect of UL32 antisense expression on the synthesis of viral products was specific. As a consequence of the inhibitory effect, virus yield was significantly reduced in antisense mRNA cell lines. Ultrastructural comparison of control and antisense cells revealed no difference in nucleocapsid forms in the nucleus. However, in the cytoplasm of antisense cells, DNA-containing C capsids and virions were absent and abnormal forms of non-infectious enveloped particles were observed. The data suggest the involvement of pp150 either in the transport of DNA-containing particles through the nuclear envelope or in the stabilization of capsids in the cytoplasm. Thus, UL32 antisense mRNA appears to interfere strongly with virus maturation during the late phase of the infectious cycle.

Horst F. Kern

Papers

Intracellular localization and expression of the human cytomegalovirus matrix phosphoprotein pp71 (ppUL82): evidence for its translocation into the nucleus.

Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppul32)

Human cytomegalovirus late-phase maturation is blocked by stably expressed UL32 antisense mRNA in astrocytoma cells