The three-dimensional structure of the intact human cytomegalovirus (HCMV) was determined to 18-A resolution by electron cryomicroscopy and computer reconstruction. Its capsid shell is composed of pentons, hexons, and triplexes arranged on a T = 16 icosahedral lattice and is identical to that of the B-capsid isolated from host cell nuclei. An icosahedrally ordered tegument layer formed by 960 copies of filamentous density is also visualized, which interacts with the pentons, hexons, and triplexes of the underlying capsid. The observed structural similarities and differences of HCMV with those of herpes simplex virus offer insights into the significance of the different tegument components for their infection processes while maintaining similar capsids.

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals.

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus which causes mild or subclinical diseases in immunocompetent adults but may lead to severe morbidity or mortality in neonates and immunocompromised individuals (2, 24). Disseminated HCMV infection, common in AIDS patients and organ transplant recipients, is usually associated with gastroenteritis, pneumonia, and retinitis (12,29). Studies on the functions of viral genes in HCMV replication in vivo are essential for understanding viral pathogenesis and developing new strategies to combat the viral infection. However, there are currently no suitable animal models for HCMV infection. HCMV only propagates in human cells and grows slowly due to a long lytic replication cycle (24). These properties of HCMV have hampered the studies of HCMV pathogenesis and gene function.Infection of the mouse with murine CMV (MCMV) provides a valuable in vivo model for studying the biology of CMV infection. This is because infection of mice by MCMV resembles in many ways its human counterpart with respect to pathogenesis during acute infection, establishment of latency, and reactivation after immunosuppression, transfusion, or transplantation (2,15,17,24). Its genome of 230 kb is predicted to encode more than 170 open reading frames, 78 of which have extensive homology to those of HCMV (5, 32). However, many of these MCMV genes remained uncharacterized and their functions in viral pathogenesis have not been investigated.One of the most powerful appr...

Human cytomegalovirus (HCMV) is the most genetically and structurally complex human herpesvirus and is composed of an envelope, a tegument, and a dsDNA-containing capsid. HCMV tegument plays essential roles in HCMV infection and assembly. Using cryo-electron tomography (cryoET), here we show that HCMV tegument compartment can be divided into two sub-compartments: an inner and an outer tegument. The inner tegument consists of densely-packed proteins surrounding the capsid. The outer tegument contains those components that are loosely packed in the space between the inner tegument and the pleomorphic glycoprotein-containing envelope. To systematically characterize the inner tegument proteins interacting with the capsid, we used chemical treatment to strip off the entire envelope and most tegument proteins to obtain a tegumented capsid with inner tegument proteins. SDS-polyacrylamide gel electrophoresis analyses show that only two tegument proteins, UL32-encoded pp150 and UL48-encoded high molecular weight protein (HMWP), remains unchanged in their abundance in the tegumented capsids as compared to their abundance in the intact particles. 3D reconstructions by single particle cryo-electron microscopy (cryoEM) reveal that the net-like layer of icosahedrally-ordered tegument densities are also the same in the tegumented capsid and in the intact particles. CryoET reconstruction of the tegumented capsid labeled with an anti-pp150 antibody is consistent with the biochemical and cryoEM data in localizing pp150 within the ordered tegument. Taken together, these results suggest that pp150, a betaherpesvirus-specific tegument protein, is a constituent of the net-like layer of icosahedrally-ordered capsid-bound tegument densities, a structure lacking similarities in alpha and gammaherpesviruses.

A pool of murine cytomegalovirus (MCMV) mutants was previously generated by using a Tn3-based transposon mutagenesis approach (X. Zhan, M. Lee, J. Xiao, and F. Liu, J. Virol. 74:7411-7421, 2000). In this study, one of the MCMV mutants, Rvm155, which contained the transposon insertion in open reading frame m155, was characterized in vitro for its replication in tissue culture and in vivo for its growth and virulence in immunodeficient SCID mice. Compared to the wild-type strain and a rescued virus that restored the m155 region, the mutant is significantly deficient in growth in many organs of the infected animals. At 21 days postinfection the titers of Rvm155 in the salivary glands, lungs, spleens, livers, and kidneys of the intraperitoneally infected SCID mice were lower than the titers of the wild-type virus and the rescued virus by 50-, 1,000-, 500-, 100-, and 500-fold, respectively. Moreover, the viral mutant was attenuated in killing the SCID mice, as none of the SCID mice that were intraperitoneally infected with Rvm155 died until 38 days postinfection while all the animals infected with the wild-type and rescued viruses died at 27 days postinfection. Our results provide the first direct evidence that a disruption of m155 expression leads to attenuation of viral virulence and growth in animals. Moreover, these results suggest that m155 is a viral determinant for optimal MCMV growth and virulence in vivo.Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes mild or subclinical diseases in immunocompetent adults but may lead to severe complications in neonates and immunocompromised individuals (18, 23). Disseminated HCMV infection, common in AIDS patients, is usually associated with gastroenteritis, pneumonia, and retinitis (8,22). HCMV infection continues to be a major cause of morbidity and mortality in bone marrow and solid-organ transplant recipients (18, 23). Studies on the functions of viral genes in HCMV replication in vivo are essential for understanding viral pathogenesis and developing new strategies to combat the viral infection. However, there are presently no suitable animal models for HCMV infection. HCMV only propagates in human cells and grows slowly due to a long lytic replication cycle (18,23). These properties of HCMV have hampered the studies of HCMV pathogenesis and gene function.Infection of the mouse with murine cytomegalovirus (MCMV) provides a valuable in vivo model for studying the biology of CMV infection. This is because infection of mice by MCMV resembles in many ways its human counterpart with respect to pathogenesis (11,12,18,23). For example, tropism for the spleen and liver is believed to be important in the infection of both HCMV and MCMV, as the spleen and liver are the sites for primary acute infections as well as persistent and latent infections (18,23). The MCMV genome is 230 kb long and is predicted to encode more than 170 open reading frames, 78 of which have extensive homology to those of HCMV (4,25). A clear understanding of the biology of MCMV and the ...