HCMV viral particles

One aspect of viral proteomics has been the characterization of virus particles and virally infected cells. Characterization of purified virions has led to the identification of viral proteins that were not originally identified with the virion as well as the identification of cellular proteins associated with the purified virus. For example, analysis of HCMV viral particles identified 12 additional ORFs not previously known to reside in virions as well as the identification of 71 cellular proteins [6]. The importance of these cellular and viral proteins in viral replication or pathogenesis awaits further analysis. Additionally, 12 unique polypeptides were identified that did not correspond to previously identified ORFs [6], illustrating the fact that despite intensive sequence analysis, sequence characteristics of viral promoters and ORFs are still not entirely understood. Analysis of virally infected cells has also led to the characterization of events leading to EBV-induced transformation [9,10,47], identification of cellular proteins induced in HIV-infected... 

Viral particles of HCMV and Kaposi s sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) have recently been examined. During the her-pesviral replicative cycle, different viral particles are formed. For HCMV, this includes mature, infectious virions, noninfectious enveloped particles, and dense bodies [6]. Similarly for KSHV, only a portion of the produced virus particles is infectious [40]. Therefore, analysis of infectious virions requires their separation from the noninfectious and immature forms. Density ultacentrifugation gradients are typically used to separate the various forms. Each fraction can be analyzed by electron microscopy to determine the level of purity [6,41] or by assaying for viral DNA and an envelope glycoprotein [40]. 

Members of the herpesvirus family acquire their envelope in the final stages of assembly by wrapping of Golgi, TGN, or endosomal membranes around nucleo-capsids (Brack et al. 2000 McMillan and Johnson 2001 Sanchez et al. 2000a,b). Thus, as virus particles bud into the TGN or endosomes. viral membrane glycoproteins, tegument, and capsid proteins are incorporated into the endosomal system. With HCMV the exact site of envelopment in the cytoplasm is less well defined than for the cx-herpesviruses, but it is likely that envelopment occurs into... 

More recently, the use of liquid chromatography and tandem MS (LC/MS/MS) has also eased purification and recovery methods. For instance, Vamum and colleagues utilized gel-free two-dimensional capillary LC/MS/MS and Fourier transform ion cyclotron resonance MS to identily and determine the relative abundances of viral and cellular proteins in purified HCMV virions and dense bodies. Analysis of the proteins Irom purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. They identified more than 71 HCMV-encoded proteins and 70 host cellular proteins in HCMV virions, which included cellular structural proteins, enzymes, and chaperones [6]. Another study using LC/MS/MS for the adenovirus type 5 pro-teome found a total of 11 protein species from 154 peptides, at a sensitivity of 10 copies per virus and a detection limit of 70 finol for two proteins [36]. 

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