O L

hiGH iHi

Tandem repeats

13 18\ -fTTTTTTTTTMnTTTTTl-

900bp 1300bp

Tandem repeats 18 13

nnmHm^

1300bp 900bp

FIGURE 6.2 Schematic representation of the DNA of vaccinia virus. Upper part: linear double-stranded DNA with terminal hairpins and inverted repeats (not to scale). Center line is the HindlH restriction map (* indicates the TK gene). Color coding of the HindlH fragments is the same as that used in Tables 6.4 and 6.5. Lower line diagrams the internal structure of the terminal repeats. [From Fenner et al. (1988).]

pathway by which midcycle genes and finally late genes are expressed, Early and mid-cycle functions include interference with host defense mechanisms and the replication of the viral genome, whereas late genes are primarily involved with formation of progeny virions,

Poxviruses have large genomes, from 130 to 380 kb, and encode hundreds of proteins, A schematic of the vaccinia virus genome is shown in Fig, 6,2, It is double stranded and linear but the ends of the genome are covalently closed so that the genome consists, in essence, of a very large single-stranded circular molecule that is self-complementary, The ends of the genome possess inverted terminal repeats that are involved in the initiation of DNA replication,

Because virus replication, including DNA replication, occurs in the cytoplasm, poxviruses must encode all enzymes required for DNA replication and production of mRNAs, Thus the encoded proteins include DNA and RNA polymerases, a poly(A) polymerase to polyadenylate mRNAs, a capping enzyme, several enzymes with functions in nucleotide metabolism, protein kinases, DNA topoiso-merases, as well as the proteins that form components of the virion and proteins that interfere with antiviral host defense mechanisms, Table 6,4 lists many enzymatic proteins encoded by vaccinia virus and Table 6,5 lists structural proteins and proteins without enzymatic activity, Poxvirus-encoded molecules that interfere with host defenses are discussed in Chapter 8,

A model for vaccinia DNA replication is shown in Fig, 6,3, The mechanisms by which DNA replication is initiated have not been completely worked out, but it is thought that a viral enzyme specifically nicks the DNA in or near the terminal repetitions, and the 3' end of the nicked DNA forms a primer for DNA synthesis, Continued elonga tion of the DNA chain leads to production of concatenated progeny DNA, These concatamers must subsequently be resolved into genome-length segments whose ends are then covalently closed,

Replication of DNA and assembly of progeny virions occurs in what have been called factories in the cytoplasm, These are electron-dense areas that contain viral DNA and membranes, Progeny DNA is assembled into cores and assembly of virions occurs by condensation of membranes around the viral core, It is not clear whether the membranes that are used to assemble virions are derived from previously existing cell membranes or are newly synthesized, Intracellular forms of the virus, which are infectious, are surrounded by a lipid-containing membrane, but extracellular virions have an additional lipid-containing envelope, It is not known how the envelope is acquired, Poxvirions are illustrated schematically in Fig, 2,20,

Interactions with the Host

The poxvirus genome encodes many proteins that are not required for replication of the virus in cell culture, These include proteins that subvert the antiviral defenses of the host and that may extend the host range of the virus, Vaccinia virus, variola virus, and other poxviruses encode proteins that interfere with the complement system and the interferon system, that prevent an inflammatory response to the virus or the induction of apoptosis (programmed cell death), and that interfere with the activity of TNF (tumor necrosis factor), among others, The discovery of these proteins that interfere with host antiviral defenses is recent, and more study is necessary to understand the full extent of viral inhibition of host

TABLE 6.4 Vaccinia-Encoded Enzymes

Functional group

ORF"

kDa

Properties

name of enzyme

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