Alphavirus replication

Perhaps as a consequence of their adaptation to dissimilar hosts in nature, the alphaviruses replicate readily, and generally to very high titers, in a wide range of cell types and culture conditions in vitro. Virus titers of 1 billion infectious units per milliliter are not unusual, and the viruses are stable in storage and in a variety of laboratory procedures. Because of the relative ease with which these viruses can be manipulated in the laboratory, they have long served as model systems by which to study various aspects of virus replication, pathogenesis, induction of immune responses, and virus-vector relationships. As a result, the alphaviruses are well described and their characteristics well defined.1213... 

Alphavirus replication requires three RNA synthesis activities a minus strand replicase to produce full-length minus strands using the plus strand as a template, a plus-strand replicase to produce full length plus strands from the minus strands, and a transcriptase to produce the 26 S mRNA for the structural proteins. Each of these activities appears to be independently regulated and different recognition sites for the corresponding enzymes are utilized. These enzymatic activities apparently reside in four different rionstructural polypeptides (Keranen and Ruohonen, 1983 Strauss and Strauss, 1983 Strauss et al., 1983). 

In this section, I will discuss the evidence that host cell components are directly involved in the alphavirus replication cycle during acute cytolytic infection. 

Another example of host cell involvement in alphavirus replication comes from the results of Mento and Siminovitch (1981), who isolated mutant CHO cell lines in which SIN replicated without causing cytopathic effects. The mutants were selected in one step from mu-tagenized wild-type cells. In one of the mutant cell lines, the host cell block to virus replication appears to be at the level of virus mRNA translation. Although they could detect only very small amounts of viral antigens in the infected mutant cells, viral mRNAs made in these... 

The structure of an alphavirus particle is simpler than that of all known cellular organelles, but it is built according to the same principles. This is because the viral genome is small and the virus must use for its construction those cellular components normally engaged in the biogenesis of host cell membranes. This means that studies of viral replication can be exploited to study cellular functions at the molecular level. Naturally viral infections also perturb cellular physiology, but there is usually enough time early in infection for studies to be carried out before cellular malfunction becomes a source of error. 

Studies of the alphavirus life cycle have revealed how heavily the virus relies on cellular processes for replication. The paucity of functions that seem unique to the virus is striking. The binding of the virus to the cell surface and the fusion of its membrane intracellularly depend on the viral spike glycoproteins. RNA-dependent RNA polymerases specific for the virus catalyze the replication of the viral RNA. Exit from the cell requires the interaction of the viral spike proteins with the viral capsid... 

Strauss JH, Strauss EG (1994), The alphaviruses gene expression, replication, and evolution, Microbiol. Rev. 58 491-562. 

Wengler, G. (1980). Effects of alphaviruses on host cell macromolecular synthesis. In The Togaviruses Biology, Structure, Replication (R. W. Schlesinger, Ed.), pp. 459-472. Academic Press, New York. 

Fig. 1 Schematic representation of a typical DNA vaccine plasmid. Shown are the promoter (pCMVIE), transcription terminator (BGHpa), bacterial origin of replication (ori), and antibiotic resistance gene (Kan ). Several different types of inserts may be included in a DNA vaccine. Shown are ones containing a discrete open reading frame and those containing an alphavirus RNA replicon. SG = subgenomic promoter.

STRUCTURE AND REPLICATION OF ALPHAVIRUSES Virion Structure Replication... 

Maximal amounts of virus are typically produced from mammalian cells within 8 to 10 hours after infection, and disintegration of the infected cell is likely due to programmed cell death (apoptosis) rather than direct effects of the virus on cellular function.87 In contrast, alphaviruses initially replicate to high titer in arthropod cells with little or no evidence of cytopathology. The surviving cells continue to produce lesser amounts of virus, often for weeks or months. The ability of the virus to replicate without causing cell death in arthropod cells may be critical for maintenance of the virus in the mosquito vector in nature. 

An unresolved problem with the use of TC-83, and presumably with other live-attenuated alpha-virus vaccines, is the phenomenon of vaccine interference, in which prior immunity to heterologous alphaviruses inhibits vaccine virus replication and subsequent immune responses. This occurrence has been observed in horses,169 170 in which preexisting antibodies to EEE and WEE may have interfered with TC-83 vaccination. Interference has also been observed in humans, in whom prior vaccination with Chikungunya virus has reduced the response to TC-83, and vice versa (D.J.McC., unpublished research, 1994). 

Strauss EG, Strauss JH. Structure and replication of the alphavirus genome. In Schlesinger S, Schlesinger MJ, eds. The Togaviridae and Flaviviridae. New York, NY Plenum Press 1986 350-390. 

The replication of alphaviruses and flaviviruses is supported by both vertebrate and invertebrate cells in tissue culture. Infection by either virus group in permissive vertebrate cells produces infectious viruses and inhibits host cell macromolecular synthesis leading to eventual host cell death, whereas, in permissive arthropod cells, in-... 

The replication of alphaviruses is relatively well understood at the molecular level and extensive review articles on these viruses have recently appeared (Strauss and Strauss, 1977, 1983 Kaariainen and Soderlund, 1978 Schlesinger and Kaariainen, 1980 Garoff et al., 1982). In particular, an excellent review concerning the effects of alphavirus infection on host cell macromolecular synthesis has been written by Wengler (1980), and I will concentrate on recent progress in this field. 

It is important to know whether viral-specified proteins are involved in the inhibition of host protein synthesis and ts mutants have been used to try to answer this question. RNA t mutants did not inhibit cellular protein synthesis at the restrictive temperature (Atkins, 1976). Under these conditions, RNA / mutants of alphaviruses make neither viral encoded proteins nor viral RNAs (Keranen and Kaariainen, 1975 Hashimoto and Simizu, 1978). From these findings, we can conclude that initiation of viral RNA replication is necessary for inhibition of host protein synthesis and also that viral components introduced into cells by the infecting virions are not directly responsible for this inhibition. 

In alphavirus infection, the rate of this inhibition depends on the multiplicity of infection, and is closely related to viral replication. This inhibition does not result from degradation of cellular DNA, nor suppression of precursor uptake (Simizu et al., 1976). 

Infection of A. albopictus cells with alphaviruses leads to the synthesis of maximum titers of infectious virus by approximately 24 hr postinfection (acute phase). At the acute phase, up to 85% of the cells released infectious virus (Davey and Dalgarno, 1974), and the rate of synthesis of intracellular 42 S and 26 S viral RNA and structural proteins is maximal. By 48 hr postinfection, viral 26 S RNA and protein syntheses are inhibited, and the proportion of cells releasing virus is dramatically reduced (Davey and Dalgarno, 1974 Eaton, 1979). Inhibition of 42 S RNA synthesis occurs at 3 days after infection (start of chronic phase see Fig. 2). The nature of the factor(s) responsible for inhibiting viral replication in infected mosquito cells at the chronic phase is not known. The fact that large amounts of 42 S RNA are made in infected mosquito cells 48 hr after infection, at a time when viral structural protein synthesis is inhibited, suggests that the replication inhibition factor may act initially at the level of viral protein synthesis (Eaton, 1979). Eaton demonstrated that viral structural protein synthesis is inhibited before a decrease in 26 S RNA synthesis is detected in SIN-infected A. albopictus cells. Thus, the biphasic nature of alphavirus infection is also observed in the case of flavivirus infection in mosquito cells (Paul et al., 1969). 

D. F. Bowers et al. Replication and tissue tropism of the alphavirus Sindbis in the mosquito Aedes albopictus. Virology 212, 1, 1995. 

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