Subviral Agents
This chapter considers a number of infectious agents that
Defective interfering viruses are a special class of defec-
are subcellular, but that are not viruses in the strict sense
tive viruses that arise by recombination and rearrangement of
of the term. Some of these are not capable of independent
viral genomes during replication. DIs are defective because
replication but require a helper virus, in which case the agent
they have lost essential functions required for replication.
is effectively a parasite of a parasite. Others replicate inde-
Thus, they require the simultaneous infection of a cell by
pendently but use unconventional means to achieve their
a helper virus, which is normally the parental wild-type virus
replication and spread. Many of the agents discussed here
from which the DI arose. They interfere with the replication
cause important diseases in plants or animals, including
of the parental virus by competition for resources within the
cell. These resources include the machinery that replicates
The agents to be considered include defective interfer-
the viral nucleic acid, which is in part encoded by the helper
ing (DI) viruses that arise by deletions and rearrangements
virus, and the proteins that encapsidate the viral genome to
in the genome of a virus. DIs require coinfection by a
form virions.
helper virus to replicate. They may play an important role
DIs of many RNA viruses have been the best studied.
in modulation of viral disease or they may simply be arti-
Because DI RNAs must retain all cis-acting sequences
facts that arise in laboratory studies. Related to DIs, at least
required for the replication of the RNA and its encapsidation
conceptually, are satellites of viruses, which can replicate
into progeny particles, sequencing of such DI RNAs can pro-
only in the presence of a helper. Satellites are known for
vide clues as to the identity of these sequences. Identification
many plant viruses and are known to influence the viru-
of cis-acting sequences is important for the construction of
lence of the helper viruses. Completely different are agents
virus vectors used to express a particular gene of interest,
called viroids. Viroids consist of small, naked RNA mol-
whether in a laboratory experiment or for gene therapy.
ecules that are capable of directing their own replication.
The most highly evolved DI RNAs are often not trans-
They do not encode protein, but instead contain promoter
lated and consist of deleted and rearranged versions of the
elements that cause cellular enzymes to replicate them.
parental genome. In the case of alphaviruses, whose genome
Many are important plant pathogens. Related to viroids
is about 12 kb (Chapter 3), DI RNAs have been described
are virusoids, which are satellites of viruses that resemble
that are about 2 kb in length. However, they have a sequence
packaged viroids. There are also agents that combine the
complexity of only 600 nucleotides, because sequences are
attributes of both satellites and viroids, such as hepatitis
repeated one or more times. The sequences of two such DI
delta virus, which is an important human pathogen. Finally,
RNAs of Semliki Forest virus (SFV) are illustrated schemat-
prion diseases, caused by infectious agents whose identity
ically in Fig. 9.1. From the sequences of these DIs as well as
is controversial but which may consist only of protein, are
DIs of other alphaviruses, specific functions for the elements
found in these DIs have been proposed. Other approaches
length = 2286; complexity = 1169
Sequences in DIb expanded 4x
Subgenomic Promoter
Nonstructural Proteins
Structural Proteins
Sequences in DIa expanded 4x
length = 1652; complexity = 680
Schematic representation of DIs (defective interfering particles) found after high multiplicity infection
of Semliki Forest virus. The central block shows the genome of the nondefective virus, with vertical lines demarking
the four nonstructural and five structural polypeptides. The blocks of sequence found in two different DIs are expanded
fourfold below and above. Their location in the DI genome is illustrated with blocks of identical shading. Note that some
blocks of unique sequence are repeated three times in DIa and one block is repeated four times in DIb. Adapted from
Strauss and Strauss (1997), Figure 1.
3end of the antigenomic RNA is stronger than the promoter
have then been used to confirm the hypotheses derived from
such sequence studies. Thus, the 3end of the parental RNA,
at the 3end of the genomic RNA. Thus, it is not surprising
that some DI RNAs have the stronger promoter at the 3ends
which is retained in all alphavirus DI RNAs, forms a pro-
moter for the initiation of minus-strand RNA synthesis from
of both (+) and (-)RNA (as in Class II DIs), ensuring more
the plus-strand genome. The 5end of the RNA is also pre-
rapid replication of the DI RNAs. The DI RNAs may have
served in many DI RNAs, such as those illustrated in Fig.
the luxury of doing this because they are not translated nor
9.1. Surprisingly, however, it has been replaced by a cellular
do they serve as templates for the synthesis of mRNAs.
tRNA in some DI RNAs. The complement of this sequence
The well-studied alphavirus DI RNAs and the VSV DI
is present at the 3end of the minus strand, where it forms a
RNAs are not translated. For many DI RNAs, however,
promoter for initiation of genomic RNA synthesis. The find-
translation is required for efficient DI RNA replication. The
ing that the DI RNAs with the tRNA as the 5terminus have
best studied examples of this are DIs of poliovirus and of
a selective advantage over the parental genome during RNA
coronaviruses (these viruses are described in Chapter 3). DI
replication suggests that this promoter is a structural element
RNAs of poliovirus are uncommon and contain deletions in
recognized by the viral replicase. It also suggests that the
the structural protein region. It has been suggested that in
element present in the genomic RNA is suboptimal, perhaps
this case it is the translation product that is required for effi-
because the genomic RNA must be translated as well as rep-
cient replication of the RNA (the replicase translated from
licated. Finally, repeated sequences from two regions of the
the RNA may preferentially use as a template for replication
genome are present in all alphavirus DI RNAs. It is thought
the RNA from which it was translated). In contrast, for at
that one sequence (shown as red patterned blocks in Fig. 9.1)
least one well-studied DI of a coronavirus, translation of
is an enhancer element for RNA replication and the second
the RNA is required for efficient replication, but the transla-
(shown as yellow and green patterned blocks) is a packaging
Yon product is not important. In this case, translation may
signal. Repetition of these elements may increase the efficiency
stabilize the DI RNA, since there appears to be a cellular
of replication and packaging of the DI RNA.
pathway to rid the cell of mRNAs that are not translatable.
Vesicular stomatitis virus (VSV) (Chapter 4) DI RNAs
If so, it is uncertain how DI RNAs that are not translated
vary in size from a third to half the length of the virion RNA.
avoid this pathway. Some representative naturally occurring
Some DI RNAs are simply deleted RNA genomes, but others
DIs of mouse hepatitis virus, a murine coronavirus, are
have rearrangements at the ends of the RNAs. Representative
illustrated in Fig. 9.2B.
examples are illustrated in Fig. 9.2A. During replication of
Because DI RNAs are replicated by the helper virus
the RNA, the sequences at the ends must contain promoter
machinery and encapsidated by the capsid proteins of
elements for initiation of RNA synthesis. More genomic
the helper virus, they interfere with the parental virus by
RNA (minus strand that is packaged in virions) is made than
diverting these resources to the production of DI particles
antigenomic RNA (which functions only as a template for
rather than to the production of infectious virus particles. It
genomic RNA synthesis) and therefore the promoter at the
was the first noted by von Magnus in the early 1950s that
Vesicular stomatitis virus (VSV) and DIs derived from it
tr  VSV viral RNA
Strand le
Types of VSV DIs
Class I -Panhandle
Class II -Hairpin
Class III -Simple internal deletion
Class IV - Mosaic
tr Trailer
Minus-strand sequences
Plus-strand sequences
Complement of trailer
le Leader
Murine hepatitis virus (MHV) and DIs derived from it
MHV viral RNA
(~30 kb)
DIssA (~25 kb)
An DI-a (5.5 kb)
An DIssF (3.6 kb)
An DIssE (2.2 kb)
An B36 (2.2 kb)
FIGURE 9.2  Types of DIs generated from a rhabdovirus and a coronavirus. Upper panel: diagrammatic representation
of the VSV genome and members of the four classes of DI particles. The leader and trailer are shown as patterned
blocks. The genome is shown 3to 5for the minus strand (ochre underline). The parts of the DIs corresponding to the
complement of the minus strand are underlined in green. A red triangle marks the internal deletion in the L gene, which
is found in Class III and Class IV DIs. Adapted from Whelan and Wertz (1997). Lower panel: structures of naturally
occurring DI RNAs of MHV (a murine coronavirus). DIssA, DI-a, etc. were isolated from MHV-infected cells. The
bottom line shows a synthetic DI replicon called B36. Sequences in the DIs are color coded by their region of origin in
the parental virus genome. Adapted from Brian and Spaan (1997) Figure 1.
influenza virus, passed at high multiplicity for many pas-
yields of virus. Thus, the yield of infectious virus continues
sages, produced yields that cycled between high and low.
to fluctuate.
This effect is illustrated schematically in Fig. 9.3A. We
In a laboratory setting, at least, DIs can drive the evolu-
now know that this is due to the presence of DI particles.
tion of the wild-type virus. This is shown schematically in
In early passages virus yields are high. When DIs arise,
Fig. 9.3B. When virus is passed at high multiplicity for very
they depress the yield of virus. Because high multiplici-
many generations, mutants often arise that have altered pro-
ties of infection are required to maintain DI replication, so
moters that are recognized by mutant replication proteins.
that cells are infected with both the helper and the DI, low
Such mutants are resistant to the DIs that are in the popula-
yields of virus lead to a reduction in DI replication in the
tion at the time, because the mutant replication proteins do
next passage or two. Reduced DI replication leads to higher
not recognize the promoters in the DIs. The mutant virus
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