The RCAS vectors are simple, replication-competent avian retroviruses. They adhere to the rules that govern retroviral replication. For this reason, the RCAS vectors are widely used as tools to study retroviral replication. Including a scorable (GFP, AP, etc.) or a selectable (puro, neo, etc.) marker in the vector makes it much simpler to monitor the viral titer and viral replication. The retroviral life cycle is shown schematically in Figure 1 below. The life cycle begins with the binding of the envelope glycoprotein on the surface of the virus to a cognate receptor on the surface of the target cell. This interaction leads to the fusion of the viral and cellular membranes. Although in the figure this process is depicted as occurring at the plasma membrane, recent data suggest that the ASLVs may use a pathway similar to that used by influenza virus and enter the cell through a low-pH mechanism (Mothes et al., 2000).
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Figure 1. A diagrammatic representation of the retroviral life cycle.
After viral and cellular membranes fuse, the viral core is in the host cytoplasm, where the single-stranded RNA genome of the virus is converted into double-stranded linear DNA by the enzyme reverse transcriptase. Understanding the mechanics of the reverse transcription process is important for good vector design (see Overview – What to Avoid in Vector Design). However, the details of the reverse transcription process are beyond the scope of this website. If you would like to learn more about reverse transcription, please refer to Retroviruses (edited by John M. Coffin, Stephen H. Hughes, and Harold E. Varmus, 1997, Cold Spring Harbor Laboratory Press), which is now available as an online publication through the National Library of Medicine website.
Once the double-strand viral DNA is synthesized, it must move from
the cytoplasm to the nucleus. The viral DNA of some complex retroviruses,
such as HIV-1, can transit the nuclear membrane. The viral DNA of
some simple retroviruses, including MLV, does not transit the nuclear
membrane and these viruses cannot infect nondividing cells. However, it was
recently shown that ASLVs (including RCAS vectors) can infect nondividing
cells in culture, albeit at a reduced efficiency relative to infection
of dividing cells (Hatziioannou
and Goff, 2001; Katz
et al., 2002).
Once the viral DNA has access to the host genome, integration can
take place. Integration links the ends of the linear viral DNA to
host DNA. The reaction is specific with respect to the viral sequences
involved and nonspecific with respect to the host sequences. The site
of integration in the host genome influences the expression of the
integrated provirus. Although the provirus has its own promoter, the
activity of the viral promoter is influenced by the adjacent host
sequences. Conversely, the insertion of the provirus can also influence
the expression of host genes. The provirus is an insertional mutagen:
it can disrupt a gene by inserting into it, or can either enhance
or decrease the expression of gene by inserting next to it. Because higher eukaryotes are diploid, this ordinarily does not matter; however,
in some cases, insertion near a cellular oncogene can lead to oncogenesis
in infected animals. Because the viral DNA becomes part of the host
genome, infection is permanent. The infected cells, and all their
progeny, will carry the inserted viral sequence. If the infection
takes place in a germ cell, or a germ cell precursor, the resulting
animal will carry the virus as a transgene (usually called an endogenous
It is sometimes useful, in studying viral replication, to recover either unintegrated or integrated viral DNA. With conventional retroviruses (or retroviral vectors), recovery usually involves PCR amplification and/or cloning. However, it is possible to generate
retroviral shuttle vectors that can be propagated either as viral stocks or as plasmids in E. coli. This makes it easy to recover either unintegrated or integrated viral DNA. We have prepared a set of RCASBP(A)-derived shuttle vectors, the RSVPs. These vectors are listed in Table 2; a more complete description is provided in Oh et al., 2002.
Once embedded in the host genome, the provirus behaves like a cellular gene. For simple retroviruses, such as ASLVs, expression is entirely under the control of the host’s machinery. The RNA is transcribed by the host’s DNA-dependent RNA polymerase. Unless the experimentalist has inserted an internal promoter, there is, for simple retroviruses, one primary transcript that begins at the U3 R junction in the upstream LTR and terminates at the R U5 junction in the downstream LTR. This full-length RNA is exported from the nucleus and serves as genomic RNA and as the message for the Gag and Gag-Pol polyproteins. A portion of the full-length RNA is spliced to produce the message for the Env protein, and in the case of RSV and the RCAS vectors, there is a second spliced message (see Figure 2B below), which leads to the expression of the src gene in RSV or the inserted gene in RCAS.
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Figure 2. RSV virion structure and expression. Panel A
shows a diagrammatic representation of a mature Rous sarcoma virus
(RSV) virion. The legend at the right identifies individual proteins
found in the mature virion. The outer membrane of the virion contains
the transmembrane protein (TM), which is associated with the surface
protein (SU). The matrix protein (MA) lies just under this outer
membrane. The core of the virion is structurally delimited by the
capsid protein (CA). Inside the capsid are two viral RNA genomes,
shown partially covered with nucleocapsid protein (NC). The two
genomic RNAs are hydrogen bonded near their 5' ends. The core also
contains reverse transcriptase (RT), integrase (IN), and protease
(PR). Panel B shows the relationship of the proviral DNA, the open
reading frames, viral RNAs, and proteins of RSV. The LTRs of the
provirus are shown as a series of three boxes (U3, R, and U5). The
viral genome is divided into gag, pol, env,
and src genes. Adjacent host DNA is shown as a wavy double
line. In the case of RSV, the gag and env genes are
in the same reading frame; pol and src are in a different
reading frame. Host DNA-dependent RNA polymerase transcribes the
provirus, yielding a full-length RNA that serves both as the viral
genome and as the message for the gag and gag-pol
polyproteins. This RNA is capped (indicated by pppG) and polyadenylated
(AAAn). Approximately 5% of the time full-length RSV RNA is translated, there is a frameshift event that allows the ribosome to synthesize the gag-pol polyprotein.
The gag and gag-pol polyproteins are processed by
the viral PR to yield the proteins found in the mature virion. The
relative positions of these components in the polyproteins are shown.
The RSV polyproteins contain a p10 protein whose function is not
well understood. A portion of the full-length viral RNA is processed
by the host splicing machinery, which gives rise to the env
and src mRNAs. The env mRNA is translated into the
envelope glycoprotein, which is cleaved into SU and TM by a host
cell protease. The src mRNA is translated into the src
The messages are translated in the cytoplasm. The gag gene is the 5'-most gene in unspliced RNA. In the case of the ASLVs, the pol gene is in a different reading frame. During ~5% of Gag translation, a frameshift suppression event causes a ribosome near the gag-pol boundary to slip over into the pol gene. This leads to the synthesis of the Gag-Pol polyprotein. Gag and Gag-Pol self-assemble at the plasma membrane, picking up two copies of viral genomic RNA and some small host RNAs — in particular, the tRNA trp, which is used to initiate reverse transcription. The assembled virus buds through the plasma membrane of the host cell. In so doing, it not only acquires a membrane, but picks up the envelope glycoprotein along with it. Either during or shortly after budding, the viral protease cleaves the Gag and Gag-Pol polyproteins, giving rise to infectious virus. The newly budded virus cannot (usually) reinfect the cell that released it. Ordinarily, the infected cell produces enough of the envelope glycoprotein to block its own receptors, a process called receptor interference.
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