Virus Replication

As with other retroviruses, little is known about the processes that govern the entry and exit of MMTV from infected host cells. However, it is clear that the surface glycoprotein gp52 (SU) mediates the attachment of the virus to the host-encoded viral receptor

(also known as MTVR). Monoclonal antibodies against gp52 will block viral entry. Following entry and partial uncoating due to adsorptive endocytosis, the virally encoded reverse transcriptase produces a double-stranded (ds) DNA copy (also known as the provirus) of the viral RNA. The viral RT synthesizes a minus-strand DNA primed by lysyl-tRNA to form an RNA-DNA hybrid. Using the virally encoded ribonuclease H, the majority of the virion RNA is degraded, and the plus-stranded DNA is completed to form the provirus that, in contrast to the template RNA, contains a long terminal repeat composed of U3, R and U5 regions (Fig. 1).

The provirus then enters the nucleus subsequent to nuclear membrane breakdown during mitosis, and viral DNA integrates using the MMTV-encoded integrase (IN) protein, one of the products of the pol gene. The IN protein introduces an asymmetric cut 2 bp from the linear ends of the provirus as well as an asymmetric break exactly 6 bp apart on opposite DNA strands of the host cell chromosome. Although there appear to be no discrete sites for proviral integration, like other retroviruses, it is possible that integration occurs preferentially in regions that have an open chromatin structure, e.g. transcriptionally active sites. Following joining events, the repair of the virus—cell junction generates a direct repeat of cellular DNA that, in turn, flanks the viral LTRs. This structure resembles the transposable elements of bacteria, yeast and Drosophila.

The integrated provirus contains all the signals necessary for recognition by RNA polymerase II, and many of these signals are present in the U3 region of the LTR. Generally, transcription from the standard promoter is initiated in the 5' LTR starting at the U3/ R junction and terminating at the R/U5 junction in the 3' LTR (Fig. 1). Because of the terminal redundancy, the polyadenylation signals are present in both LTRs, but the sequence in the 5' LTR is ignored because of the multipartite nature of the poly(A) signal. Termination appears to be reasonably inefficient, and a number of MMTV transcripts probably terminate in the adjacent cellular DNA. Transcripts are capped by cellular enzymes in the nucleus. A portion of this RNA leaves the nucleus in the unspliced form, whereas the remainder is spliced in at least two alternative ways to generate the envelope mRNA and, in some cases, the superantigen or sag mRNA.

Full-length transcripts (8.7 kb) are translated into Gag, Gag-Pro and Gag-Pro-Pol precursor proteins. Since pro (the viral protease gene) and pol (the polymerase gene) are out-of-frame with respect to gag and each other, two independent ribosomal frameshifts are required to obtain Gag-Pro and then

Gag-Pro-Pol. Because of the ribosomal frameshifting events, the viral PR and RT are made at a fraction of the amounts of Gag synthesized. The viral envelope mRNA is translated into a precursor protein on membrane-bound polyribosomes. The protein precursor is modified by glycosylation in both the endoplasmic reticulum and the Golgi, and protein cleavage to SU and TM also occurs in the latter compartment. The sag mRNA (approximately 1.4 kb) appears to be translated into a type II transmembrane protein of 36 kDa; this protein is glycosylated and reportedly cleaved to generate a C-terminal 18 kDa fragments. Sag is associated with major histocompatibility complex (MHC) type II protein. Unlike the other virally encoded proteins, Sag is not believed to be a structural component of virions (see below).

Recent evidence suggests that sag mRNA can be initiated from at least two other MMTV promoters (Fig. 1). One of these promoters is located within the U3 region of the LTR, resulting in RNA initiation approximately 500 bp upstream of the U3/R junction. This RNA can be processed to form both singly and doubly spliced RNAs containing only the sag open reading frame. In addition, a promoter within the envelope region gives a spliced RNA with a different splice donor, but the same splice acceptor as that originally described for spliced sag RNAs from the standard promoter. The use of the standard promoter, the internal U3 promoter or the envelope promoter for synthesis of Sag protein appears to be specific for different strains of MMTV. The amount of spliced sag mRNA produced from any of these promoters appears to be very low compared to the amount of gag-pol or env mRNAs produced.

The precursors for Gag, Gag-Pro, and Gag-Pro-Pol aggregate within the cell cytoplasm into procapsids (approximately 70 nm in diameter) that are referred to as intracytoplasmic A particles. This process is distinct from the maturation of C-type particles which assemble the Gag precursors at the cell surface membrane concomitant with the budding process. Presumably the precursor proteins are folded so that nucleocapsid and RT proteins are sequestered inside the particle to interact with a dimer of viral RNA. The viral PR, which is present in a fraction of the Gag precursors, is apparently responsible for the cleavage events that produce the mature virion proteins MA, p21 (function unknown), CA and NC. The procapsid very likely initiates budding through viral glycopro-tein-modified cellular membranes following interaction between the MA protein and the cytoplasmic tail of the TM protein. RT appears to be inactive in newly forming virions since enzyme activation requires several cleavage events by PR to form the infectious mature B-particles.

Figure 2 Life cycle of milk-borne MMTV. Newborn pups ingest virus-infected milk from their mothers, and virus particles pass through the stomach to encounter lymphoid cells associated with the gut epithelial cells. B cells become infected and express Sag at the cell surface in conjunction with MHC class II protein. The Sag-MHC complex is recognized by specific T cell subsets that respond by the release of cytokines. These cytokines stimulate the proliferation of bystander B and T cells that may be the target for additional MMTV integrations. Cytokines also amplify the number of previously infected cells. The mechanism for transfer of MMTV infection by lymphoid cells to the mammary gland is unclear.

Figure 2 Life cycle of milk-borne MMTV. Newborn pups ingest virus-infected milk from their mothers, and virus particles pass through the stomach to encounter lymphoid cells associated with the gut epithelial cells. B cells become infected and express Sag at the cell surface in conjunction with MHC class II protein. The Sag-MHC complex is recognized by specific T cell subsets that respond by the release of cytokines. These cytokines stimulate the proliferation of bystander B and T cells that may be the target for additional MMTV integrations. Cytokines also amplify the number of previously infected cells. The mechanism for transfer of MMTV infection by lymphoid cells to the mammary gland is unclear.

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