Retroviruses can be classified as simple or complex. The genomes of simple retroviruses, such as murine leukemia virus (MLV), encode only the virion proteins and enzymes required for viral replication. In contrast, complex retroviruses, human immunodeficiency virus (HIV)-1 or human T cell leukemia virus (HTLV) 1, encode in addition a variety of non-structural proteins that facilitate various steps of the replication pathway or counteract cellular and immunological anti-viral host responses. While MMTV was initially classified as a simple retrovirus, it is now clear that it probably lies somewhere in between viruses like MLV and HIV-1 in complexity.
The MMTV genome is approximately 9 kb in size. At least five transcripts are generated from the viral genome, four of which initiate in the 5′ long terminal repeat (LTR) and terminate in the 3′ LTR; the different transcripts are generated by alternative splicing (). The LTR also contains binding sites for transcription factors that determine hormone-responsive and tissue-specific transcription, both of which are necessary for in vivo
infection and optimal virus production. Specifically, the LTRs encode sites that regulate both mammary epithelial and lymphoid cell-specific expression, as well as glucorticoid/progesterone response elements that cause increased virus transcription during pregnancy and lactation, when virions are shed into milk (4
). Because the MMTV LTR encodes transcriptional regulatory elements that direct high level expression in mammary epithelial cells, it has been widely used to drive transgene expression in mouse mammary tissue (reviewed in XXX, this volume).
MMTV proviral genome and gene products.
Like all retroviruses, the full-length, unspliced MMTV RNA serves two functions. First, two copies are packaged into virions and thus provide the viral genome. Second, the full-length transcript serves as the mRNA for the gene products encoded by the gag
). The gag
translation product is a polyprotein precursor that is processed by the viral protease, PR or Pro, into the capsid (CA) and nucleocapsid (NC) proteins, as well as several other peptides of unknown function. Both the Dut-Pro and Pol polyproteins are translated from the same mRNA as Gag, but in different reading frames, by a process termed ribosomal frameshifting. The pro
gene encodes the viral protease and dut,
a dUTPase, whose role in virus infection is not known. However, for other retroviruses that encode a dUTPase, such as equine infectious anemia virus (EIAV), it is believed that this protein contributes to pathogenesis by maintaining adequate nucleotide pools and thereby facilitating productive viral replication in non-dividing cells (10
). Since MMTV infects dendritic cells (DCs), which are non-dividing in vivo
, the dUTPase could play a similar role. The pol
gene codes for reverse transcriptase (RT), needed to generate the double-stranded DNA, and the integrase (IN), which is required for integration of this DNA into the host chromosome.
A singly spliced mRNA is translated from the envelope (env
) gene, which is cleaved by host furin enzymes to yield two polypeptides, the surface (SU) and transmembrane (TM) domains of the Env protein, required for binding of the virions to cell surface receptor(s) that mediate cell entry (9
). The SU domain carries the receptor binding site (RBS), while the TM domain mediates virion-cell membrane fusion required for entry. The MMTV entry receptor is transferrin receptor 1 (TfR1) (11
). TfR1 belongs to a class of cell surface receptors that traffic to the acidic endosome upon ligand binding. Unlike MLV or HIV-1, which enter cells via the surface or a neutral compartment, MMTV entry occurs in a late endosomal compartment and probably requires co-trafficking of virions with receptor. The identification of TfR1 as the MMTV entry receptor explains in part the in vivo tissue-specific tropism of this virus, since activated cells of the immune system and dividing mammary epithelial cells express some of the highest levels of this protein in vivo
). However, cell-type restriction in vivo
is also probably due to post-entry events. For example, the enhancer elements in the LTR function predominantly in mammary epithelia and lymphoid cells and thus, MMTV is not transcribed in many tissues (15
Retroviral Env proteins can have other activities in addition to mediating cellular entry and recent work has indicated that the MMTV Env protein may play additional roles in in vivo
infection and MMTV-mediated tumorigenesis. In addition to interacting with TfR1 to mediate viral entry, the Env protein has been shown to activate antigen presenting cells, like DCs and B cells, via Toll-like receptor 4 (TLR4) (16
). TLR4 is a member of a family of receptors that contribute to innate immune responses to pathogens (18
). Env interaction with TLR4 may facilitate initial infection of cells of the immune system (see next section). The Env protein may also participate in the transformation of mammary epithelial cells (see below).
At least two other proteins required for efficient MMTV infection are encoded by additional alternatively spliced mRNAs (). These include a viral protein with HIV-1 rev-like activity required for efficient transport of unspliced MMTV mRNA from the nucleus, termed regulator of export of MMTV (Rem) (19
) and the viral superantigen (Sag), encoded in the 3′ LTR, which is dispensable for in vitro
infection but plays a critical role in virus dissemination in vivo
(see next section). Several other alternatively spliced mRNAs originating from the MMTV genome have also been described, but their gene products and role in infection have not been well-studied (21
In summary, since MMTV encodes a number of accessory proteins, as well as an Env that plays multiple roles, it is clearly not a simple retrovirus. Unlike HIV-1 and HTLV-1, viral gene products equivalent to tat and tax, respectively, which function as transcriptional activators of virus and host transcription have not been found in the MMTV genome. However, MMTV does encode proteins that allow it to alter the host immune system, as well as a protein that facilitates transport of unspliced viral RNA out of the nucleus. MMTV may encode these accessory proteins, particularly Sag, because its in vivo infection pathway requires trafficking through diverse cell types, as described in the next section.